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1.  An analysis of MIF structural features that control functional activation of CD74 
Chemistry & biology  2015;22(9):1197-1205.
For more than fifteen years, the tautomerase active site of macrophage migration inhibitory factor (MIF) and its catalytic residue Pro1 have been being targeted for the development of therapeutics that block activation of its cell surface receptor, CD74. Neither the biological role of the MIF catalytic site nor the mechanistic details of CD74 activation are well understood. The inherently unstable structure of CD74 remains the biggest obstacle in structural studies with MIF for understanding the basis of CD74 activation. Using a novel approach, we elucidate the mechanistic details that control activation of CD74 by MIF surface residues and identify structural parameters of inhibitors that reduce CD74 biologic activation. We also find that N-terminal mutants located deep in the catalytic site affect surface residues immediately outside the catalytic site, which are responsible for reduction of CD74 activation.
Graphical Abstract
PMCID: PMC4575896  PMID: 26364929
2.  The Airway Microbiome at Birth 
Scientific Reports  2016;6:31023.
Alterations of pulmonary microbiome have been recognized in multiple respiratory disorders. It is critically important to ascertain if an airway microbiome exists at birth and if so, whether it is associated with subsequent lung disease. We found an established diverse and similar airway microbiome at birth in both preterm and term infants, which was more diverse and different from that of older preterm infants with established chronic lung disease (bronchopulmonary dysplasia). Consistent temporal dysbiotic changes in the airway microbiome were seen from birth to the development of bronchopulmonary dysplasia in extremely preterm infants. Genus Lactobacillus was decreased at birth in infants with chorioamnionitis and in preterm infants who subsequently went on to develop lung disease. Our results, taken together with previous literature indicating a placental and amniotic fluid microbiome, suggest fetal acquisition of an airway microbiome. We speculate that the early airway microbiome may prime the developing pulmonary immune system, and dysbiosis in its development may set the stage for subsequent lung disease.
PMCID: PMC4973241  PMID: 27488092
3.  BPD Following Preterm Birth: A Model for Chronic Lung Disease and a Substrate for ARDS in Childhood 
It has been suggested that pediatric acute respiratory distress syndrome (PARDS) may be a different entity, vis-à-vis adult acute respiratory distress syndrome (ARDS), based on its epidemiology and outcomes. A more pediatric-specific definition of PARDS to include the subgroup of patients with underlying lung (and heart) disease has been proposed. Epidemiological data suggest that up to 13% of the children with ARDS have a history of prematurity and/or underlying chronic lung disease. However, the specific contribution of bronchopulmonary dysplasia (BPD), the most common chronic lung disease in infants, to the development of PARDS is not known. BPD leads to damaged lungs with long-term consequences secondary to disordered growth and immune function. These damaged lungs could potentially act as a substrate, which given the appropriate noxious stimuli, can predispose a child to PARDS. Interestingly, similar biomarkers [KL-6, interleukin (IL)-6, IL-8, sICAM-1, angiopoietin-2, and matrix metalloproteinase-8 and -9] of pulmonary injury have been associated both with BPD and ARDS. Recognition of a unique pattern of clinical symptomatology and/or outcomes of PARDS, if present, could potentially be useful for investigating targeted therapeutic interventions.
PMCID: PMC4908128  PMID: 27379219
BPD; pediatric ARDS; lung diseases; biomarkers; lung development
4.  Particle streak velocimetry-optical coherence tomography: a novel method for multidimensional imaging of microscale fluid flows: erratum 
Biomedical Optics Express  2016;7(6):2360-2361.
A table summarizing existing methods for velocimetry using OCT miscategorized Lee et al.’s work on DLS-OCT as being intensity-based, when it was actually field-based. This erratum report gives the corrected table. Also, the acknowledgments section was missing, which we include here.
PMCID: PMC4918588  PMID: 27375950
(110.4500) Optical coherence tomography; (120.7250) Velocimetry;  (110.4153) Motion estimation and optical flow; (170.3880) Medical and biological imaging; (280.2490) Flow diagnostics
5.  Particle streak velocimetry-optical coherence tomography: a novel method for multidimensional imaging of microscale fluid flows 
Biomedical Optics Express  2016;7(4):1590-1603.
We present a new OCT method for flow speed quantification and directional velocimetry: particle streak velocimetry-OCT (PSV-OCT). PSV-OCT generates two-dimensional, 2.5-vector component (vx,|vy|,vz) maps of microscale flow velocity fields. Knowledge of 2.5-vector components also enables the estimation of total flow speed. The enabling insight behind PSV-OCT is that tracer particles in sparsely-seeded fluid flow trace out streaks in (x,z,t)-space. The streak orientations in x-t and z-t yield vx and vz, respectively. The in-plane (x-z plane) residence time yields the out-of-plane speed |vy|. Vector component values are generated by fitting streaks to a model of image formation that incorporates equations of motion in 3D space. We demonstrate cross-sectional estimation of (vx,|vy|,vz) in two important animal models in ciliary biology: Xenopus embryos (tadpoles) and mouse trachea.
PMCID: PMC4929663  PMID: 27375926
(110.4500) Optical coherence tomography; (120.7250) Velocimetry; (110.4153) Motion estimation and optical flow; (170.3880) Medical and biological imaging; (280.2490) Flow diagnostics
6.  Role of Nitric Oxide Isoforms in Vascular and Alveolar Development and Lung Injury in Vascular Endothelial Growth Factor Overexpressing Neonatal Mice Lungs 
PLoS ONE  2016;11(1):e0147588.
The role of vascular endothelial growth factor (VEGF)-induced 3 different nitric oxide synthase (NOS) isoforms in lung development and injury in the newborn (NB) lung are not known. We hypothesized that VEGF-induced specific NOS pathways are critical regulators of lung development and injury.
We studied NB wild type (WT), lung epithelial cell-targeted VEGF165 doxycycline-inducible overexpressing transgenic (VEGFTG), VEGFTG treated with a NOS1 inhibitor (L-NIO), VEGFTG x NOS2-/- and VEGFTG x NOS3+/- mice in room air (RA) for 7 postnatal (PN) days. Lung morphometry (chord length), vascular markers (Ang1, Ang2, Notch2, vWF, CD31 and VE-cadherin), cell proliferation (Ki67), vascular permeability, injury and oxidative stress markers (hemosiderin, nitrotyrosine and 8-OHdG) were evaluated.
VEGF overexpression in RA led to increased chord length and vascular markers at PN7, which were significantly decreased to control values in VEGFTG x NOS2−/− and VEGFTG x NOS3+/- lungs. However, we found no noticeable effect on chord length and vascular markers in the VEGFTG / NOS1 inhibited group. In the NB VEGFTG mouse model, we found VEGF-induced vascular permeability in the NB murine lung was partially dependent on NOS2 and NOS3-signaling pathways. In addition, the inhibition of NOS2 and NOS3 resulted in a significant decrease in VEGF-induced hemosiderin, nitrotyrosine- and 8-OHdG positive cells at PN7. NOS1 inhibition had no significant effect.
Our data showed that the complete absence of NOS2 and partial deficiency of NOS3 confers protection against VEGF-induced pathologic lung vascular and alveolar developmental changes, as well as injury markers. Inhibition of NOS1 does not have any modulating role on VEGF-induced changes in the NB lung. Overall, our data suggests that there is a significant differential regulation in the NOS-mediated effects of VEGF overexpression in the developing mouse lung.
PMCID: PMC4723240  PMID: 26799210
7.  Understanding the Impact of Infection, Inflammation, and Their Persistence in the Pathogenesis of Bronchopulmonary Dysplasia 
The concerted interaction of genetic and environmental factors acts on the preterm human immature lung with inflammation being the common denominator leading to the multifactorial origin of the most common chronic lung disease in infants – ­bronchopulmonary dysplasia (BPD). Adverse perinatal exposure to infection/inflammation with added insults like invasive mecha nical ventilation, exposure to hyperoxia, and sepsis causes persistent immune dysregulation. In this review article, we have attempted to analyze and consolidate current knowledge about the role played by persistent prenatal and postnatal inflammation in the pathogenesis of BPD. While some parameters of the early inflammatory response (neutrophils, cytokines, etc.) may not be detectable after days to weeks of exposure to noxious stimuli, they have already initiated the signaling pathways of the inflammatory process/immune cascade and have affected permanent defects structurally and functionally in the BPD lungs. Hence, translational research aimed at prevention/amelioration of BPD needs to focus on dampening the inflammatory response at an early stage to prevent the cascade of events leading to lung injury with impaired healing resulting in the pathologic pulmonary phenotype of alveolar simplification and dysregulated vascularization characteristic of BPD.
PMCID: PMC4685088  PMID: 26734611
premature newborn; chronic lung disease; cytokines; sepsis; hyperoxia; mechanical ventilation
8.  The Effect of Modified Ultrafiltration on Angiopoietins in Pediatric Cardiothoracic Surgery 
The Annals of thoracic surgery  2014;98(5):1699-1704.
Cardiopulmonary bypass subjects patients’ blood to hemodilution and nonphysiologic conditions resulting in a systemic inflammatory response. Modified ultrafiltration (MUF) counteracts hemodilution and has also been postulated to improve outcomes by pro-inflammatory cytokine removal. The objective of this study was to investigate whether the benefits of MUF include the removal of pro-inflammatory mediators, such as angiopoietin-2 (angpt-2). We hypothesize that some of the clinical benefits of MUF are related to the preferential removal of angpt-2.
We performed a prospective cohort study in children ≤ 18 years of age undergoing cardiopulmonary bypass. Serum samples were obtained from each patient: 1. Preoperative, 2. post cardiopulmonary bypass, and 3. upon intensive care unit admission. A fluid sample from the MUF effluent was also analyzed. Angpt-1, angpt-2, interleukin-8 and interleukin-10 levels were determined by enzyme-linked immunosorbent assay.
Thirty-one subjects were enrolled. Angpt-1 levels significantly decreased across all time points (p<0.01). Angpt-2 concentrations were significantly elevated at intensive care unit admission when compared to both preoperative and post cardiopulmonary bypass levels (p<0.01). The angpt-2/1 ratio significantly increased post cardiopulmonary bypass to intensive care unit admission (p<0.01). There was no significant difference between the angpt-2 or angpt- 1 percent extraction within MUF effluent. Interleukin-8 and interleukin-10 significantly increased from preoperative to intensive care unit admission (both p<0.01).
The results of this study demonstrate that MUF removes both pro- and anti-inflammatory mediators equally. This study suggests that the clinical benefits of MUF cannot be attributed to removal of larger quantities of pro-inflammatory mediators such as angpt-2 and interleukin-8.
PMCID: PMC4253712  PMID: 25258157
Cardiopulmonary bypass; Inflammatory mediators; Perioperative care
9.  Setting research priorities to improve global newborn health and prevent stillbirths by 2025 
Yoshida, Sachiyo | Martines, José | Lawn, Joy E | Wall, Stephen | Souza, Joăo Paulo | Rudan, Igor | Cousens, Simon | Aaby, Peter | Adam, Ishag | Adhikari, Ramesh Kant | Ambalavanan, Namasivayam | Arifeen, Shams EI | Aryal, Dhana Raj | Asiruddin, Sk | Baqui, Abdullah | Barros, Aluisio JD | Benn, Christine S | Bhandari, Vineet | Bhatnagar, Shinjini | Bhattacharya, Sohinee | Bhutta, Zulfiqar A | Black, Robert E | Blencowe, Hannah | Bose, Carl | Brown, Justin | Bührer, Christoph | Carlo, Wally | Cecatti, Jose Guilherme | Cheung, Po–Yin | Clark, Robert | Colbourn, Tim | Conde–Agudelo, Agustin | Corbett, Erica | Czeizel, Andrew E | Das, Abhik | Day, Louise Tina | Deal, Carolyn | Deorari, Ashok | Dilmen, Uğur | English, Mike | Engmann, Cyril | Esamai, Fabian | Fall, Caroline | Ferriero, Donna M | Gisore, Peter | Hazir, Tabish | Higgins, Rosemary D | Homer, Caroline SE | Hoque, DE | Irgens, Lorentz | Islam, MT | de Graft–Johnson, Joseph | Joshua, Martias Alice | Keenan, William | Khatoon, Soofia | Kieler, Helle | Kramer, Michael S | Lackritz, Eve M | Lavender, Tina | Lawintono, Laurensia | Luhanga, Richard | Marsh, David | McMillan, Douglas | McNamara, Patrick J | Mol, Ben Willem J | Molyneux, Elizabeth | Mukasa, G. K | Mutabazi, Miriam | Nacul, Luis Carlos | Nakakeeto, Margaret | Narayanan, Indira | Olusanya, Bolajoko | Osrin, David | Paul, Vinod | Poets, Christian | Reddy, Uma M | Santosham, Mathuram | Sayed, Rubayet | Schlabritz–Loutsevitch, Natalia E | Singhal, Nalini | Smith, Mary Alice | Smith, Peter G | Soofi, Sajid | Spong, Catherine Y | Sultana, Shahin | Tshefu, Antoinette | van Bel, Frank | Gray, Lauren Vestewig | Waiswa, Peter | Wang, Wei | Williams, Sarah LA | Wright, Linda | Zaidi, Anita | Zhang, Yanfeng | Zhong, Nanbert | Zuniga, Isabel | Bahl, Rajiv
Journal of Global Health  null;6(1):010508.
In 2013, an estimated 2.8 million newborns died and 2.7 million were stillborn. A much greater number suffer from long term impairment associated with preterm birth, intrauterine growth restriction, congenital anomalies, and perinatal or infectious causes. With the approaching deadline for the achievement of the Millennium Development Goals (MDGs) in 2015, there was a need to set the new research priorities on newborns and stillbirth with a focus not only on survival but also on health, growth and development. We therefore carried out a systematic exercise to set newborn health research priorities for 2013–2025.
We used adapted Child Health and Nutrition Research Initiative (CHNRI) methods for this prioritization exercise. We identified and approached the 200 most productive researchers and 400 program experts, and 132 of them submitted research questions online. These were collated into a set of 205 research questions, sent for scoring to the 600 identified experts, and were assessed and scored by 91 experts.
Nine out of top ten identified priorities were in the domain of research on improving delivery of known interventions, with simplified neonatal resuscitation program and clinical algorithms and improved skills of community health workers leading the list. The top 10 priorities in the domain of development were led by ideas on improved Kangaroo Mother Care at community level, how to improve the accuracy of diagnosis by community health workers, and perinatal audits. The 10 leading priorities for discovery research focused on stable surfactant with novel modes of administration for preterm babies, ability to diagnose fetal distress and novel tocolytic agents to delay or stop preterm labour.
These findings will assist both donors and researchers in supporting and conducting research to close the knowledge gaps for reducing neonatal mortality, morbidity and long term impairment. WHO, SNL and other partners will work to generate interest among key national stakeholders, governments, NGOs, and research institutes in these priorities, while encouraging research funders to support them. We will track research funding, relevant requests for proposals and trial registers to monitor if the priorities identified by this exercise are being addressed.
PMCID: PMC4576458  PMID: 26401272
10.  Three-dimensional, three-vector-component velocimetry of cilia-driven fluid flow using correlation-based approaches in optical coherence tomography 
Biomedical Optics Express  2015;6(9):3515-3538.
Microscale quantification of cilia-driven fluid flow is an emerging area in medical physiology, including pulmonary and central nervous system physiology. Cilia-driven fluid flow is most completely described by a three-dimensional, three-component (3D3C) vector field. Here, we generate 3D3C velocimetry measurements by synthesizing higher dimensional data from lower dimensional measurements obtained using two separate optical coherence tomography (OCT)-based approaches: digital particle image velocimetry (DPIV) and dynamic light scattering (DLS)-OCT. Building on previous work, we first demonstrate directional DLS-OCT for 1D2C velocimetry measurements in the sub-1 mm/s regime (sub-2.5 inch/minute regime) of cilia-driven fluid flow in Xenopus epithelium, an important animal model of the ciliated respiratory tract. We then extend our analysis toward 3D3C measurements in Xenopus using both DLS-OCT and DPIV. We demonstrate the use of DPIV-based approaches towards flow imaging of Xenopus cerebrospinal fluid and mouse trachea, two other important ciliary systems. Both of these flows typically fall in the sub-100 μm/s regime (sub-0.25 inch/minute regime). Lastly, we develop a framework for optimizing the signal-to-noise ratio of 3D3C flow velocity measurements synthesized from 2D2C measures in non-orthogonal planes. In all, 3D3C OCT-based velocimetry has the potential to comprehensively characterize the flow performance of biological ciliated surfaces.
PMCID: PMC4574676  PMID: 26417520
(110.4500) Optical coherence tomography; (120.7250) Velocimetry; (170.3340) Laser Doppler velocimetry; (170.6480) Spectroscopy, speckle
11.  Corrigendum: The Human Neonatal Gut Microbiome: A Brief Review 
PMCID: PMC4485073  PMID: 26176006
newborn; microbiome; gut; probiotics; necrotizing enterocolitis
12.  The Human Neonatal Gut Microbiome: A Brief Review 
The field of genomics has expanded into subspecialties such as metagenomics over the course of the last decade and a half. The development of massively parallel sequencing capabilities has allowed for increasingly detailed study of the genome of the human microbiome, the microbial super organ that resides symbiotically within the mucosal tissues and integumentary system of the human host. The gut microbiome, and particularly the study of its origins in neonates, has become subtopics of great interest within the field of genomics. This brief review seeks to summarize recent literature regarding the origins and establishment of the neonatal gut microbiome, beginning in utero, and how it is affected by neonatal nutritional status (breastfed versus formula fed) and gestational age (term versus preterm). We also explore the role of dysbiosis, a perturbation within the fragile ecosystem of the microbiome, and its role in the origin of select pathologic states, specifically, obesity and necrotizing enterocolitis (NEC) in preterm infants. We discuss the evidence supporting enteral pre- and pro-biotic supplementation of commensal organisms such as Bifidobacterium and Lactobacillus in the neonatal period, and their role in the prevention and amelioration of NEC in premature infants. Finally, we review directions to consider for further research to promote human health within this field.
PMCID: PMC4350424  PMID: 25798435
newborn; preterm; microbiota; probiotics; dysbiosis
Exposure to hyperoxia, invasive mechanical ventilation and systemic/local sepsis are important antecedents of postnatal inflammation in the pathogenesis of bronchopulmonary dysplasia (BPD). This review will summarize information obtained from animal (baboon, lamb/sheep, rat and mouse) models that pertain to the specific inflammatory agents and signaling molecules that predispose a premature infant to BPD.
PMCID: PMC4023567  PMID: 24578018
invasive ventilation; infection; hyperoxia; lung; newborn
14.  Conditional overexpression of TGFβ1 promotes pulmonary inflammation, apoptosis and mortality via TGFβR2 in the developing mouse lung 
Respiratory Research  2015;16(1):4.
Earlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.
We utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.
Our data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.
Our study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.
PMCID: PMC4307226  PMID: 25591994
Transforming growth factor; Oxygen; Inflammation; Cell death; Angiopoietin; Newborn; Pulmonary; Bronchopulmonary dysplasia
15.  The Temporal Kinetics of Circulating Angiopoietin Levels in Children with Sepsis 
Capillary integrity continues to challenge critical care physicians worldwide when treating children with sepsis. Vascular growth factors, specifically angiopoietin (angpt)-1 and angpt-2, play opposing roles in capillary stabilization in septic patients, respectively. We aim to determine whether pediatric patients with severe sepsis/shock have persistently high angpt-2/1 ratios when compared to non-septic pediatric intensive care unit (PICU) patients over a 7-day period.
Prospective, observational study. Patients were classified within 24h of admission into: non-systemic inflammatory response syndrome (non-SIRS), SIRS/sepsis, or severe sepsis/shock. Plasma levels of angpt-1 and angpt-2 were measured via ELISA. The angpt-2/1 ratio was graphically plotted and determined whether patients fell into ‘constant’ or ‘variable’ patterns.
Tertiary care center PICU.
Critically ill pediatric patients with varying sepsis severity.
Measurements and Main Results
Forty five patients were enrolled (n=9 non-SIRS, n=19 SIRS/sepsis, and n=17 severe sepsis/shock). Gender, age, weight, comorbidities and PICU length of stay were not significantly different between the groups. Admission pediatric risk stratification scores and net fluid ins/outs were significantly elevated in the severe sepsis/shock group when compared (all p<0.05). Admission angpt-2 levels and angpt-2/1 ratios were significantly different in the severe sepsis/shock group when all groups were compared (both p<0.05). Additionally, the latter were significantly elevated in the severe sepsis/shock group at multiple time points (all p≤0.05) with the peak occurring on day 2 of illness. In a separate analysis, 32% of SIRS/sepsis and 82% of severe sepsis/shock had ‘variable’ angpt-2/1 ratio patterns compared to none in the control group (p<0.001).
Pediatric patients with severe sepsis and septic shock possess significantly elevated angpt-2/1 ratios during their first 3 days of illness which peak at day 2 of illness. A subset of these patients demonstrated ‘variable’ angpt-2/1 ratio patterns.
PMCID: PMC3947338  PMID: 24141659
Pediatric; capillary leak; shock; intensive care
16.  Angiopoietin-1, Angiopoietin-2 and Bicarbonate as Diagnostic Biomarkers in Children with Severe Sepsis 
PLoS ONE  2014;9(9):e108461.
Severe pediatric sepsis continues to be associated with high mortality rates in children. Thus, an important area of biomedical research is to identify biomarkers that can classify sepsis severity and outcomes. The complex and heterogeneous nature of sepsis makes the prospect of the classification of sepsis severity using a single biomarker less likely. Instead, we employ machine learning techniques to validate the use of a multiple biomarkers scoring system to determine the severity of sepsis in critically ill children. The study was based on clinical data and plasma samples provided by a tertiary care center's Pediatric Intensive Care Unit (PICU) from a group of 45 patients with varying sepsis severity at the time of admission. Canonical Correlation Analysis with the Forward Selection and Random Forests methods identified a particular set of biomarkers that included Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), and Bicarbonate (HCO) as having the strongest correlations with sepsis severity. The robustness and effectiveness of these biomarkers for classifying sepsis severity were validated by constructing a linear Support Vector Machine diagnostic classifier. We also show that the concentrations of Ang-1, Ang-2, and HCO enable predictions of the time dependence of sepsis severity in children.
PMCID: PMC4178003  PMID: 25255212
17.  Drug Therapy Trials for the Prevention of Bronchopulmonary Dysplasia: Current and Future Targets 
PMCID: PMC4110623  PMID: 25121076
newborn; clinical trials; drug therapy; lung; chronic lung disease
18.  Hyperoxia and Interferon-γ–Induced Injury in Developing Lungs Occur via Cyclooxygenase-2 and the Endoplasmic Reticulum Stress–Dependent Pathway 
We noted a marked increase in cyclooxygenase-2 (Cox2) and the activation of the endoplasmic reticulum (ER) stress pathway in newborn murine lung on exposure to hyperoxia and IFN-γ. We sought to evaluate Cox2-mediated ER stress pathway activation in hyperoxia-induced and IFN-γ–mediated injury in developing lungs. We applied in vivo genetic gain-of-function and genetic/chemical inhibition, as well as in vitro loss-of-function genetic strategies. Hyperoxia-induced and IFN-γ–mediated impaired alveolarization was rescued by Cox2 inhibition, using celecoxib. The use of small interfering RNA against the ER stress pathway mediator, the C/EBP homologous protein (CHOP; also known as growth arrest and DNA damage–inducible gene 153/GADD153), alleviated cell death in alveolar epithelial cells as well as in hyperoxia-induced and IFN-γ–mediated murine models of bronchopulmonary dysplasia (BPD). In addition, CHOP siRNA also restored alveolarization in the in vivo models. Furthermore, as evidence of clinical relevance, we show increased concentrations of Cox2 and ER stress pathway mediators in human lungs with BPD. Cox2, via CHOP, may significantly contribute to the final common pathway of hyperoxia-induced and IFN-γ–mediated injury in developing lungs and human BPD.
PMCID: PMC3727872  PMID: 23470621
newborn; oxygen; BPD; CHOP; cell death
19.  Basic Science: Obstetrics Nucleated Red Blood Cells are a Direct Response to Mediators of Inflammation in Newborns with Early Onset Neonatal Sepsis 
To test the hypothesis that inflammation modulates fetal erythroblastosis and/or the release of NRBCs independent of hypoxia or fetal stress. We sought to determine if fetal inflammation is associated with an elevation in neonatal NRBC count in the setting of inflammation-associated preterm birth.
Study Design
The relationships between peripheral NRBC count, histological chorioamnionitis, umbilical cord interleukin-6 (IL-6), erythropoietin (EPO), cortisol and acid-base status were analyzed in 68 preterm singletons, born to mothers who had an amniocentesis to rule out infection. Proteomic profiling of amniotic fluid identified presence of intra-amniotic inflammation according to established parameters. NRBC counts were assessed within 1-hour of birth. Early-onset neonatal sepsis (EONS) was established based on hematological and microbiological indices. IL-6, EPO and cortisol levels were measured by immunoassays. Fetal acid-base status was determined within 10 minutes of delivery. Parametric or nonparametric statistics was employed.
Fetuses with EONS (n=19) were delivered at earlier gestational ages (mean±SD: 27.1±2.8 weeks, P=0.001) and more often by mothers with intra-amniotic inflammation (P=0.022) and histological chorioamnionitis (P<0.001). Neonates with EONS had higher absolute NRBCs counts (P=0.011). NRBC counts were directly correlated with cord blood IL-6 levels (P<0.001) but not with EPO, cortisol or parameters of acid-base status levels regardless of EONS status. These relationships remained following correction for gestational age, diabetes, intrauterine growth restriction (IUGR) and steroid exposure.
In the setting of inflammation-associated preterm birth and in the absence of hypoxia, elevations in NRBCs in the early neonatal period may be a direct response to exposure to inflammatory mediators in utero.
PMCID: PMC4023236  PMID: 18395034
infection; inflammation; NRBC preterm birth; chorioamnionitis
20.  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.
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.
Southern Alberta regional center located at high altitude.
Preterm infants weighing ≤1250 grams with no BPD, BPD, and BPD with chronic oxygen dependency.
Main outcome measures
Neurodevelopmental and growth outcomes.
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)].
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.
PMCID: PMC3960119  PMID: 24646665
21.  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.
PMCID: PMC3872744  PMID: 24421769
mitochondria; reactive oxygen species; mitophagy; apoptosis; pulmonary disease
22.  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.
PMCID: PMC3867385  PMID: 24367543
23.  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.
PMCID: PMC3855965  PMID: 24347826
To evaluate the fetal renal artery impedance in the context of inflammation-associated preterm birth (PTB).
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.
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.
The fetus is capable of sustaining normal renal artery impedance despite inflammation. Resistance in the renal vascular bed affects urine output independent of inflammation.
PMCID: PMC3791328  PMID: 19185102
25.  Fetal Inflammatory Response in Women with Proteomic Biomarkers Characteristic of Intra-Amniotic Inflammation and Preterm Birth 
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.
Prospective observational cohort.
Tertiary referral University hospital
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.
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.
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.
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.
PMCID: PMC3791329  PMID: 18947340
Proteomics; biomarkers; amniotic fluid; inflammation; defensin; calgranulin; umbilical cord blood; interleukin-6

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