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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.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  The effects of aggressive vs. conservative phototherapy on the brainstem auditory evoked responses of extremely-low-birth-weight infants 
Pediatric Research  2012;71(1):77-84.
INTRODUCTION
This study was a two-center, stratified, parallel-group randomized trial comparing the effects of aggressive vs. conservative phototherapy on brainstem auditory evoked response (BAER) latencies in infants with extremely low birth weight (ELBW, ≤ 1,000 g).
RESULTS
BAER latencies of 751–1,000 g birth-weight infants were shorter by 0.37 ms (95% confidence interval (CI) = 0.02, 0.73) for wave V, 0.39 ms (0.08, 0.70) for wave III, and 0.33 ms (0.01, 0.65) for wave I after aggressive phototherapy at one center. Interwave intervals did not differ significantly. Similar nonsignificant trends were recorded for 501–750 g birth-weight infants. At the other participating center, no significant differences were recorded, cautioning against overgeneralizing these results.
DISCUSSION
The effects of bilirubin on the auditory pathway in ELBW infants depend on a complex interaction of bilirubin exposure, newborn characteristics, and clinical management.
METHODS
Aggressive phototherapy was initiated sooner and continued at lower bilirubin levels than conservative phototherapy. A total of 174 ELBW infants were enrolled in the study; 111 infants were successfully tested at 35 weeks postmenstrual age (PMA); 57 died; and 6 were not successfully tested.
doi:10.1038/pr.2011.17
PMCID: PMC3326602  PMID: 22289854
14.  HEMOLYSIS AND HYPERBILIRUBINEMIA IN ABO BLOOD GROUP HETEROSPECIFIC NEONATES 
The Journal of pediatrics  2010;157(5):772-777.
Objective
We quantified hemolysis and determined the incidence of hyperbilirubinemia in direct antiglobulin titer (DAT) positive, ABO heterospecific neonates and compared variables among O-A and O-B subgroups. Study design Plasma total bilirubin (PTB) was determined predischarge and more frequently if clinically warranted, in DAT positive, blood group A or B neonates of group O mothers. Heme catabolism (and therefore bilirubin production) was indexed by blood carboxyhemoglobin corrected for inspired carbon monoxide (COHbc). Hyperbilirubinemia was defined as any PTB concentration >95th percentile on the hour-of-life-specific bilirubin nomogram.
Results
Of 164 neonates, 111 were O-A and 53 O-B. Overall, 85 (51.8%) developed hyperbilirubinemia, which tended to be more prevalent in the O-B than O-A neonates (62.3% vs. 46.8% respectively, p=0.053). Importantly, more O-B than O-A newborns developed hyperbilirubinemia at <24 hours (93.9% vs. 48.1%, p<0.0001). COHbc values were globally higher than our previously published newborn values. Babies who developed hyperbilirubinemia had higher COHbc values than the already high values of those non-hyperbilirubinemic, and O-B newborns tended to have higher values than O-A counterparts.
Conclusions
DAT positive, ABO heterospecificity is associated with increased hemolysis and a high incidence of neonatal hyperbilirubinemia. O-B heterospecificity tends to confer even higher risk than O-A counterparts.
doi:10.1016/j.jpeds.2010.05.024
PMCID: PMC2951500  PMID: 20598320
Direct antiglobulin titer; bilirubin; ABO heterospecificity; hemolysis; hyperbilirubinemia; carboxyhemoglobin
15.  Metalloporphyrins in the management of neonatal hyperbilirubinemia 
Summary
Neonatal jaundice in the first week of life is a common problem in newborns. It is due to an imbalance of bilirubin production and its elimination, which can lead to significantly elevated levels of circulating bilirubin or hyperbilirubinemia. Use of phototherapy and/or exchange transfusion are the current modes for treating neonatal hyperbilirubinemia and preventing any neurologic damage. These strategies, however, only remove bilirubin that has already been formed. Preventing the production of excess bilirubin may be a more logical approach. Synthetic heme analogs, metalloporphyrins, are competitive inhibitors of heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, and their use has been proposed as an attractive alternative strategy for preventing or treating severe hyperbilirubinemia.
doi:10.1016/j.siny.2009.11.004
PMCID: PMC2859976  PMID: 20006567
Bilirubin; Heme oxygenase; Hyperbilirubinemia metalloporphyrin; Neonatal jaundice
16.  Heme Oxygenase-1 Deletion Affects Stress Erythropoiesis 
PLoS ONE  2011;6(5):e20634.
Background
Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1) deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.
Methodology/Principal Findings
We used a transplant model to induce stress conditions. In irradiated recipients that received hmox+/− or hmox+/+ bone marrow cells, we evaluated (i) the erythrocyte parameters in the peripheral blood; (ii) the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii) the patterns of histological iron staining; and (iv) the number of Mac-1+-cells expressing TNF-α. In the spleens of mice that received hmox+/− cells, we show (i) decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii) increases in the insoluble iron levels and decreases in the soluble iron levels; (iii) increased numbers of Mac-1+-cells expressing TNF-α; and (iv) decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.
Conclusions/Significance
As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.
doi:10.1371/journal.pone.0020634
PMCID: PMC3105104  PMID: 21655188
17.  Influence of Clinical Status on the Association Between Plasma Total and Unbound Bilirubin and Death or Adverse Neurodevelopmental Outcomes in Extremely Low Birth Weight Infants 
Objectives
To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18–22 months corrected age in extremely low birth weight infants.
Method
Total plasma biirubin and unbound biirubin were measured in 1,101 extremely low birth weight infants at 5±1 day of age. Clinical criteria were used to classify infants as clinically stable or unstable. Survivors were examined at 18–22 months corrected age by certified examiners. Outcome variables were death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death prior to follow-up. For all outcomes, the interaction between bilirubin variables and clinical status was assessed in logistic regression analyses adjusted for multiple risk factors.
Results
Regardless of clinical status, an increasing level of unbound bilirubin was associated with higher rates of death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss and death before follow-up. Total plasma bilirubin values were directly associated with death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death before follow-up in unstable infants, but not in stable infants. An inverse association between total plasma bilirubin and death or cerebral palsy was found in stable infants.
Conclusions
In extremely low birth weight infants, clinical status at 5 days of age affects the association between total plasma and unbound bilirubin and death or adverse neurodevelopmental outcomes at 18–22 months of corrected age. An increasing level of UB is associated a higher risk of death or adverse neurodevelopmental outcomes regardless of clinical status. Increasing levels of total plasma bilirubin are directly associated with increasing risk of death or adverse neurodevelopmental outcomes in unstable, but not in stable infants.
doi:10.1111/j.1651-2227.2010.01688.x
PMCID: PMC2875328  PMID: 20105142
Plasma bilirubin; unbound bilirubin; Extremely low birth weight infants; Neurodevelopmental outcomes
18.  EFFECT OF HEME OXYGENASE-1 DEFICIENCY ON PLACENTAL DEVELOPMENT 
Placenta  2009;30(10):861-868.
Heme oxygenase (HO) is the rate-limiting enzyme in the heme catabolic pathway and highly expressed in the placenta. Deficiencies in HO-1, the inducible isoform, have been associated with pregnancy disorders, such as recurrent miscarriages, intrauterine growth retardation, and pre-eclampsia. The aim of this study was to identify if a deficiency in HO-1 affects placental development using a mouse model. When HO-1 heterozygote (Het, HO-1+/−) mice were cross-bred, an extremely low birth rate in homozygote (Mut, HO-1−/−) offspring (2.4%) and small litter sizes were observed. Placentas and fetuses from Het cross-breedings were relatively smaller and weighed less than those from wild-type (WT) cross-breedings at E12.5 and E15.5. Furthermore, Het placentas had significantly less HO-1 mRNA and protein levels than WT placentas, but no significant differences in placental HO activity. Interestingly, HO-2, the constitutive HO isoform, as well as iNOS and eNOS expression were significantly upregulated in Het placentas. Histological examination showed that the junctional zone (JZ) of Het placentas were markedly thinner than those of WT placentas and appeared to be due to an increase in apoptosis. Immunohistochemistry revealed that HO-1-expressing cells were located primarily in the JZ of Het placentas, specifically in the spongiotrophoblast layer. In addition, diastolic blood pressures and plasma soluble VEGFR-1 (sFlt-1) levels were significantly elevated in pregnant Het mice. We conclude that a partial deficiency in HO-1 is associated with morphological changes in the placenta and elevations in maternal diastolic blood pressure and plasma sFlt-1 levels, despite a compensatory increase in HO-2 expression.
doi:10.1016/j.placenta.2009.07.012
PMCID: PMC2771543  PMID: 19699520
19.  Dermal Carbon Monoxide (CO) Excretion in Neonatal Rats During Light Exposure 
Pediatric research  2009;66(1):66-69.
Total body, head, and trunk carbon monoxide (CO) excretion rates were measured separately by gas chromatography in 1–7 day-old Wistar rat pups exposed to the dark and to mixed blue (1 Special Blue – F20T12/BB) and white (2 Cool White – F20T12/CW) fluorescent light or blue light-emitting diode (LED) sources. During 48-min cycled exposures to the dark and to either light source, total body CO excretion rapidly increased 1.9- and 1.4-fold, respectively, over dark control levels. When CO excretion rates from the head and trunk were measured separately during exposure to either light source, CO excretion from the head did not change significantly; however, a large mean 4.4-fold increase in CO excretion from the trunk was observed. When light intensity delivered by the blue LED source was varied, we found that trunk CO excretion increased with increasing light intensities. In the presence of riboflavin (10 µmol/kg), total body CO excretion increased 2.8- and 2.1-fold during exposure to the mixed fluorescent light and blue LED sources, respectively. We conclude that light-induced elevations in total body CO excretion may be caused by transdermally-excreted CO, which is most likely produced through endogenous photosensitizer-mediated photo-oxidation of dermal biomolecules.
doi:10.1203/PDR.0b013e3181a7be77
PMCID: PMC2714864  PMID: 19342986
20.  Aggressive vs. Conservative Phototherapy for Infants with Extremely Low Birth Weight 
Background
It is unclear whether aggressive phototherapy to prevent neurotoxic effects of bilirubin benefits or harms infants with extremely low birth weight (1000 g or less).
Methods
We randomly assigned 1974 infants with extremely low birth weight at 12 to 36 hours of age to undergo either aggressive or conservative phototherapy. The primary outcome was a composite of death or neurodevelopmental impairment determined for 91% of the infants by investigators who were unaware of the treatment assignments.
Results
Aggressive phototherapy, as compared with conservative phototherapy, significantly reduced the mean peak serum bilirubin level (7.0 vs. 9.8 mg per deciliter [120 vs. 168 μmol per liter], P<0.01) but not the rate of the primary outcome (52% vs. 55%; relative risk, 0.94; 95% confidence interval [CI], 0.87 to 1.02; P = 0.15). Aggressive phototherapy did reduce rates of neurodevelopmental impairment (26%, vs. 30% for conservative phototherapy; relative risk, 0.86; 95% CI, 0.74 to 0.99). Rates of death in the aggressive-phototherapy and conservative-phototherapy groups were 24% and 23%, respectively (relative risk, 1.05; 95% CI, 0.90 to 1.22). In preplanned subgroup analyses, the rates of death were 13% with aggressive phototherapy and 14% with conservative phototherapy for infants with a birth weight of 751 to 1000 g and 39% and 34%, respectively (relative risk, 1.13; 95% CI, 0.96 to 1.34), for infants with a birth weight of 501 to 750 g.
Conclusions
Aggressive phototherapy did not significantly reduce the rate of death or neurodevelopmental impairment. The rate of neurodevelopmental impairment alone was significantly reduced with aggressive phototherapy. This reduction may be offset by an increase in mortality among infants weighing 501 to 750 g at birth. (ClinicalTrials. gov number, NCT00114543.)
doi:10.1056/NEJMoa0803024
PMCID: PMC2821221  PMID: 18971491
21.  Predictors of Death or Bronchopulmonary Dysplasia in Preterm Infants with Respiratory Failure 
Objectives
To identify the variables that predict death/physiologic BPD in preterm infants with severe respiratory failure.
Study Design
The study was a secondary analysis of data from the NICHD Neonatal Research Network trial of inhaled nitric oxide (iNO) in preterm infants. Stepwise logistic regression models and Classification and Regression Tree (CART) models were developed for the outcome of death or physiologic BPD (O2 at 36 weeks’ postmenstrual age).
Results
Death and/or BPD was associated with lower birth weight, higher oxygen requirement, male gender, additional surfactant doses, higher oxygenation index, and outborn status, but not the magnitude of response in PaO2 to iNO. The positive predictive value of the CART model was 82% at 95% sensitivity.
Conclusions
The major factors associated with death/BPD were an increased severity of respiratory failure, lower birth weight, male gender, and outborn status, but not the magnitude of initial response to iNO.
doi:10.1038/jp.2008.18
PMCID: PMC2776028  PMID: 18337740
Logistic models; Predictive value of tests; ROC curve
22.  INTEROBSERVER RELIABILITY AND ACCURACY OF CRANIAL ULTRASOUND INTERPRETATION IN PREMATURE INFANTS 
The Journal of pediatrics  2007;150(6):592-596.e5.
Objective
To assess interobserver reliability between two central readers of cranial ultrasound (CUS) and accuracy of local compared with central interpretations.
Study design
A retrospective analysis of CUS data from the NICHD trial of inhaled nitric oxide for premature infants. Interobserver reliability of two central readers was assessed by kappa or weighted kappa. Accuracy of local compared with central interpretations was assessed by sensitivity and specificity.
Results
Cranial US from 326 infants had both central reader and local interpretations. Central reader agreement for grade 3/4 IVH, grade 3/4 IVH or PVL, grade of IVH, and degree of ventriculomegaly was very good (kappa=0.84, 0.81, 0.79, and 0.75, respectively). Agreement was poor for lower grade IVH and for PVL alone. Local interpretations were highly accurate for grade 3/4 IVH or PVL (sensitivity 87–90%, specificity 92–93%), but sensitivity was poor to fair for grade 1/2 IVH (48–68%) and PVL (20–44%).
Conclusions
Our findings demonstrate reliability and accuracy of highly unfavorable CUS findings, but suggest caution when interpreting mild to moderate IVH or white matter injury.
doi:10.1016/j.jpeds.2007.02.012
PMCID: PMC2757063  PMID: 17517240
intraventricular; hemorrhage; leukomalacia; central reader; neurodevelopmental; brain
23.  Inhaled Nitric Oxide for Preterm Premature Rupture of Membranes, Oligohydramnios, and Pulmonary Hypoplasia 
American journal of perinatology  2008;26(4):317-322.
We sought to determine if inhaled nitric oxide (iNO) administered to preterm infants with premature rupture of membranes (PPROM), oligohydramnios, and pulmonary hypoplasia improved oxygenation, survival, or other clinical outcomes. Data were analyzed from infants with suspected pulmonary hypoplasia, oligohydramnios, and PPROM enrolled in the National Institute of Child Health and Development Neonatal Research Network Preemie Inhaled Nitric Oxide (PiNO) trial, where patients were randomized to receive placebo (oxygen) or iNO at 5 to 10 ppm. Outcome variables assessed were PaO2 response, mortality, bronchopulmonary dysplasia (BPD), and severe intraventricular hemorrhage (IVH) or periventricular leukomalacia (PVL). Twelve of 449 infants in the PiNO trial met criteria. Six infants received iNO and six received placebo. The iNO group had a mean increase in PaO2 of 39±50 mm Hg versus a mean decrease of 11±15 mm Hg in the control group. Mortality was 33% versus 67%, BPD (2/5) 40% versus (2/2) 100%, and severe IVH or PVL (1/5) 20% versus (1/2) 50% in the iNO and control groups, respectively. None of these changes were statistically significant. Review of a limited number of cases from a large multicenter trial suggests that iNO use in the setting of PPROM, oligohydramnios, and suspected pulmonary hypoplasia improves oxygenation and may decrease the rate of BPD and death without increasing severe IVH or PVL. However, the small sample size precludes definitive conclusions. Further studies are required to determine if iNO is of benefit in this specific patient population.
doi:10.1055/s-0028-1104743
PMCID: PMC2676224  PMID: 19067285
Nitric oxide; pulmonary hypoplasia; oligohydramnios; PPROM; bronchopulmonary dysplasia
24.  The Role of Bach1 in the Induction of Heme Oxygenase by Tin Mesoporphyrin 
Tin mesoporphyrin (SnMP), a competitive heme oxygenase (HO) inhibitor, also induces HO-1 mRNA and protein expression by a mechanism that is not fully understood. We examined whether the induction by SnMP is mediated by a de-repression of Bach1, a transcription factor that suppresses the HO-1 gene. Incubation of NIH3T3-HO-1-luc cells with SnMP attenuated HO activity with a concomitant increase in HO-1 mRNA and protein and a decrease in Bach1 and HO-2 proteins, which was not due to transcriptional down-regulation, but accelerated protein decay. Similarly, HO-1 protein degradation was increased by SnMP, despite of an elevation in HO-1 transcription. Transfection of Bach1 shRNA in Hepa cells raised basal HO-1 expression significantly, and SnMP treatment further increased HO-1 mRNA. In conclusion, SnMP induces HO-1 expression not only by de-repressing the HO-1 promoter by binding Bach1, but also by accelerating Bach1 degradation.
doi:10.1016/j.bbrc.2007.01.050
PMCID: PMC1805812  PMID: 17257585
Bach1; heme oxygenase; isozymes; metalloporphyrins; tin mesoporphyrin
25.  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

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