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1.  Reduction in Red Blood Cell Transfusions Among Preterm Infants: Results of a Randomized Trial With an In-Line Blood Gas and Chemistry Monitor 
Pediatrics  2005;115(5):1299-1306.
Background
Critically ill, extremely premature infants develop anemia because of intensive laboratory blood testing and undergo multiple red blood cell (RBC) transfusions in the early weeks of life. To date, researchers have had only limited success in finding ways to reduce transfusions significantly in this patient population.
Objective
To reduce RBC transfusions for these infants by using a point-of-care bedside monitor that returns analyzed blood to the patient.
Design, Setting, and Patients
This was a prospective, 2-center, randomized, open, controlled, clinical trial with a 1:1 assignment of extremely low birth weight infants (weighing 500–1000 g at birth) to control or monitor groups and analysis with the intention-to-treat approach. Predefined RBC transfusion criteria were applied uniformly in the 2 groups.
Interventions
Clinical treatment of study subjects with an in-line, ex vivo, bedside monitor that withdraws blood through an umbilical artery catheter, analyzes blood gases and sodium, potassium, and hematocrit levels, and returns the sample to the patient.
Main Outcome Measures
The total volume and number of RBC transfusions during the first 2 weeks of life and the total volume of blood removed for laboratory testing.
Results
The trial was terminated prematurely when one center's NICU changed its standard method of laboratory testing. In the first 2 weeks of life, there was a nonsignificant 17% lower cumulative RBC transfusion volume in the monitor group (n = 46), compared with the control group (n = 47). However, data from the first week only (the period of greater catheter use) demonstrated a significant 33% lower cumulative RBC transfusion volume in the monitor group. Cumulative phlebotomy loss was ~25% less in the monitor group throughout the 2-week study period. There was no difference between groups in neonatal mortality, morbidity, and neurodevelopmental outcome rates at 18 to 24 months. This is the first randomized trial documenting that RBC transfusions administered to neonates can by reduced by decreasing laboratory phlebotomy loss.
Conclusions
As long as an umbilical artery catheter is available for blood sampling with an in-line blood gas and chemistry monitor, significant reductions in neonatal RBC transfusions can be achieved. The patients most likely to benefit from monitor use are the smallest, most critically ill newborns.
doi:10.1542/peds.2004-1680
PMCID: PMC2867083  PMID: 15867038
2.  Investigation of maternal environmental exposures in association with self-reported preterm birth 
Identification of maternal environmental factors influencing preterm birth risks is important to understand the reasons for the increase in prematurity since 1990. Here, we utilized a health survey, the US National Health and Nutrition Examination Survey (NHANES) to search for personal environmental factors associated with preterm birth. 201 urine and blood markers of environmental factors, such as allergens, pollutants, and nutrients were assayed in mothers (range of N: 49 to 724) who answered questions about any children born preterm (delivery <37 weeks). We screened each of the 201 factors for association with any child born preterm adjusting by age, race/ethnicity, education, and household income. We attempted to verify the top finding, urinary bisphenol A, in an independent study of pregnant women attending Lucile Packard Children’s Hospital. We conclude that the association between maternal urinary levels of bisphenol A and preterm birth should be evaluated in a larger epidemiological investigation.
doi:10.1016/j.reprotox.2013.12.005
PMCID: PMC4316205  PMID: 24373932
environmental exposure; environment-wide association study; preterm birth
3.  Heme oxygenase-1 in pregnancy and cancer: similarities in cellular invasion, cytoprotection, angiogenesis, and immunomodulation 
Pregnancy can be defined as a “permissible” process, where a semi-allogeneic fetus and placenta are allowed to grow and survive within the mother. Similarly, in tumor growth, antigen-specific malignant cells proliferate and evade into normal tissues of the host. The microenvironments of the placenta and tumors are amazingly comparable, sharing similar mechanisms exploited by fetal or cancer cells with regard to surviving in a hypoxic microenvironment, invading tissues via degradation and vasculogenesis, and escaping host attack through immune privilege. Heme oxygease-1 (HO-1) is a stress-response protein that has antioxidative, anti-apoptotic, pro-angiogenic, and anti-inflammatory properties. Although a large volume of research has been published in recent years investigating the possible role(s) of HO-1 in pregnancy and in cancer development, the molecular mechanisms that regulate these “yin-yang” processes have still not been fully elucidated. Here, we summarize and compare pregnancy and cancer development, focusing primarily on the function of HO-1 in cellular invasion, cytoprotection, angiogenesis, and immunomodulation. Due to the similarities of both processes, a thorough understanding of the molecular mechanisms of each process may reveal and guide the development of new approaches to prevent not only pregnancy disorders; but also, to study cancer.
doi:10.3389/fphar.2014.00295
PMCID: PMC4294126  PMID: 25642189
Placenta; trophoblast invasion; angiogenesis;  immunosuppression; tolerogenic dendritic cells (tDC); alternatively activated macrophage (M2)
4.  Death or Neurodevelopmental Impairment at 18 To 22 Months in a Randomized Trial of Early Dexamethasone to Prevent Death or Chronic Lung Disease in Extremely Low Birth Weight Infants 
The Journal of pediatrics  2013;164(1):34-39.e2.
Objective
To evaluate the incidence of death or neurodevelopmental impairment (NDI) at 18 to 22 months corrected age in subjects enrolled in a trial of early dexamethasone treatment to prevent death or chronic lung disease in extremely low birth weight infants.
Methods
Evaluation of infants at 18 to 22 months corrected age included anthropomorphic measurements, a standard neurological examination, and the Bayley Scales of Infant Development-II, including the Mental Developmental Index (MDI) and the Psychomotor Developmental Index (PDI). NDI was defined as moderate or severe cerebral palsy, MDI or PDI less than 70, blindness, or hearing impairment.
Results
Death or NDI at 18 to 22 months corrected age was similar in the dexamethasone and placebo groups (65 vs 66 percent, p= 0.99 among those with known outcome). The proportion of survivors with NDI was also similar, as were mean values for weight, length, and head circumference and the proportion of infants with poor growth (50 vs 41 percent, p=0.42 for weight less than 10th percentile). Forty nine percent of infants in the placebo group received treatment with corticosteroid compared to 32% in the dexamethasone group (p=0.02).
Conclusion
The risk of death or NDI and rate of poor growth were high but similar in the dexamethasone and placebo groups. The lack of a discernible effect of early dexamethasone on neurodevelopmental outcome may be due to frequent clinical corticosteroid use in the placebo group.
doi:10.1016/j.jpeds.2013.07.027
PMCID: PMC4120744  PMID: 23992673
neurodevelopmental outcome; growth; bronchopulmonary dysplasia; cerebral palsy; neonatal follow-up
5.  Immunogenicity of Haemophilus influenzae Type b Protein Conjugate Vaccines in Very Low Birth Weight Infants 
doi:10.1097/01.inf.0000437263.04493.7c
PMCID: PMC3960569  PMID: 24569312
Infant; premature; infant; very low birth weight; Haemophilus influenzae vacines; immunization; vaccines
6.  Prevention of Traumatic Stress in Mothers With Preterm Infants: A Randomized Controlled Trial 
Pediatrics  2013;132(4):e886-e894.
OBJECTIVE:
The current study evaluates a treatment intervention developed with the goal of reducing symptoms of posttraumatic stress, depression, and anxiety in parents of premature infants.
METHODS:
A total of 105 mothers of preterm infants (25–34 weeks’ gestational age; >600 g) were randomized to receive a 6-session intervention developed to target parental trauma as well as facilitate infant redefinition (n = 62) or to an active comparison group (n = 43). Mothers in the intervention group received a combination of trauma-focused treatments, including psychoeducation, cognitive restructuring, progressive muscle relaxation, and development of their trauma narrative. The intervention also incorporated material targeting infant redefinition, defined as the process of changing the mother’s negative perceptions of her infant and the parenting experience.
RESULTS:
Mothers in the intervention group reported a greater reduction in both trauma symptoms (Cohen’s d = 0.41, P = .023) and depression (Cohen’s d = 0.59, P < .001) compared with the comparison group. Patients under both conditions improved significantly in terms of anxiety, with no differences between groups. Results of the moderator analysis showed that mothers with higher ratings of baseline NICU stress benefited more from the intervention compared with mothers who had lower ratings (P = .036).
CONCLUSIONS:
This short, highly manualized intervention for mothers of preterm infants statistically significantly reduced symptoms of trauma and depression. The intervention is feasible, can be delivered with fidelity, and has high ratings of maternal satisfaction. Given that improvements in mothers’ distress may lead to improved infant outcomes, this intervention has the potential for a high public health impact.
doi:10.1542/peds.2013-1331
PMCID: PMC3784295  PMID: 23999956
intervention; neonatal intensive care; premature infants; posttraumatic stress disorder
7.  Raltegravir In Vitro Effect on Bilirubin Binding 
The Pediatric infectious disease journal  2013;32(9):10.1097/INF.0b013e31829044a8.
Drugs that displace bilirubin from albumin may increase the risk of kernicterus in neonates. We evaluated the effect of raltegravir on bilirubin-albumin binding in pooled neonatal serum using the peroxidase method. Raltegravir had minimal effect on bilirubin-albumin binding at concentrations of 5 and 10 μM, caused a small but statistically significant increase in unbound bilirubin at 100 μM, and caused potentially harmful increases at 500 and 1000 μM. Our data suggest that the effect of raltegravir on neonatal bilirubin binding is unlikely to be clinically significant at typical peak concentrations reached with usual dosing.
doi:10.1097/INF.0b013e31829044a8
PMCID: PMC3856729  PMID: 23470680
raltegravir; bilirubin binding; neonate
8.  A Genome-Wide Association Study (GWAS) for Bronchopulmonary Dysplasia 
Pediatrics  2013;132(2):290-297.
OBJECTIVE:
Twin studies suggest that heritability of moderate-severe bronchopulmonary dysplasia (BPD) is 53% to 79%, we conducted a genome-wide association study (GWAS) to identify genetic variants associated with the risk for BPD.
METHODS:
The discovery GWAS was completed on 1726 very low birth weight infants (gestational age = 250–296/7 weeks) who had a minimum of 3 days of intermittent positive pressure ventilation and were in the hospital at 36 weeks’ postmenstrual age. At 36 weeks’ postmenstrual age, moderate-severe BPD cases (n = 899) were defined as requiring continuous supplemental oxygen, whereas controls (n = 827) inhaled room air. An additional 795 comparable infants (371 cases, 424 controls) were a replication population. Genomic DNA from case and control newborn screening bloodspots was used for the GWAS. The replication study interrogated single-nucleotide polymorphisms (SNPs) identified in the discovery GWAS and those within the HumanExome beadchip.
RESULTS:
Genotyping using genomic DNA was successful. We did not identify SNPs associated with BPD at the genome-wide significance level (5 × 10−8) and no SNP identified in previous studies reached statistical significance (Bonferroni-corrected P value threshold .0018). Pathway analyses were not informative.
CONCLUSIONS:
We did not identify genomic loci or pathways that account for the previously described heritability for BPD. Potential explanations include causal mutations that are genetic variants and were not assayed or are mapped to many distributed loci, inadequate sample size, race ethnicity of our study population, or case-control differences investigated are not attributable to underlying common genetic variation.
doi:10.1542/peds.2013-0533
PMCID: PMC3727675  PMID: 23897914
genome-wide association study (GWAS); chronic lung disease; genetic predisposition to disease; premature; very low birth weight infant
9.  Neonatal physiological correlates of near-term brain development on MRI and DTI in very-low-birth-weight preterm infants 
NeuroImage : Clinical  2014;5:169-177.
Structural brain abnormalities identified at near-term age have been recognized as potential predictors of neurodevelopment in children born preterm. The aim of this study was to examine the relationship between neonatal physiological risk factors and early brain structure in very-low-birth-weight (VLBW) preterm infants using structural MRI and diffusion tensor imaging (DTI) at near-term age.
Structural brain MRI, diffusion-weighted scans, and neonatal physiological risk factors were analyzed in a cross-sectional sample of 102 VLBW preterm infants (BW ≤ 1500 g, gestational age (GA) ≤ 32 weeks), who were admitted to the Lucile Packard Children's Hospital, Stanford NICU and recruited to participate prior to routine near-term brain MRI conducted at 36.6 ± 1.8 weeks postmenstrual age (PMA) from 2010 to 2011; 66/102 also underwent a diffusion-weighted scan. Brain abnormalities were assessed qualitatively on structural MRI, and white matter (WM) microstructure was analyzed quantitatively on DTI in six subcortical regions defined by DiffeoMap neonatal brain atlas. Specific regions of interest included the genu and splenium of the corpus callosum, anterior and posterior limbs of the internal capsule, the thalamus, and the globus pallidus. Regional fractional anisotropy (FA) and mean diffusivity (MD) were calculated using DTI data and examined in relation to neonatal physiological risk factors including gestational age (GA), bronchopulmonary dysplasia (BPD), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), and sepsis, as well as serum levels of C-reactive protein (CRP), glucose, albumin, and total bilirubin.
Brain abnormalities were observed on structural MRI in 38/102 infants including 35% of females and 40% of males. Infants with brain abnormalities observed on MRI had higher incidence of BPD (42% vs. 25%) and sepsis (21% vs. 6%) and higher mean and peak serum CRP levels, respectively, (0.64 vs. 0.34 mg/dL, p = .008; 1.57 vs. 0.67 mg/dL, p= .006) compared to those without. The number of signal abnormalities observed on structural MRI correlated to mean and peak CRP (rho = .316, p = .002; rho = .318, p= .002). The number of signal abnormalities observed on MRI correlated with thalamus MD (left: r= .382, p= .002; right: r= .400, p= .001), controlling for PMA-at-scan. Thalamus WM microstructure demonstrated the strongest associations with neonatal risk factors. Higher thalamus MD on the left and right, respectively, was associated with lower GA (r = −.322, p = .009; r= −.381, p= .002), lower mean albumin (r = −.276, p= .029; r= −.385, p= .002), and lower mean bilirubin (r = −.293, p= .020; r= −.337 p= .007).
Results suggest that at near-term age, thalamus WM microstructure may be particularly vulnerable to certain neonatal risk factors. Interactions between albumin, bilirubin, phototherapy, and brain development warrant further investigation. Identification of physiological risk factors associated with selective vulnerability of certain brain regions at near-term age may clarify the etiology of neurodevelopmental impairment and inform neuroprotective treatment for VLBW preterm infants.
Highlights
•Biomarkers of inflammation in preterm infants correlated with brain abnormalities detected on near-term structural MRI.•Biomarkers of inflammation in preterm infants correlated with near-term WM microstructure assessed on DTI.•Signal abnormalities observed on near-term structural MRI correlated with increased thalamus MD.
doi:10.1016/j.nicl.2014.05.013
PMCID: PMC4110350  PMID: 25068107
MRI; Diffusion tensor imaging; White matter microstructure; Brain development; Risk factors; Preterm infants; VLBW, very-low-birth-weight; GA, gestational age; PMA, post-menstrual age; DTI, diffusion tensor imaging; FA, fractional anisotropy; MD, mean diffusivity; CC, corpus callosum; IC, internal capsule; ALIC, anterior limb of the internal capsule; PLIC, posterior limb of the internal capsule; GloP, globus pallidus
10.  Association of Early Preterm Birth with Abnormal Levels of Routinely Collected First and Second Trimester Biomarkers 
American journal of obstetrics and gynecology  2013;208(6):492.e1-492.e11.
Objective
To examine the relationship between typically measured prenatal screening biomarkers and early preterm birth in euploid pregnancies.
Study Design
Included were 345 early preterm cases (< 30 weeks) and 1,725 controls drawn from a population-based sample of California pregnancies that all had both first and second trimester screening results. Logistic regression analyses were used to compare patterns of biomarkers in cases and controls and to develop predictive models. Replicability of the biomarker-early preterm relationships revealed by the models was evaluated by examining the frequency and associated adjusted relative risks (RRsadj) for early preterm birth and for preterm birth in general (< 37 weeks) in pregnancies with identified abnormal markers compared to those without these markers in a subsequent independent California cohort of screened pregnancies (n = 76,588).
Results
The final model for early preterm birth included first trimester pregnancy-associated plasma protein A (PAPP-A) ≤ the 5th percentile, second trimester alpha-fetoprotein (AFP) ≥ the 95th percentile, and second trimester inhibin (INH) ≥ the 95th percentile (odds ratios 2.3 to 3.6). In general, pregnancies in the subsequent cohort with a biomarker pattern found to be associated with early preterm delivery in the first sample were at an increased risk for early preterm birth and preterm birth in general (< 37 weeks) (RRsadj 1.6 to 27.4). Pregnancies with two or more biomarker abnormalities were at particularly increased risk (RRsadj 3.6 to 27.4).
Conclusion
When considered across cohorts and in combination, abnormalities in routinely collected biomarkers reveal predictable risks for early preterm birth.
doi:10.1016/j.ajog.2013.02.012
PMCID: PMC3672244  PMID: 23395922
Preterm Birth; Prenatal Screening; Biomarkers
11.  Population-Level Correlates of Preterm Delivery among Black and White Women in the U.S 
PLoS ONE  2014;9(4):e94153.
Objective
This study examined the ability of social, demographic, environmental and health-related factors to explain geographic variability in preterm delivery among black and white women in the US and whether these factors explain black-white disparities in preterm delivery.
Methods
We examined county-level prevalence of preterm delivery (20–31 or 32–36 weeks gestation) among singletons born 1998–2002. We conducted multivariable linear regression analysis to estimate the association of selected variables with preterm delivery separately for each preterm/race-ethnicity group.
Results
The prevalence of preterm delivery varied two- to three-fold across U.S. counties, and the distributions were strikingly distinct for blacks and whites. Among births to blacks, regression models explained 46% of the variability in county-level risk of delivery at 20–31 weeks and 55% for delivery at 32–36 weeks (based on R-squared values). Respective percentages for whites were 67% and 71%. Models included socio-environmental/demographic and health-related variables and explained similar amounts of variability overall.
Conclusions
Much of the geographic variability in preterm delivery in the US can be explained by socioeconomic, demographic and health-related characteristics of the population, but less so for blacks than whites.
doi:10.1371/journal.pone.0094153
PMCID: PMC3989227  PMID: 24740117
12.  Treatment of neonatal jaundice with filtered sunlight in Nigerian neonates: study protocol of a non-inferiority, randomized controlled trial 
Trials  2013;14:446.
Background
Severe neonatal jaundice and its progression to kernicterus is a leading cause of death and disability among newborns in poorly-resourced countries, particularly in sub-Saharan Africa. The standard treatment for jaundice using conventional phototherapy (CPT) with electric artificial blue light sources is often hampered by the lack of (functional) CPT devices due either to financial constraints or erratic electrical power. In an attempt to make phototherapy (PT) more readily available for the treatment of pathologic jaundice in underserved tropical regions, we set out to test the hypothesis that filtered sunlight phototherapy (FS-PT), in which potentially harmful ultraviolet and infrared rays are appropriately screened, will be as efficacious as CPT.
Methods/design
This prospective, non-blinded randomized controlled non-inferiority trial seeks to enroll infants with elevated total serum/plasma bilirubin (TSB, defined as 3 mg/dl below the level recommended by the American Academy of Pediatrics for high-risk infants requiring PT) who will be randomly and equally assigned to receive FS-PT or CPT for a total of 616 days at an inner-city maternity hospital in Lagos, Nigeria. Two FS-PT canopies with pre-tested films will be used. One canopy with a film that transmits roughly 33% blue light (wavelength range: 400 to 520 nm) will be used during sunny periods of a day. Another canopy with a film that transmits about 79% blue light will be used during overcast periods of the day. The infants will be moved from one canopy to the other as needed during the day with the goal of keeping the blue light irradiance level above 8 μW/cm2/nm.
Primary outcome: FS-PT will be as efficacious as CPT in reducing the rate of rise in bilirubin levels. Secondary outcome: The number of infants requiring exchange transfusion under FS-PT will not be more than those under CPT.
Conclusion
This novel study offers the prospect of an effective treatment for infants at risk of severe neonatal jaundice and avoidable exchange transfusion in poorly-resourced settings without access to (reliable) CPT in the tropics.
Trial registration
ClinicalTrials.gov Identifier: NCT01434810
doi:10.1186/1745-6215-14-446
PMCID: PMC3879162  PMID: 24373547
Filtered sunlight phototherapy; Hyperbilirubinemia; Developing country; Low-cost technologies; Irradiance; Africa
13.  Integrating multiple ‘omics’ analyses identifies serological protein biomarkers for preeclampsia 
BMC Medicine  2013;11:236.
Background
Preeclampsia (PE) is a pregnancy-related vascular disorder which is the leading cause of maternal morbidity and mortality. We sought to identify novel serological protein markers to diagnose PE with a multi-’omics’ based discovery approach.
Methods
Seven previous placental expression studies were combined for a multiplex analysis, and in parallel, two-dimensional gel electrophoresis was performed to compare serum proteomes in PE and control subjects. The combined biomarker candidates were validated with available ELISA assays using gestational age-matched PE (n=32) and control (n=32) samples. With the validated biomarkers, a genetic algorithm was then used to construct and optimize biomarker panels in PE assessment.
Results
In addition to the previously identified biomarkers, the angiogenic and antiangiogenic factors (soluble fms-like tyrosine kinase (sFlt-1) and placental growth factor (PIGF)), we found 3 up-regulated and 6 down-regulated biomakers in PE sera. Two optimal biomarker panels were developed for early and late onset PE assessment, respectively.
Conclusions
Both early and late onset PE diagnostic panels, constructed with our PE biomarkers, were superior over sFlt-1/PIGF ratio in PE discrimination. The functional significance of these PE biomarkers and their associated pathways were analyzed which may provide new insights into the pathogenesis of PE.
doi:10.1186/1741-7015-11-236
PMCID: PMC4226208  PMID: 24195779
2D gel (two-dimensional gel electrophoresis); LCMS; Multiplex analysis; Preeclampsia; Proteomic profile
14.  Peptidomic Identification of Serum Peptides Diagnosing Preeclampsia 
PLoS ONE  2013;8(6):e65571.
We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.
doi:10.1371/journal.pone.0065571
PMCID: PMC3686758  PMID: 23840341
15.  High Quality Genome-Wide Genotyping from Archived Dried Blood Spots without DNA Amplification 
PLoS ONE  2013;8(5):e64710.
Spots of blood are routinely collected from newborn babies onto filter paper called Guthrie cards and used to screen for metabolic and genetic disorders. The archived dried blood spots are an important and precious resource for genomic research. Whole genome amplification of dried blood spot DNA has been used to provide DNA for genome-wide SNP genotyping. Here we describe a 96 well format procedure to extract DNA from a portion of a dried blood spot that provides sufficient unamplified genomic DNA for genome-wide single nucleotide polymorphism (SNP) genotyping. We show that SNP genotyping of the unamplified DNA is more robust than genotyping amplified dried blood spot DNA, is comparable in cost, and can be done with thousands of samples. This procedure can be used for genome-wide association studies and other large-scale genomic analyses that require robust, high-accuracy genotyping of dried blood spot DNA.
doi:10.1371/journal.pone.0064710
PMCID: PMC3667813  PMID: 23737996
16.  Molecular mechanism and functional consequences of lansoprazole-mediated heme oxygenase-1 induction 
AIM: To investigate the molecular mechanism and functional consequences of heme oxygenase-1 (HO-1) activation by lansoprazole in endothelial cells and macrophages.
METHODS: Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species (ROS) formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed using in vivo bioluminescence imaging.
RESULTS: Lansoprazole increased HO-1 mRNA levels in endothelial cells and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH-mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity.
CONCLUSION: Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002.
doi:10.3748/wjg.15.4392
PMCID: PMC2747059  PMID: 19764090
Antioxidants; Ferritin; Heme oxygenase-1; Lansoprazole; Reactive oxygen species
17.  Risk of Bronchopulmonary Dysplasia by Second Trimester Maternal Serum Levels of Alpha-fetoprotein, Human Chorionic Gonadotrophin, and Unconjugated Estriol 
Pediatric research  2012;71(4 0 1):399-406.
Although maternal serum alpha-fetoprotein (AFP), human chorionic gonandotrophin (hCG), and estriol play important roles in immunomodulation and immunoregulation during pregnancy, their relationship to the development of bronchopulmonary dysplasia (BPD) in young infants is unknown despite BPD being associated with pre- and postnatal inflammatory factors. The objective of this population-based study was to examine whether second trimester levels of AFP, hCG, and unconjugated estriol (uE3) were associated with an increased risk of BPD. We found that these serum biomarkers were associated with an increased risk of BPD. Risks were especially high when AFP and/or hCG levels were above the 95th percentile and/or when uE3 levels were below the 5th percentile (relative risks (RRs) 3.1 to 6.7). Risks increased substantially when two or more biomarker risks were present (RRs 9.9 to 75.9). Data suggested that pregnancies which had a biomarker risk and yielded an offspring with BPD were more likely to have other factors present that suggested early intrauterine fetal adaptation to a stress including maternal hypertension and asymmetric growth restriction.
doi:10.1038/pr.2011.73
PMCID: PMC3616500  PMID: 22391642
18.  Panhematin provides a therapeutic benefit in experimental pancreatitis 
Gut  2010;60(5):671-679.
Background and aim
Acute pancreatitis (AP) can result in pancreatic necrosis and inflammation, with subsequent multi-organ failure. AP is associated with increased neutrophil recruitment and rise in pro-inflammatory cytokines such as TNFα. Pretreatment with hemin, results in recruitment of hemeoxygenase-1 (HO-1)+ macrophages and protects from experimental pancreatitis. It is not clear whether modulation of HO-1 after onset of disease has a protective role. In this study, we tested the utility of Panhematin, a water-soluble hemin formulation, in activating and inducing pancreatic HO-1, and as a therapeutic agent in treating mouse acute pancreatitis.
Methods
We defined the distribution of radiolabeled hemin, then used in-vivo HO-1-luciferase bioluminescence imaging and CO2-release-assay to test Panhematin-induced upregulation of HO-1 transcription and activity, respectively. Using two well-defined AP murine models, we tested the therapeutic benefit of Panhematin, and quantified cytokine release using a luminex assay.
Results
Intravenously-administered Panhematin induces rapid recruitment of HO-1+ cells to the pancreas within 2h and de novo splenic HO-1 transcription by 12h. Despite high baseline spleen HO-1 activity, the pancreas is particularly responsive to Panhematin-mediated HO-1 induction. Panhematin-treated mice, at various time points after AP induction had significant reduction in mortality, pancreatic injury, together with up-regulation of HO-1 and down-regulation of pro-inflammatory cytokines and CXCL1, a potent neutrophil chemoattractant.
Conclusions
Despite AP-associated mortality and morbidity, no effective treatment other than supportive care exists. We demonstrate that Panhematin leads to: i) rapid induction and activation of pancreatic HO-1 with recruitment of HO-1+ cells to the pancreas, ii) amelioration of AP even when given late during the course of disease, and iii) a decrease in leukocyte infiltration and pro-inflammatory cytokines including CXCL1. The utility of Panhematin at modest doses as a therapeutic in experimental pancreatitis, coupled with its current use and safety in humans, raises the potential of its applicability to human pancreatitis.
doi:10.1136/gut.2010.217208
PMCID: PMC3580958  PMID: 21159893
Acute pancreatitis; heme-oxygenase 1; CXCL1
19.  Effects of Sample Dilution, Peroxidase Concentration, and Chloride Ion on the Measurement of Unbound Bilirubin in Premature Newborns 
Clinical biochemistry  2006;40(3-4):261-267.
Objectives
To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl-) on plasma unbound bilirubin (Bf) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns.
Design and Methods
Bf was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with Bf measured at a 2-fold sample dilution using a FloPro Analyzer. Bf was measured at two peroxidase concentrations to determine whether the peroxidase steady state Bf (Bfss) measurements were significantly less than the equilibrium Bf (Bfeq), in which case it was necessary to calculate Bfeq from the two Bfss measurements. Bf was also measured before and after adding 100 mmol/L Cl- to the UB Analyzer assay buffer.
Results
Bfeq at the 42-fold dilution was nearly 10-fold less than but correlated significantly with Bfeq at the 2-fold dilution (mean 8.2±5.2 nmol/L versus 73.5 ±70 nmol/L, respectively, p<0.0001; correlation r=0.6). The two UB Analyzer Bfss measurements were significantly less than Bfeq in 42 of 74 (57%) samples, and Cl- increased Bfeq in 66 of 74 (89%) samples by a mean of 82±67%.
Conclusions
Bfss measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl- and a single peroxidase concentration is significantly less than the Bfeq in undiluted plasma. Accurate Bf measurements can be made only in minimally diluted serum or plasma.
doi:10.1016/j.clinbiochem.2006.09.006
PMCID: PMC1945224  PMID: 17069786
newborn jaundice; hyperbilirubinemia; unbound bilirubin; peroxidase test; bilirubin/albumin binding; free bilirubin
20.  Cytokines and Neurodevelopmental Outcomes in Extremely Low Birth Weight Infants 
The Journal of pediatrics  2011;159(6):919-925.e3.
Objective
To determine if selected pro-inflammatory and anti-inflammatory cytokines/mediators of inflammation reported to be related to development of cerebral palsy predict neurodevelopmental outcome in extremely low birth weight infants.
Study design
Infants with birth weights ≤ 1000 g (n=1067) had blood samples collected at birth and on days 3±1, 7±1, 14±3, and 21±3 to examine the association between cytokines and neurodevelopmental outcomes. The analyses were focused on five cytokines (IL-1β, IL-8, TNF-α, RANTES, and IL-2) reported to be most predictive of CP in term and late preterm infants.
Results
IL-8 was higher on days 0–4 and subsequently in infants who developed CP compared with infants who did not develop CP in both unadjusted and adjusted analyses. Other cytokines (IL-12, IL-17, TNF-β, SIL-rα, MIP-1β) were found to be altered on days 0–4 in infants who developed CP.
Conclusions
CP in former preterm infants may, in part, have a late perinatal and/or early neonatal inflammatory origin.
doi:10.1016/j.jpeds.2011.05.042
PMCID: PMC3215787  PMID: 21798559
21.  Is phototherapy exposure associated with better or worse outcomes in 501–1000 gram birth weight infants? 
Aim
To compare risk-adjusted outcomes at 18–22 months corrected age for extremely low birth weight (ELBW) infants who never received phototherapy (NoPTx) to those who received any phototherapy (PTx) in the NICHD Neonatal Research Network randomized trial of Aggressive vs. Conservative Phototherapy.
Methods
Outcomes at 18–22 months corrected age included death, neurodevelopmental impairment (NDI), and Bayley Scales Mental Developmental Index (MDI). Regression models evaluated the independent association of PTx with adverse outcomes controlling for center and other potentially confounding variables.
Results
Of 1972 infants, 216 were NoPTx and 1756 were PTx. For the entire 501–1000 g BW cohort, PTx was not independently associated with death or NDI (OR 0.85, 95% CI 0.60 –1.20), death, or adverse neurodevelopmental endpoints. However, among infants 501–750 g BW, the rate of significant developmental impairment with MDI<50 was significantly higher for NoPTx (29%) than PTx (12%) (p=0.004).
Conclusions
Phototherapy did not appear to be independently associated with death or NDI for the overall ELBW group. Whether PTx increases mortality could not be excluded due to bias from deaths before reaching conservative treatment threshold. The higher rate of MDI<50 in the 501–750g BW NoPTx group is concerning, and consistent with NRN Trial results.
doi:10.1111/j.1651-2227.2011.02175.x
PMCID: PMC3505994  PMID: 21272067
22.  Maternal Heme Oxygenase 1 Regulates Placental Vasculature Development via Angiogenic Factors in Mice1 
Biology of Reproduction  2011;85(5):1005-1012.
The placental vasculature is critical for nutrient, gas, and waste exchange between the maternal and fetal systems. Its development depends on the proper expression and interaction of angiogenesis and associated growth factors. Heme oxygenase (HMOX), the enzyme for heme degradation, plays a role in angiogenesis and is highly expressed in the placenta. To evaluate the role of maternal HMOX1, the inducible HMOX isozyme, on placental vasculature formation, mice with a partial deficiency in Hmox1 (Hmox1+/−) were used. Three-dimensional images of placental vasculatures as well as spiral arteries from Hmox1+/+ or Hmox1+/− placentas were created by vascular corrosion casting technique and imaged by micro-computerized tomography (microCT). The structures and morphologies of fetomaternal interfaces were observed by histological staining and the ultrastructure of uterine natural killer (uNK) cells, a major regulator in spiral artery remodeling, was analyzed by transmission electron microscopy. A group of growth factors and angiogenic factors from the decidua/mesometrial lymphoid aggregate of pregnancy (MLAp) as well as labyrinth regions were quantified using an angiogenesis PCR array kit and compared between Hmox1+/+ or Hmox1+/− placentas. In conclusion, a partial deficiency of maternal Hmox1 resulted in the malformation of fetomaternal interface, insufficiency of spiral artery remodeling, and alteration of uNK cell differentiation and maturation. These changes were independent of the fetal genotype, but relied on the maternal HMOX1 level, which determined the balance of expression levels of pro- and antiangiogenic factors in the decidua/MLAp region. These results implied that Hmox1 polymorphisms among the human population might contribute to some unexplained cases of pregnancy disorders, such as fetal growth retardation and preeclampsia.
Maternal HMOX1 is essential for the formation of the fetomaternal interface, remodeling of the uterine spiral arteries, and differentiation and maturation of uNK cells; its effects are primarily mediated through the regulation of a group of pro- and anti-angiogenesis factors in the decidua/MLAp region.
doi:10.1095/biolreprod.111.093039
PMCID: PMC3197918  PMID: 21778140
angiogenesis; heme oxygenase 1; intrauterine growth restriction (IUGR); placenta; spiral artery remodeling; uterine natural killer (uNK) cell
23.  Effects of Zinc Deuteroporphyrin Bis Glycol on Newborn Mice After Heme-Loading 
Pediatric research  2011;70(5):467-472.
Infants with hemolytic diseases frequently develop hyperbilirubinemia, but standard phototherapy only eliminates bilirubin after its production. A better strategy might be to directly inhibit heme oxygenase (HO), the rate-limiting enzyme in bilirubin production. Metalloporphyrins (Mps) are heme analogs that competitively inhibit HO activity in vitro and in vivo and suppress plasma bilirubin levels in vivo. A promising Mp, zinc deuteroporphyrin bis glycol (ZnBG), is orally absorbed and effectively inhibits HO activity at relatively low doses. We determined the I50 (the dose needed to inhibit HO activity by 50%) of orally administered ZnBG in vivo and then evaluated ZnBG’s effects on in vivo bilirubin production, HO activity, HO protein levels, and HO-1 gene expression in newborn mice following heme-loading, a model analogous to a hemolytic infant. The I50 of ZnBG was found to be 4.0 μmol/kg body weight (BW). At a dose of 15-μmol/kg BW, ZnBG reduced in vivo bilirubin production, inhibited heme-induced liver HO activity and spleen HO activity to and below baseline, respectively, transiently induced liver and spleen HO-1 gene transcription, and induced liver and spleen HO-1 protein levels. We conclude that ZnBG may be an attractive compound for treating severe neonatal hyperbilirubinemia caused by hemolytic disease.
doi:10.1203/PDR.0b013e31822e1675
PMCID: PMC3189293  PMID: 21785387
24.  Predictive Value of an Early Amplitude Integrated Electroencephalogram and Neurologic Examination 
Pediatrics  2011;128(1):e112-e120.
OBJECTIVE:
To examine the predictive validity of the amplitude integrated electroencephalogram (aEEG) and stage of encephalopathy among infants with hypoxic-ischemic encephalopathy (HIE) eligible for therapeutic whole-body hypothermia.
DESIGN:
Neonates were eligible for this prospective study if moderate or severe HIE occurred at <6 hours and an aEEG was obtained at <9 hours of age. The primary outcome was death or moderate/severe disability at 18 months.
RESULTS:
There were 108 infants (71 with moderate HIE and 37 with severe HIE) enrolled in the study. aEEG findings were categorized as normal, with continuous normal voltage (n = 12) or discontinuous normal voltage (n = 12), or abnormal, with burst suppression (n = 22), continuous low voltage (n = 26), or flat tracing (n = 36). At 18 months, 53 infants (49%) experienced death or disability. Severe HIE and an abnormal aEEG were related to the primary outcome with univariate analysis, whereas severe HIE alone was predictive of outcome with multivariate analysis. Addition of aEEG pattern to HIE stage did not add to the predictive value of the model; the area under the curve changed from 0.72 to 0.75 (P = .19).
CONCLUSIONS:
The aEEG background pattern did not significantly enhance the value of the stage of encephalopathy at study entry in predicting death and disability among infants with HIE.
doi:10.1542/peds.2010-2036
PMCID: PMC3124102  PMID: 21669899
neonatal hypoxic-ischemic encephalopathy; amplitude integrated EEG
25.  Metalloporphyrins – An Update 
Metalloporphyrins are structural analogs of heme and their potential use in the management of neonatal hyperbilirubinemia has been the subject of considerable research for more than three decades. The pharmacological basis for using this class of compounds to control bilirubin levels is the targeted blockade of bilirubin production through the competitive inhibition of heme oxygenase (HO), the rate-limiting enzyme in the bilirubin production pathway. Ongoing research continues in the pursuit of identifying ideal metalloporphyrins, which are safe and effective, by defining therapeutic windows and targeted interventions for the treatment of excessive neonatal hyperbilirubinemia.
doi:10.3389/fphar.2012.00068
PMCID: PMC3337460  PMID: 22557967
bilirubin; heme oxygenase; hemolysis; neonatal hyperbilirubinemia

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