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1.  Need for Supplemental Oxygen at Discharge in Infants with Bronchopulmonary Dysplasia Is Not Associated with Worse Neurodevelopmental Outcomes at 3 Years Corrected Age 
PLoS ONE  2014;9(3):e90843.
Objectives
To determine if chronic oxygen dependency (discharge home on supplemental oxygen) in children with bronchopulmonary dysplasia (BPD; defined as requirement for supplemental O2 at 36 weeks postmenstrual age) predicts neurodevelopmental disability rates and growth outcomes at 36 months corrected age (CA).
Study Design
Longitudinal cohort study.
Setting
Southern Alberta regional center located at high altitude.
Participants
Preterm infants weighing ≤1250 grams with no BPD, BPD, and BPD with chronic oxygen dependency.
Main outcome measures
Neurodevelopmental and growth outcomes.
Results
Of 1563 preterm infants admitted from 1995–2007, 1212 survived. Complete follow-up data were available for 1030 (85%) children. Children in BPD and BPD with chronic oxygen dependency groups had significantly lower birth weights, gestational ages, prolonged mechanical ventilation and oxygen supplementation and received more postnatal steroids, compared to those without BPD. Children with BPD and BPD with chronic oxygen dependency were more likely to be below the 5th centile in weight and height compared to those without BPD but there was little difference between the BPD and BPD with chronic oxygen dependency groups. After controlling for confounding variables, children who had BPD and BPD with chronic oxygen dependency had higher odds of neurodevelopmental disability compared to those without BPD [OR (odds ratio) 1.9 (95%CI 1.1 to 3.5) and OR 1.8 (1.1 to 2.9), respectively], with no significant difference between BPD and BPD with chronic oxygen dependency [OR 0.9 (95% CI 0.6 to 1.5)].
Conclusions
BPD and BPD with chronic oxygen dependency in children predicts abnormal neurodevelopmental outcomes at 36 months CA. However, the neurodevelopmental disability rates were not significantly higher in BPD with chronic oxygen dependency children compared to children with BPD only. Compared to those without BPD, growth is impaired in children with BPD and BPD with chronic oxygen dependency, but no difference between the latter two groups.
doi:10.1371/journal.pone.0090843
PMCID: PMC3960119  PMID: 24646665
2.  Targeting mitochondrial dysfunction in lung diseases: emphasis on mitophagy 
During mild stressful conditions, cells activate a multitude of mechanisms in an attempt to repair or re-establish homeostasis. One such mechanism is autophagic degradation of mitochondria or mitophagy to dispose damaged mitochondria. However, if stress persists beyond recovery then dysfunctional mitochondria can ignite cell death. This review article summarizes recent studies highlighting the molecular pathways that facilitate mitochondria to alter its morphological dynamics, coordinate stress responses, initiate mitophagy and activate cell death in relevance to pulmonary pathologies. Thorough understanding of how these signaling mechanisms get disrupted may aid in designing new mitochondria-based therapies to combat lung diseases.
doi:10.3389/fphys.2013.00384
PMCID: PMC3872744  PMID: 24421769
mitochondria; reactive oxygen species; mitophagy; apoptosis; pulmonary disease
3.  Which Biomarkers Reveal Neonatal Sepsis? 
PLoS ONE  2013;8(12):e82700.
We address the identification of optimal biomarkers for the rapid diagnosis of neonatal sepsis. We employ both canonical correlation analysis (CCA) and sparse support vector machine (SSVM) classifiers to select the best subset of biomarkers from a large hematological data set collected from infants with suspected sepsis from Yale-New Haven Hospital's Neonatal Intensive Care Unit (NICU). CCA is used to select sets of biomarkers of increasing size that are most highly correlated with infection. The effectiveness of these biomarkers is then validated by constructing a sparse support vector machine diagnostic classifier. We find that the following set of five biomarkers capture the essential diagnostic information (in order of importance): Bands, Platelets, neutrophil CD64, White Blood Cells, and Segs. Further, the diagnostic performance of the optimal set of biomarkers is significantly higher than that of isolated individual biomarkers. These results suggest an enhanced sepsis scoring system for neonatal sepsis that includes these five biomarkers. We demonstrate the robustness of our analysis by comparing CCA with the Forward Selection method and SSVM with LASSO Logistic Regression.
doi:10.1371/journal.pone.0082700
PMCID: PMC3867385  PMID: 24367543
4.  Hyperoxia Exacerbates Postnatal Inflammation-Induced Lung Injury in Neonatal BRP-39 Null Mutant Mice Promoting the M1 Macrophage Phenotype 
Mediators of Inflammation  2013;2013:457189.
Rationale. Hyperoxia exposure to developing lungs—critical in the pathogenesis of bronchopulmonary dysplasia—may augment lung inflammation by inhibiting anti-inflammatory mediators in alveolar macrophages. Objective. We sought to determine the O2-induced effects on the polarization of macrophages and the role of anti-inflammatory BRP-39 in macrophage phenotype and neonatal lung injury. Methods. We used RAW264.7, peritoneal, and bone marrow derived macrophages for polarization (M1/M2) studies. For in vivo studies, wild-type (WT) and BRP-39−/− mice received continuous exposure to 21% O2 (control mice) or 100% O2 from postnatal (PN) 1 to PN7 days, along with intranasal lipopolysaccharide (LPS) administered on alternate days (PN2, -4, and -6). Lung histology, bronchoalveolar lavage (BAL) cell counts, BAL protein, and cytokines measurements were performed. Measurements and Main Results. Hyperoxia differentially contributed to macrophage polarization by enhancing LPS induced M1 and inhibiting interleukin-4 induced M2 phenotype. BRP-39 absence led to further enhancement of the hyperoxia and LPS induced M1 phenotype. In addition, BRP-39−/− mice were significantly more sensitive to LPS plus hyperoxia induced lung injury and mortality compared to WT mice. Conclusions. These findings collectively indicate that BRP-39 is involved in repressing the M1 proinflammatory phenotype in hyperoxia, thereby deactivating inflammatory responses in macrophages and preventing neonatal lung injury.
doi:10.1155/2013/457189
PMCID: PMC3855965  PMID: 24347826
5.  FETAL RENAL ARTERY IMPEDANCE AS ASESSED BY DOPPLER ULTRASOUND IN PREGNANCIES COMPLICATED BY INTRA-AMNIOTIC INFLAMMATION AND PRETERM BIRTH 
OBJECTIVE
To evaluate the fetal renal artery impedance in the context of inflammation-associated preterm birth (PTB).
STUDY DESIGN
We conducted a prospective Doppler assessment of the fetal renal artery impedance in 70 singleton fetuses. The study group consisted of 56 premature fetuses (28.1 [25.3–30.6] weeks at enrollment). Gestational age (GA) reference ranges were generated based on fetuses with uncomplicated pregnancies (n=14). Doppler studies included renal artery pulsatility index (PI), resistance index (RI), systolic/diastolic (S/D) ratio and presence-or-absence of end-diastolic blood flow. We assessed amniotic fluid (AF) inflammation by proteomic profiling (SELDI-TOF). Data were interpreted in relationship to amniotic fluid index (AFI), cord blood interleukin-6 (IL-6) and erythropoietin (EPO) levels. The cardiovascular and metabolic profiles of the neonates were investigated in the first 24 hours of life.
RESULTS
Fetuses delivered by mothers with intra-amniotic inflammation had higher cord blood IL-6 but not EPO levels. Fetal inflammation did not affect either renal artery PI,RI,S/D ratio or end-diastolic blood flow. Neonates delivered in the context of intraamniotic inflammation had higher serum blood urea nitrogen levels, which correlated significantly with AF IL-6 levels. The renal artery RI and SD ratio were inversely correlated with the AFI independent of GA, cord blood IL-6 and status of the membranes.
CONCLUSION
The fetus is capable of sustaining normal renal artery impedance despite inflammation. Resistance in the renal vascular bed affects urine output independent of inflammation.
doi:10.1016/j.ajog.2008.11.001
PMCID: PMC3791328  PMID: 19185102
6.  Fetal Inflammatory Response in Women with Proteomic Biomarkers Characteristic of Intra-Amniotic Inflammation and Preterm Birth 
Objective
To determine the relationship between presence of amniotic fluid (AF) biomarkers characteristic of inflammation (defensins 2 and 1, calgranulins C and A) and fetal inflammatory status at birth.
Design
Prospective observational cohort.
Setting
Tertiary referral University hospital
Population
132 consecutive mothers (gestational age, median [interquartile range]: 29.6 [24.1-33.6] weeks), who had a clinically indicated amniocentesis to rule-out infection and their newborns.
Methods
Intra-amniotic inflammation was diagnosed by mass spectrometry SELDI-TOF. The AF proteomic fingerprint [Mass Restricted (MR) score] ranges from 0-4 (none to all biomarkers present). The intensity of intra-amniotic inflammation was graded based on the number of proteomic biomarkers: MR score 0: “no” inflammation; MR score 1-2: “minimal” inflammation; MR score 3-4: “severe” inflammation. At birth, cord blood was obtained for all cases. Severity of histological chorioamnionitis (HCA) and early onset neonatal sepsis (EONS) was based on established histological and hematological criteria. Interleukin-6 (IL-6) levels were measured by sensitive immunoassays. The cord blood-to-AF IL-6 ratio was used as an indicator of the differential inflammatory response in the fetal versus the AF compartment.
Main Outcome Measures
to relate proteomic biomarkers of intra-amniotic infection to cord blood IL-6 and to use the latter as the primary marker of fetal inflammatory response.
Results
Women with intra-amniotic inflammation delivered at an earlier gestational age (ANOVA, P<0.001) and had higher AF IL-6 levels (P<0.001). At birth, neonates of women with “severe” intra-amniotic inflammation had higher cord blood IL-6 levels (P=0.002) and a higher frequency of EONS (P=0.002). EONS was characterized by significantly elevated cord blood IL-6 levels (P<0.001). Out of the 39 neonates delivered by mothers with “minimal” intra-amniotic inflammation, 15 (39%) had umbilical cord blood IL-6 levels above the mean for the group, and 2 neonates had confirmed sepsis. The severity of the neutrophilic infiltrate in the chorionic plate (P<0.001), choriodecidua (P=0.002), umbilical cord (P<0.001), but not amnion (P>0.05) was an independent predictor of the cord blood-to-AF IL-6 ratio. Relationships were maintained following correction for gestational age, birthweight, amniocentesis-to delivery interval, cesarean delivery, status of the membranes, race, MR score, antibiotics and steroid exposure.
Conclusion
We provide evidence that presence in the AF of proteomic biomarkers characteristic of inflammation is associated with an increased inflammatory status of the fetus at birth. Neonates mount an increased inflammatory status and have positive blood cultures even in the context of “minimal” intra-amniotic inflammation.
doi:10.1111/j.1471-0528.2008.01925.x
PMCID: PMC3791329  PMID: 18947340
Proteomics; biomarkers; amniotic fluid; inflammation; defensin; calgranulin; umbilical cord blood; interleukin-6
7.  A MODEL OF GAG:MIP-2:CXCR2 INTERFACES AND ITS FUNCTIONAL EFFECTS 
Biochemistry  2012;51(28):5642-5654.
MIP-2/CXCL2 is a murine chemokine related to human chemokines that possess the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9Å resolution and created a model with its receptor murine CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G protein-coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model a GAG:MIP-2:CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum but all activity of the mutants is lost in the lung supporting the concept that GAG regulation of chemokines is tissue-dependent.
doi:10.1021/bi3001566
PMCID: PMC3511906  PMID: 22686371
8.  VEGF levels in humans and animal models with RDS and BPD: Temporal relationships 
Experimental lung research  2012;38(4):192-203.
Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) contribute significantly to neonatal morbidity and mortality. Pulmonary function depends on the interaction between alveolar microvasculature and airspace development. While it has been shown in various animal models that vascular endothelial growth factor (VEGF) and its receptors increase in normal animal lung development, its pathophysiological role in neonatal respiratory failure is not yet entirely clear. Current animal and human studies exhibit controversial results. Though animal models are invaluable tools in the study of human lung disease, inherent differences in physiology mandate clarification of the timing of these studies to ensure that they appropriately correlate with the human stages of lung development. The purpose of this review article is to highlight the importance of considering the temporal relationship of VEGF and lung development in human neonates and developmentally-appropriate animal models with RDS and BPD.
doi:10.3109/01902148.2012.663454
PMCID: PMC3705912  PMID: 22394267
VEGF; bronchopulmonary dysplasia; neonatal lung disease; respiratory distress syndrome
9.  Neutrophil Cd64 as a Diagnostic Marker in Neonatal Sepsis 
We assessed neutrophil CD64 as a diagnostic marker for neonatal sepsis. For early-onset sepsis, the CD64 index with a cut-point value of 2.38 had sensitivity, specificity and a negative predictive values of 100%, 68%, and 100%, respectively. For late-onset sepsis, the respective values were 3.62, 75%, 77%, and 96%. Neutrophil CD64 index can be incorporated as a valuable marker for excluding neonatal sepsis.
doi:10.1097/INF.0b013e318256fb07
PMCID: PMC3375383  PMID: 22481422
Sensitivity; specificity; infection; newborn; CD64; diagnosis of sepsis
10.  Cord blood erythropoietin and interleukin-6 for prediction of intraventricular hemorrhage in the preterm neonate 
Objective
To evaluate cord blood erythropoietin (EPO) and interleukin-6 (IL-6) levels to predict preterm infants at risk of developing intraventricular hemorrhage (IVH).
Methods
Levels of umbilical cord EPO, acid–base status and IL-6 were analyzed in 116 consecutive, preterm newborns (GA at delivery: 29 [23–34] weeks) born to mothers who had a clinically indicated amniocentesis to rule out infection. Early-onset neonatal sepsis (EONS) was diagnosed using symptoms, hematological criteria and blood cultures.
Results
IVH was diagnosed by cranial ultrasounds. The prevalence of IVH in our population was 25% (29/116). There was a direct relationship between cord blood EPO and cord blood IL-6 concentration (r = 0.225, p = 0.014), independent of GA at birth. Elevated cord blood EPO levels (r = 0.182, p = 0.016) and GA birth at birth (r = –0.236, p = 0.004) remained significant independent factors associated with the risk of IVH, when evaluated with stepwise logistic regression analyses. Cord blood IL-6, pH, and EONS were not associated with IVH. These relationships remained following correction for GA at birth (p = 0.027).
Conclusions
Our results suggest that elevation in cord blood EPO may predict newborns at risk for IVH, independent of fetal inflammatory status. Further studies are warranted to confirm this association.
doi:10.3109/14767058.2010.520048
PMCID: PMC3683649  PMID: 20937006
Erythropoietin; IL-6; premature; newborn; IVH
11.  Increased Hyperoxia-Induced Lung Injury in Nitric Oxide Synthase 2 Null Mice Is Mediated via Angiopoietin 2 
Supplemental oxygen is frequently prescribed. However, prolonged exposure to high concentrations of oxygen causes hyperoxic acute lung injury (HALI), which manifests as acute respiratory distress syndrome in adults and leads to bronchopulmonary dysplasia in newborns (NBs). Nitric oxide (NO), NO synthases (NOSs), and angiopoietin (Ang) 2 have been implicated in the pathogenesis of HALI. However, the mechanisms of the contributions of NOS/NO and the relationship(s) between NOS/NO and Ang2 have not been addressed. In addition, the relevance of these moieties in adults and NBs has not been evaluated. To address these issues, we compared the responses in hyperoxia of wild-type (NOS [+/+]) and NOS null (−/−) young adult and NB mice. When compared with NOS2+/+ adult controls, NOS2−/− animals manifest exaggerated alveolar–capillary protein leak and premature death. These responses were associated with enhanced levels of structural cell death, enhanced expression of proapoptotic regulatory proteins, and Ang2. Importantly, silencing RNA knockdown of Ang2 decreased the levels of cell death and the expression of proapoptotic mediators. These effects were at least partially NOS2 specific, and were development dependent, because survival was similar in adult NOS3+/+ and NOS3−/− mice and NB NOS2+/+ and NOS2−/− mice, respectively. These studies demonstrate that NOS2 plays an important protective role in HALI in adult animals. They also demonstrate that this response is mediated, at least in part, by the ability of NOS2 to inhibit hyperoxia-induced Ang2 production and thereby decrease Ang2-induced tissue injury.
doi:10.1165/rcmb.2011-0074OC
PMCID: PMC3359903  PMID: 22227562
cytokines; hyperoxia; lung
12.  Risk Factors Associated with Biliary Pancreatitis in Children 
Little is known about risk factors for biliary pancreatitis in children.
Objectives
We characterized cases of pediatric biliary pancreatitis, compared biliary with non-biliary cases, examined differences in presentation between younger and older children, and studied features distinguishing gallstone- from sludge-induced pancreatitis.
Methods
We evaluated 76 episodes of biliary pancreatitis from 271 cases of acute pancreatitis in children admitted to a tertiary care hospital from 1994 to 2007.
Results
Of the 76 cases, 55% had gallstones, 21% had sludge, and 24% had structural defects. Hispanic children had a 2.85 (p=0.01) and 5.59 (p=0.003) higher probability for biliary pancreatitis than white and black children, respectively. Median serum amylase and lipase in children with biliary pancreatitis were 64% and 49% higher, respectively, compared to other etiologies (p<0.05). In multiple logistic regression, aspartate aminotransferase (AST) was an independent predictor of biliary pancreatitis (OR=6.69, p=0.001). When comparing gallstone- with sludge-induced etiologies, obesity was an independent predictor (38% more prevalent, p<0.01) of gallstone cases.
Conclusions
Hispanic ethnicity is a risk factor and AST is a biomarker for biliary pancreatitis over other etiologies. Furthermore, obesity can distinguish gallstone- from sludge-induced pancreatitis. These findings may spur prospective studies to determine the optimal evaluation and management of children with biliary pancreatitis.
doi:10.1097/MPG.0b013e31823a897d
PMCID: PMC3626418  PMID: 22002481
Acute pancreatitis; children; pediatric; biliary; gallstone; sludge; Hispanic ethnicity; obesity
13.  Novel Characterization of Drug-associated Pancreatitis in Children 
Background and Objectives
Medications are a major cause of acute pancreatitis; however, little is known about their influence in children. Our primary aims were to identify common comorbidities and concomitant pancreatitis etiologies in children with drug-associated pancreatitis. Our secondary aims were to identify the most commonly associated drugs in the different age groups, evaluate management practices, and compare drug-associated cases with non–drug-associated cases.
Patients and Methods
In the present study, we examined children (ages 0–20 years) admitted to Yale-New Haven Children’s Hospital with pancreatitis between 1994 and 2007.
Results
Of a total of 271 pediatric cases, drugs were associated with pancreatitis in 25.6% (55). The 3 most common comorbidities in children with drug-associated pancreatitis were seizure disorders, acute lymphocytic leukemia, and Crohn disease. One third of drug-associated cases had an additional pancreatitis etiology. The most commonly associated drugs were valproic acid and corticosteroids. Compared with non–drug-associated cases, children with drug-associated cases were more likely to undergo CT scanning (54.5% vs 28.4%; P < 0.001), stay in the hospital longer (10 vs 4 days; P < 0.001), and transition to parenteral nutrition from a nil per os status (37.5% vs 21.2%; P < 0.05). There was a higher frequency of valproic acid–associated cases in children younger than 11 years (29.4% vs 9.5% in the 11- to 20-year-old age group).
Conclusions
Our study underscores the importance of considering drugs as a cause and a contributor to pancreatitis in children, particularly valproic acid in young children.
doi:10.1097/MPG.0b013e318228574e
PMCID: PMC3626448  PMID: 21681111
acute pancreatitis; children; drug; medications; pediatric; valproic acid
14.  Comparative Microbial Analysis of Paired Amniotic Fluid and Cord Blood from Pregnancies Complicated by Preterm Birth and Early-Onset Neonatal Sepsis 
PLoS ONE  2013;8(2):e56131.
Background
16S rRNA-based genomic analyses have revolutionized our understanding of infectious diseases. Many cases which were recognized as “idiopathic” are now known to have an infectious etiology. Here, we present a proof-of-concept study to examine the microbial link between intra-amniotic infection (IAI) and early-onset neonatal sepsis (EONS).
Results
Using culture independent methods, we analyzed paired amniotic fluid (AF) and cord blood (CB) samples from 36 singleton pregnancies complicated by preterm birth (PTB), IAI, and/or EONS. PTB cases were grouped as 1) Group 1– neonatal blood culture-positive EONS (n = 6). 2) Group 2– neonatal blood culture-negative presumed EONS with positive IAI (n = 16). 3) Group 3– neonatal blood culture-negative presumed EONS with no IAI (n = 7); 4) Group 4– no EONS or IAI (n = 7). In addition, samples from term healthy deliveries (n = 8) served as technical controls. A total of 31 species (15 non-redundant) were identified in AF, of which only 1/3 were cultivated. Significantly fewer microorganisms were detected in CB, with a total of 18 species (7 non-redundant) identified, of which only 2 (Escherichia coli, Streptococcus agalactiae) were cultivated. Of those, Bergeyella, Fusobacterium nucleatum, and Sneathia sanguinegens had not been detected in EONS before. The novel species identified in AF by PCR include Peptoniphilus harei and Lachnospiraceae sp. The majority (72%) of CB species were also detected in the matching AF, with E. coli and F. nucleatum as the most prevalent. The 16S rRNA sequences of paired AF and CB were 99.9–100% identical, while no identical sequences were found between different pregnancies.
Conclusions
Previously unrecognized, uncultivated or difficult-to-cultivate species are implicated in EONS. Microbial species in paired AF and CB likely share the same infectious origin. Given its prevalence in EONS, F. nucleatum should be placed on the same importance scale as E. coli.
doi:10.1371/journal.pone.0056131
PMCID: PMC3577789  PMID: 23437088
15.  SNIPPV vs. NIPPV: DOES SYNCHRONIZATION MATTER? 
Background
Use of nasal intermittent positive pressure ventilation (NIPPV) in the neonatal intensive care unit (NICU) has shown promise with better clinical outcomes in premature neonates. It is not known if synchronization makes a significant clinical impact when using this technique.
Objective
To compare clinical outcomes of premature infants on synchronized NIPPV (SNIPPV) vs. NIPPV in the NICU.
Design/Methods
Retrospective data were obtained (1/04 to 12/09) of infants who received NIPPV anytime in the NICU. SNIPPV (Infant Star with StarSync) was utilized from 2004–06, while NIPPV (Bear Cub) was used from 2007–09. BPD was defined using the NIH consensus definition. Unadjusted associations between potential risk factors and BPD/death were assessed using the chi-square or Wilcoxon Rank Sum test. Adjusted analyses were performed using generalized linear mixed models, taking into account correlation among infants of multiple gestation.
Results
There was no significant difference in the mean gestational age and birth weight in the 2 groups: SNIPPV (n=172; 27.0w; 1016g), NIPPV (n=238; 27.7w; 1117g). There were no significant differences in maternal demographics, use of antenatal steroids, gender, multiple births, SGA, or Apgar scores in the 2 groups. More infants in the NIPPV group were given resuscitation in the delivery room (SNIPPV vs. NIPPV: 44.2% vs. 63%, p<0.001). Surfactant use (84.4% vs. 70.2%; p<0.001) was significantly higher in the SNIPPV group. There were no differences in the rate of PDA, IVH, PVL, ROP, and NEC in the 2 groups. After adjusting for the significant variables, use of NIPPV vs. SNIPPV (OR 0.74; 95%CI: 0.42, 1.30) was not associated with BPD/death.
Conclusions
These data suggest that use of SNIPPV vs. NIPPV is not significantly associated with a differential impact on clinical outcomes.
doi:10.1038/jp.2011.117
PMCID: PMC3534723  PMID: 22116527
Premature newborn; non invasive ventilation; BPD
16.  Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung 
Nature medicine  2004;10(10):1095-1103.
Exaggerated levels of VEGF (vascular endothelial growth factor) are present in persons with asthma, but the role(s) of VEGF in normal and asthmatic lungs has not been defined. We generated lung-targeted VEGF165 transgenic mice and evaluated the role of VEGF in T-helper type 2 cell (TH2)-mediated inflammation. In these mice, VEGF induced, through IL-13–dependent and –independent pathways, an asthma-like phenotype with inflammation, parenchymal and vascular remodeling, edema, mucus metaplasia, myocyte hyperplasia and airway hyper-responsiveness. VEGF also enhanced respiratory antigen sensitization and TH2 inflammation and increased the number of activated DC2 dendritic cells. In antigen-induced inflammation, VEGF was produced by epithelial cells and preferentially by TH2 versus TH1 cells. In this setting, it had a critical role in TH2 inflammation, cytokine production and physiologic dysregulation. Thus, VEGF is a mediator of vascular and extravascular remodeling and inflammation that enhances antigen sensitization and is crucial in adaptive TH2 inflammation. VEGF regulation may be therapeutic in asthma and other TH2 disorders.
doi:10.1038/nm1105
PMCID: PMC3434232  PMID: 15378055
17.  A Role for Matrix Metalloproteinase 9 in IFNγ-Mediated Injury in Developing Lungs 
We noted a marked increase in IFNγ mRNA in newborn (NB) murine lungs after exposure to hyperoxia. We sought to evaluate the role of IFNγ in lung injury in newborns. Using a unique triple-transgenic (TTG), IFNγ-overexpressing, lung-targeted, externally regulatable NB murine model, we describe a lung phenotype of impaired alveolarization, resembling human bronchopulmonary dysplasia (BPD). IFNγ-mediated abnormal lung architecture was associated with increased cell death and the upregulation of cell death pathway mediators caspases 3, 6, 8, and 9, and angiopoietin 2. Moreover, an increase was evident in cathepsins B, H, K, L, and S, and in matrix metalloproteinases (MMPs) 2, 9, 12, and 14. The IFNγ-mediated abnormal lung architecture was found to be MMP9-dependent, as indicated by the rescue of the IFNγ-induced pulmonary phenotype and survival during hyperoxia with a concomitant partial deficiency of MMP9. This result was concomitant with a decrease in caspases 3, 6, 8, and 9 and angiopoietin 2, but an increase in the expression of angiopoietin 1. In addition, NB IFNγ TTG mice exhibited significantly decreased survival during hyperoxia, compared with littermate controls. Furthermore, as evidence of clinical relevance, we show increased concentrations of the downstream targets of IFNγ chemokine (C-X-C motif) ligands (CXCL10 and CXCL11) in baboon and human lungs with BPD. IFNγ and its downstream targets may contribute significantly to the final common pathway of hyperoxia-induced injury in the developing lung and in human BPD.
doi:10.1165/rcmb.2010-0058OC
PMCID: PMC3095982  PMID: 21216975
newborn; hyperoxia; BPD; IFNγ; MMP9
18.  The Impact of Environmental and Genetic Factors on Neonatal Late-Onset Sepsis 
The Journal of pediatrics  2010;158(2):234-238.e1.
Objective
To assess the genetic contribution to late-onset sepsis in twins in the newborn intensive care unit (NICU).
Study design
A retrospective cohort analysis of twins born from 1994 to 2009 was performed on data collected from the NICUs at Yale University and the University of Connecticut. Sepsis concordance rates were compared between monozygotic and dizygotic twins. Mixed effects logistic regression (MELR) analysis was performed to determine the impact of selected non-genetic factors on late-onset sepsis. The influence of additive genetic and common and residual environmental effects were analyzed and quantified.
Results
170 monozygotic and 665 dizygotic twin pairs were analyzed and sepsis identified in 8.9%. Mean gestational age and birth weight of the cohort was 31.1 weeks and 1637 grams, respectively. MELR determined birth weight (regression coefficient=−0.001; 95% CI: −0.003–0.000; p=0.028), respiratory distress syndrome (regression coefficient=1.769; 95% CI: 0.943–2.596; p<0.001) and duration of total parenteral nutrition (regression coefficient=0.041; 95% CI: 0.017–0.064; p<0.001) as significant non-genetic factors. Further analysis determined 49.0% (p=0.002) of the variance in liability to late-onset sepsis was due to genetic factors alone, and 51.0% (p=0.001) the result of residual environmental factors.
Conclusions
Our data support significant genetic susceptibility to late-onset sepsis in the NICU population.
doi:10.1016/j.jpeds.2010.07.060
PMCID: PMC3008342  PMID: 20850766
premature newborn; infection; twins
19.  TGF-β2 Suppresses Macrophage Cytokine Production and Mucosal Inflammatory Responses in the Developing Intestine 
Gastroenterology  2010;140(1):242-253.
Background & Aims
Premature neonates are predisposed to necrotizing enterocolitis (NEC), an idiopathic, inflammatory bowel necrosis. We investigated the hypothesis that NEC occurs in the preterm intestine due to incomplete ‘non-inflammatory’ differentiation of intestinal macrophages, which increases the risk of a severe mucosal inflammatory response to bacterial products.
Methods
We compared inflammatory properties of human/murine fetal, neonatal, and adult intestinal macrophages. To investigate gut-specific macrophage differentiation, we next treated monocyte-derived macrophages with conditioned media from ex planted human fetal and adult intestinal tissues. Transforming growth factor-beta (TGF-β) expression and bioactivity were measured in fetal/adult intestine and in NEC. Finally, we used wild-type and transgenic mice to investigate the effects of deficient TGF-β signaling on NEC-like inflammatory mucosal injury.
Results
Intestinal macrophages in the human preterm intestine (fetus/premature neonate), but not in full-term neonates and adults, expressed inflammatory cytokines. Macrophage cytokine production was suppressed in the developing intestine by TGF-β, particularly the TGF-β2 isoform. NEC was associated with decreased tissue expression of TGF-β2 and decreased TGF-β bioactivity. In mice, disruption of TGF-β signaling worsened NEC-like inflammatory mucosal injury, whereas enteral supplementation with recombinant TGF-β2 was protective.
Conclusions
Intestinal macrophages progressively acquire a non-inflammatory profile during gestational development. TGF-β, particularly the TGF-β2 isoform, suppresses macrophage inflammatory responses in the developing intestine and protects against inflammatory mucosal injury. Enterally-administered TGF-β2 protected mice from experimental NEC-like injury.
doi:10.1053/j.gastro.2010.09.043
PMCID: PMC3008335  PMID: 20875417
necrotizing enterocolitis; macrophage; newborn; inflammation; TGF-β
20.  A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-β1-mediated injury in the developing murine lung 
BMC Cell Biology  2011;12:54.
Background
Transforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung.
Results
We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia.
Conclusions
When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway.
doi:10.1186/1471-2121-12-54
PMCID: PMC3266206  PMID: 22172122
21.  Proteomics Mapping of Cord Blood Identifies Haptoglobin “Switch-On” Pattern as Biomarker of Early-Onset Neonatal Sepsis in Preterm Newborns 
PLoS ONE  2011;6(10):e26111.
Background
Intra-amniotic infection and/or inflammation (IAI) are important causes of preterm birth and early-onset neonatal sepsis (EONS). A prompt and accurate diagnosis of EONS is critical for improved neonatal outcomes. We sought to explore the cord blood proteome and identify biomarkers and functional protein networks characterizing EONS in preterm newborns.
Methodology/Principal Findings
We studied a prospective cohort of 180 premature newborns delivered May 2004-September 2009. A proteomics discovery phase employing two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry identified 19 differentially-expressed proteins in cord blood of newborns with culture-confirmed EONS (n = 3) versus GA-matched controls (n = 3). Ontological classifications of the proteins included transfer/carrier, immunity/defense, protease/extracellular matrix. The 1st-level external validation conducted in the remaining 174 samples confirmed elevated haptoglobin and haptoglobin-related protein immunoreactivity (Hp&HpRP) in newborns with EONS (presumed and culture-confirmed) independent of GA at birth and birthweight (P<0.001). Western blot concurred in determining that EONS babies had conspicuous Hp&HpRP bands in cord blood (“switch-on pattern”) as opposed to non-EONS newborns who had near-absent “switch-off pattern” (P<0.001). Fetal Hp phenotype independently impacted Hp&HpRP. A Bayesian latent-class analysis (LCA) was further used for unbiased classification of all 180 cases based on probability of “antenatal IAI exposure” as latent variable. This was then subjected to 2nd-level validation against indicators of adverse short-term neonatal outcome. The optimal LCA algorithm combined Hp&HpRP switch pattern (most input), interleukin-6 and neonatal hematological indices yielding two non-overlapping newborn clusters with low (≤20%) versus high (≥70%) probability of IAI exposure. This approach reclassified ∼30% of clinical EONS diagnoses lowering the number needed to harm and increasing the odds ratios for several adverse outcomes including intra-ventricular hemorrhage.
Conclusions/Significance
Antenatal exposure to IAI results in precocious switch-on of Hp&HpRP expression. As EONS biomarker, cord blood Hp&HpRP has potential to improve the selection of newborns for prompt and targeted treatment at birth.
doi:10.1371/journal.pone.0026111
PMCID: PMC3189953  PMID: 22028810
22.  The Chitinase-like Proteins Breast Regression Protein-39 and YKL-40 Regulate Hyperoxia-induced Acute Lung Injury 
Rationale: Prolonged exposure to 100% O2 causes hyperoxic acute lung injury (HALI), characterized by alveolar epithelial cell injury and death. We previously demonstrated that the murine chitinase-like protein, breast regression protein (BRP)–39 and its human homolog, YKL-40, inhibit cellular apoptosis. However, the regulation and roles of these molecules in hyperoxia have not been addressed.
Objectives: We hypothesized that BRP-39 and YKL-40 (also called chitinase-3–like 1) play important roles in the pathogenesis of HALI.
Methods: We characterized the regulation of BRP-39 during HALI and the responses induced by hyperoxia in wild-type mice, BRP-39–null (−/−) mice, and BRP-39−/− mice in which YKL-40 was overexpressed in respiratory epithelium. We also compared the levels of tracheal aspirate YKL-40 in premature newborns with respiratory failure.
Measurements and Main Results: These studies demonstrate that hyperoxia inhibits BRP-39 in vivo in the murine lung and in vitro in epithelial cells. They also demonstrate that BRP-39−/− mice have exaggerated permeability, protein leak, oxidation, inflammatory, chemokine, and epithelial apoptosis responses, and experience premature death in 100% O2. Lastly, they demonstrate that YKL-40 ameliorates HALI, prolongs survival in 100% O2, and rescues the exaggerated injury response in BRP-39−/− animals. In accord with these findings, the levels of tracheal aspirate YKL-40 were lower in premature infants treated with hyperoxia for respiratory failure who subsequently experienced bronchopulmonary dysplasia or death compared with those that did not experience these complications.
Conclusions: These studies demonstrate that hyperoxia inhibits BRP-39/YKL-40, and that BRP-39 and YKL-40 are critical regulators of oxidant injury, inflammation, and epithelial apoptosis in the murine and human lung.
doi:10.1164/rccm.200912-1793OC
PMCID: PMC2970863  PMID: 20558631
BRP-39; YKL-40; hyperoxygen; BPD; HALI
23.  Genetic Contribution to Patent Ductus Arteriosus in the Premature Newborn 
Pediatrics  2009;123(2):669-673.
BACKGROUND
The most common congenital heart disease in the newborn population, patent ductus arteriosus, accounts for significant morbidity in preterm newborns. In addition to prematurity and environmental factors, we hypothesized that genetic factors play a significant role in this condition.
OBJECTIVE
The objective of this study was to quantify the contribution of genetic factors to the variance in liability for patent ductus arteriosus in premature newborns.
PATIENTS AND METHODS
A retrospective study (1991–2006) from 2 centers was performed by using zygosity data from premature twins born at ≤36 weeks’ gestational age and surviving beyond 36 weeks’ postmenstrual age. Patent ductus arteriosus was diagnosed by echocardiography at each center. Mixed-effects logistic regression was used to assess the effect of specific covariates. Latent variable probit modeling was then performed to estimate the heritability of patent ductus arteriosus, and mixed-effects probit modeling was used to quantify the genetic component.
RESULTS
We obtained data from 333 dizygotic twin pairs and 99 monozygotic twin pairs from 2 centers (Yale University and University of Connecticut). Data on chorioamnionitis, antenatal steroids, gestational age, body weight, gender, respiratory distress syndrome, patent ductus arteriosus, necrotizing enterocolitis, oxygen supplementation, and bronchopulmonary dysplasia were comparable between monozygotic and dizygotic twins. We found that gestational age, respiratory distress syndrome, and institution were significant covariates for patent ductus arteriosus. After controlling for specific covariates, genetic factors or the shared environment accounted for 76.1% of the variance in liability for patent ductus arteriosus.
CONCLUSIONS
Preterm patent ductus arteriosus is highly familial (contributed to by genetic and environmental factors), with the effect being mainly environmental, after controlling for known confounders.
doi:10.1542/peds.2008-1117
PMCID: PMC3161726  PMID: 19171636
neonate; patent ductus arteriosus; twins; genetic
24.  Hyperoxia-derived lung damage in preterm infants 
Summary
Hyperoxia-induced lung injury is characterized by an influx of inflammatory cells, increased pulmonary permeability, endothelial and epithelial cell death. This review highlights the mechanistic aspects of inflammation, vascular leak and cell death. The focus will be on agents that contribute to hyperoxia-induced lung injury in developmentally appropriate animal models, and those that have been detected in human premature neonates.
doi:10.1016/j.siny.2010.03.009
PMCID: PMC2910132  PMID: 20430708
Cell death; Cytokines; Oxidants; Oxygen; Pulmonary
25.  A Comparison of Presentation and Management Trends in Acute Pancreatitis Between Infants/Toddlers and Older Children 
Objectives
Acute pancreatitis is a necroinflammatory disease that leads to 210,000 hospitalizations in the United States annually. Recent reports suggest that there may be important differences in clinical features between infants/toddlers and older children. Thus, in this study we make a direct comparison between the pediatric age groups in presentation and management trends of acute pancreatitis.
Patients and Methods
We examined all children (ages 0 to 20 years) admitted to Yale-New Haven Children’s Hospital with pancreatitis between 1994 and 2007.
Results
Two hundred seventy-one cases met inclusion criteria for acute pancreatitis. Infants and toddlers manifested fewer signs and symptoms of abdominal pain, epigastric tenderness, and nausea compared with older children (43% vs 93%; 57% vs 90%; and 29% vs 76%, respectively; P < 0.05 for all comparisons). They were more likely to be diagnosed by serum lipase than by amylase and to undergo radiographic evaluation (P < 0.05). They had a longer hospital stay (19.5 vs 4 days; P < 0.05) and were less likely to be directly transitioned to oral nutrition (14% vs 71%; P < 0.05).
Conclusions
Infants and toddlers with acute pancreatitis present with fewer classical symptoms and are managed differently from older children. We believe these data will be helpful in evaluating and understanding treatment practices in this age group.
doi:10.1097/MPG.0b013e3181cea545
PMCID: PMC3038663  PMID: 20479687
acute pancreatitis; amylase; infants; lipase; toddlers

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