Brain injury from preterm birth causes white matter injury (WMI), and leads to chronic neurological deficits including cerebral palsy, epilepsy, cognitive and behavioral delay. Immature O4+ oligodendrocytes are particularly vulnerable to WMI. Understanding how the developing brain recovers after injury is essential to finding more effective therapeutic strategies. Erythropoietin (EPO) promotes neuronal recovery after injury however its role in enhancing oligodendroglial lineage recovery is unclear. Previously we found recombinant EPO (rEPO)-treatment enhances MBP expression and functional recovery in adult rats after prenatal transient systemic hypoxia-ischemia (TSHI). We hypothesized that after injury rEPO would enhance oligodendroglial lineage cell genesis, survival, maturation and myelination.
In vitro assays were used to define how rEPO contributes to specific stages of oligodendrocyte development and recovery after TSHI.
After prenatal TSHI injury, rEPO promotes genesis of oligodendrocyte progenitors from oligodendrospheres, survival of oligodendrocyte precursor cells (OPCs) and O4+ immature oligodendrocytes, O4+ cell process extension and MBP expression. rEPO did not alter OPC proliferation.
Together, these studies demonstrate that EPO signaling promotes critical stages of oligodendroglial lineage development and recovery after prenatal TSHI injury. Erythropoietin treatment may be beneficial to preterm and other infant patient populations with developmental brain injury hallmarked by WMI.
Severe hypoxic-ischemic encephalopathy (HIE) is a devastating condition that can lead to mortality and long-term disabilities in term newborns. No rapid and reliable laboratory test exists to assess the degree of neuronal injury in these patients. We propose two possible biomarkers: 1) phosphorylated axonal neurofilament heavy chain (pNF-H) protein, one of the major subunits of neurofilaments, found only in axonal cytoskeleton of neurons and 2) Ubiquitin C-terminal hydrolase 1 (UCHL1 protein) that is heavily and specifically concentrated in neuronal perikarya and dendrites. High-serum pNF-H and UCHL1 levels are reported in subarachnoid hemorrhage and traumatic brain injury, suggesting that they are released into blood following neuronal injury. We hypothesized that serum pNF-H and UCHL1 were higher in neonates with moderate-to-severe HIE than in healthy neonates. A time-limited enrollment of 14 consecutive patients with HIE and 14 healthy controls was performed. UCHL1 and pNF-H were correlated with clinical data and brain MRI. UCHL1 and pNF-H serum levels were higher in HIE versus controls. UCHL1 showed correlation with the 10-min Apgar score, and pNF-H showed correlation with abnormal brain MRI. Our findings suggest that serum UCHL1 and pNF-H could be explored as diagnostic and prognostic tools in neonatal HIE.
Tandem mass spectrometry has been proposed as a method of diagnosing or predicting the development of common complex neonatal diseases. Our objective was to identify metabolites associated with common complications of prematurity.
We performed a retrospective analysis of medical data and metabolite measurements from routine neonatal screening on 689 preterm (<37 weeks of gestational age) neonates.
We observed higher levels of phenylalanine in infants with respiratory distress syndrome (RDS; P=1.7×10−5), the only association that was significant after correction for multiple testing. We found suggestive significance (P<0.001) of higher essential amino acids in infants with patent ductus arteriosus (PDA). Functionality of these findings was explored in the ductus arteriosus (DA) isolated from term and preterm mouse pups. None of the amino acids had a direct vasodilatory effect on the isolated DA.
We found newborns with RDS had higher levels of phenylalanine that may be due to impaired phenylalanine hydroxylase activity. We also detected marginally higher levels of all measured essential amino acids in infants with PDA. We did not find dilation of the mouse ductus for these metabolites indicating that instead of potentially causing PDA they are likely serving as markers of catabolism.
Premature infants fed formula are more likely to develop necrotizing enterocolitis (NEC) than if fed breast milk, but the mechanisms of intestinal necrosis in NEC and protection by breast milk are unknown. We hypothesized that after lipase digestion, formula, but not fresh breast milk, contains levels of unbound free fatty acids (FFAs) that are cytotoxic to intestinal cells.
We digested multiple term and preterm infant formulas or human milk with pancreatic lipase, proteases (trypsin and chymotrypsin), lipase + proteases, or luminal fluid from a rat small intestine and tested FFA levels and cytotoxicity in vitro on intestinal epithelial cells, endothelial cells, and neutrophils.
Lipase digestion of formula, but not milk, caused significant death of neutrophils (ranging from 47–99% with formulas vs. 6% with milk) with similar results in endothelial and epithelial cells. FFAs were significantly elevated in digested formula versus milk and death from formula was significantly decreased with lipase inhibitor pretreatment, or treatments to bind FFAs. Protease digestion significantly increased FFA binding capacity of formula and milk but only enough to decrease cytotoxicity from milk.
FFA-induced cytotoxicity may contribute to the pathogenesis of NEC.
free fatty acids; shock; lipase; cytotoxicity
Defensins are antimicrobial peptides expressed on mucosal surfaces that contribute to maintaining intestinal homeostasis by providing innate defense mechanisms for the epithelia. Defensin expression is altered in a number of diseases that affect mucosal surfaces, such as atopic dermatitis, allergic rhinitis, and inflammatory bowel disease. Similar to atopic dermatitis, eosinophilic esophagitis (EoE) is a chronic disease in which the squamous epithelial surface is affected by a similar TH2 microenvironment and eosinophil predominant inflammation. Therefore, we hypothesized defensin expression would be decreased in EoE.
To address this, we measured defensin expression in vitro in cell lines derived from patients with EoE (EoE1-T) or gastroesophageal reflux disease (GERD) (NES-G4T cells), and ex vivo in esophageal mucosal biopsy samples from children with EoE, GERD, and control children without esophageal disease.
IL-5 induced a decrease in human beta-defensin 1 (hBD1) and human beta-defensin 3 (hBD3) expression in EoE1-T but not in NES-G4T cells. Compared to esophageal biopsy specimens from GERD and control children, specimens from EoE pediatric patients revealed significant decrease in mRNA and protein expression for hBD1 and hBD3.
Diminished expression of hBD1 and hBD3 may make the esophageal epithelium more susceptible to the development and/or perpetuation of EoE.
Prostasin is a membrane-bound/secretive serine protease interacting with aldosterone and the epithelial sodium channel in the kidney. We and others have previously proposed the concept of stress-induced pressure natriuresis (SIPN) where increased urinary sodium excretion (UNaV) is coupled with elevated blood pressure (BP) in response to behavioral stress in normotensive adolescents. This study thus aimed to test the relationship between prostasin and pressure natriuresis using the SIPN model. A cohort of 102 normotensive black adolescents (mean age: 17.0±1.2 years; 56% females) were placed on a controlled sodium (4000±200 mg/day) and potassium (2600±200 mg/day) diet for three days before testing. The SIPN protocol consisted of a one-hour baseline period, a one-hour stress period (competitive video games), and a one-hour recovery period. During the stress period, BP elevation was coupled with an increase in UNaV. Urinary prostasin concentration had more than a two-fold reduction from baseline (38.4±32.7 ng/ml) to stress (17.2±16.0 ng/ml), and further declined during recovery (12.1±16.2 ng/ml) (p<0.001). Urinary prostasin was inversely correlated with UNaV during stress (r=−0.43, p=0.0001), even after being normalized by urinary creatinine. Our data suggest that urinary prostasin could be a novel biomarker and/or mechanism for renal pressure natriuresis in normotensive black adolescents.
prostasin; adolescents; pressure natriuresis; blood pressure; urinary sodium excretion
Term newborns with congenital heart disease (CHD) show delayed brain development as early as the third trimester, especially in single ventricle physiology (SVP). Mechanisms causing delayed brain development in CHD are uncertain, but may include impaired fetal brain blood flow. Our objective was to determine if cardiac anatomy associated with obstruction to antegrade flow in the ascending aorta is predictive of delayed brain development measured by diffusion tensor imaging (DTI) and magnetic resonance spectroscopic imaging (MRSI).
Echocardiograms (ECHO) from 36 term newborns with SVP were reviewed for presence of aortic atresia and the diameter of the ascending aorta. Quantitative MR imaging parameters measuring brain microstructural (fractional anisotropy (FA), average diffusivity (Dav)) or metabolic development (N-acetylaspartate (NAA), Lactate/choline (Lac/cho)) were recorded.
Increasing NAA/cho and white matter FA, and decreasing Dav and lactate/cho characterize normal brain development. Consistent with the hypothesis that delayed brain development is related to impaired brain perfusion, smaller ascending aortic diameter and aortic atresia were associated with higher Dav and lower white matter FA. ECHO variables were not associated with brain metabolic measures.
These observations support the hypothesis that obstruction to fetal cerebral blood flow impairs brain microstructural development.
Aminoglycoside exposure is a common cause of acute kidney injury (AKI). Delay in the diagnosis of AKI using conventional biomarkers has been one of the important obstacles in applying early effective interventions. We tested the hypothesis that urinary metabolomics could identify novel early biomarkers for toxic renal injury.
3 days old rats were divided into 3 groups; they received a single daily injection of vehicle (0.9% NaCl solution) or gentamicin at a dose of 10 or 20 mg/kg/day for 7 days. Urine and blood were collected after 3 and 7days of injections. Urinary metabolites were evaluated using High-performance liquid chromatography and gas chromatography/ mass spectrometry.
A distinct urinary metabolic profile characterized by glucosuria, phosphaturia and aminoaciduria was identified preceding changes in serum creatinine. At both gentamicin doses, urinary tryptophan was significantly (p<0.05) increased; fold change (1.91 and 2.31 after 3d, 1.81and 1.93 after 7d). Similarly, kynurenic acid, a tryptophan metabolite, showed a significant (p<0.05) decrease, fold change (0.26 and 0.24 after 3d, 0.21 and 0.52 after 7d), suggesting interruption of the normal tryptophan metabolism pathway.
We conclude that urinary metabolomic profiling provides a robust approach for identifying early and novel markers of gentamicin induced AKI.
Persistent pulmonary hypertension of the newborn (PPHN) is associated with increased oxidative stress in pulmonary arteries (PA). Betamethasone decreases the oxidative stress and improves anti-oxidant balance in PPHN. We investigated whether antenatal betamethasone improves pulmonary vasodilation and postnatal oxygenation in late preterm lambs with PPHN.
PPHN was induced by constriction of fetal ductus arteriosus from 128 to 136d gestation. Ewes were given 2 i.m. doses of betamethasone or saline at 24h and 12h before Cesarian-section delivery at 136d gestation, simulating late preterm birth. Newborn lambs were mechanically ventilated for 8h with monitoring of blood gas and hemodynamic variables. Lungs were harvested post mortem to determine oxidative stress markers and in vitro responses of PA.
Postnatal arterial partial pressure of oxygen and pH were higher and the oxygenation index and arterial partial pressure of carbon dioxide lower in betamethasone treated lambs. PA pressure was lower and systemic pressure higher in betamethasone lambs. Betamethasone decreased the oxidative stress markers and increased endothelial nitric oxide synthase expression in ventilated PPHN lungs.
Antenatal betamethasone decreases oxidative stress and improves postnatal transition in late preterm lambs with PPHN. This study suggests a potential benefit for antenatal betamethasone in late preterm births.
For nearly a decade, our research group has had the privilege of developing and mining a multi-center, microarray-based, genome-wide expression database of critically ill children (≤ 10 years of age) with septic shock. Using bioinformatic and systems biology approaches, the expression data generated through this discovery-oriented, exploratory approach have been leveraged for a variety of objectives, which will be reviewed. Fundamental observations include wide spread repression of gene programs corresponding to the adaptive immune system, and biologically significant differential patterns of gene expression across developmental age groups. The data have also identified gene expression-based subclasses of pediatric septic shock having clinically relevant phenotypic differences. The data have also been leveraged for the discovery of novel therapeutic targets, and for the discovery and development of novel stratification and diagnostic biomarkers. Almost a decade of genome-wide expression profiling in pediatric septic shock is now demonstrating tangible results. The studies have progressed from an initial discovery-oriented and exploratory phase, to a new phase where the data are being translated and applied to address several areas of clinical need.
Wound healing in the pediatric patient is of utmost clinical and social importance, since hypertrophic scarring can have aesthetic and psychological sequelae, from early childhood to late adolescence. Wound healing is a well-orchestrated reparative response affecting the damaged tissue at the cellular, tissue, organ, and system scales. While tremendous progress has been made towards understanding wound healing at the individual temporal and spatial scales, its effects across the scales remain severely understudied and poorly understood. Here we discuss the critical need for systems-based computational modeling of wound healing across the scales, from short-term to long-term and from small to large. We illustrate the state of the art in systems modeling by means of three key signaling mechanisms: oxygen tension regulating angiogenesis and revascularization; TGF-β kinetics controlling collagen deposition; and mechanical stretch stimulating cellular mitosis and extracellular matrix remodeling. The complex network of biochemical and biomechanical signaling mechanisms and the multi-scale character of the healing process make systems modeling an integral tool in exploring personalized strategies for wound repair. A better mechanistic understanding of wound healing in the pediatric patient could open new avenues in treating children with skin disorders such as birth defects, skin cancer, wounds, and burn injuries.
A greater understanding of the regulatory processes contributing to lung development could be helpful to identify strategies to ameliorate morbidity and mortality in premature infants and to identify individuals at risk for congenital and/or chronic lung diseases. Over the past decade, genomics technologies have enabled the production of rich gene expression databases providing information for all genes across developmental time or in diseased tissue. These data sets facilitate systems biology approaches for identifying underlying biological modules and programs contributing to the complex processes of normal development, and those that may be associated with disease states. The next decade will undoubtedly see rapid and significant advances in redefining both lung development and disease at the systems level.
The measurement of adipose tissue depots in-vivo requires expensive imaging methods not accessible to most clinicians and researchers. The study aim was to derive mathematical models to predict total adipose tissue (TAT) and sub-depots from total body fat derived from a dual energy x-ray absorptiometry (DXA) scan.
Models were developed to predict magnetic resonance imaging derived TAT and sub-depots subcutaneous (SAT), visceral (VAT), and intermuscular (IMAT) from DXA total body fat using cross-sectional data (T0) and validated results using 1 (T1) and 2 (T2) year follow-up data. Subjects were 176 multi-ethnic healthy children ages 5 to 17 years at T0. 22 were measured at T1 and T2. TAT was compared to fat.
At T0, TAT was greater than fat (12.5 ± 8.4 vs.12.0 ± 9.4 kg; p< 0.0001), with a quadratic relationship between TAT and fat which varied by sex. Predicted mean TAT’s were not different from measured TAT’s: T1: (9.84±4.45 kg vs. 9.50±4.37 kg; p=0.11) T2: (12.94±6.75 kg vs. 12.89±7.09 kg; p=0.76). The quadratic relationship was not influenced by race or age.
In general, the prediction equations for TAT and sub-depots were consistent with the measured values using T1 and T2 data.
Elevations or deficits in thyroid hormone levels are responsible for a wide range of neonatal and adult phenotypes. Several genome-wide, candidate gene and meta-analysis studies have examined thyroid hormones in adults; however, to our knowledge no genetic association studies have been performed with neonatal thyroid levels.
A population of Iowa neonates; term (n=827) and preterm (n=815), were genotyped for 45 single nucleotide polymorphisms. Thyroid stimulating hormone (TSH) values were obtained from the Iowa Neonatal Metabolic Screening Program. Analysis of variance was performed to identify genetic associations with TSH concentrations.
The strongest association was rs4704397 in the PDE8B gene (p=1.3×10−4), followed by rs965513 (p=6.4×10−4) on chromosome 9 upstream of the FOXE1 gene. Both of these SNPs met statistical significance after correction for multiple testing. Six other SNPs were marginally significant (p<0.05).
We demonstrated for the first time two genetic associations with neonatal TSH levels that replicate findings with adult TSH levels. These SNPs should be considered as early predictors of risk for adult diseases and conditions associated with thyroid hormone levels. Furthermore, this provides a better understanding of the thyroid profile and potential risk for thyroid disorders in newborns.
Improving fat absorption remains a challenge in cystic fibrosis (CF). Antibiotics (AB) treatment has been shown to improve body weight in CF mice. The mechanism may include improvement in fat absorption. We aimed to determine the effect of AB on fat absorption in two CF mouse models.
AB did not improve total fat absorption. Interestingly, AB accelerated the absorption of isotope-labeled fats, in both Δ/Δ and WT mice. The changes observed were not related to the solubilization capacity of bile or to changes in the bacteria in the small intestine. AB reduced the fecal excretion of cholate by ∼50% (P < 0.05) in both CF mouse models, indicating improved intestinal bile salt absorption.
In conclusion, AB treatment does not improve total fat absorption in CF mice but does decrease fecal loss of bile salts and accelerate long-chain fatty acid (LCFA) absorption.
For 3 weeks, we administered oral AB (ciprofloxacin/metronidazole) or control treatment to homozygous ΔF508 (Δ/Δ), cystic fibrosis transmembrane conductance regulator (CFTR) knockout (−/−), and wild-type (WT) mice and quantified fat absorption using a 72-h fat balance test. In Δ/Δ mice, we assessed fat absorption kinetics by administering tri-1-13C-palmitin and 1-13C-stearate intragastrically and determining the appearance of stable isotope-labeled fats in plasma. We quantified biliary and fecal bile salts (gas chromatography) and small intestinal bacteria (quantitative-PCR).
Endothelin-1 (ET-1) and rho-kinase (ROCK) increase vascular tone in experimental PPHN but whether ET-1 activates ROCK to decrease angiogenesis in the developing lung remains unknown.
Proximal PAEC were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and age matched controls. Growth and tube formation were assessed after ET-1 treatment. The effect of ET-1 antagonism on tube formation was studied using ET-1 SiRNA, ET-1 monoclonal antibody (ET-1 mAb), BQ-123 (ETA blocker) and bosentan (ETA/ETB blocker). ET-1 gene and protein and ETA/ETB receptor protein expression were measured in normal and PPHN PAECs. ET-1-ROCK interactions were assessed by measuring ROCK activity after ET-1, ET-1 SiRNA and bosentan treatments and tube formation with ET-1 and Y-27632 (ROCK inhibitor).
ET-1 had no effect on growth, but decreased tube formation in normal and PPHN PAEC. PPHN decreased tube formation in PAEC. ET-1 protein and gene expression were increased and ETB receptor protein decreased in PPHN PAECs. ET-1 SiRNA, ET-1mAb, and bosentan, but not BQ-123, increased tube formation. ROCK activity was increased in PPHN PAECs, and decreased with ET-1 SiRNA and bosentan treatments. Y-27632 prevented the decrease in tube formation with ET-1.
ET-1 impairs angiogenesis of fetal PAECs through ROCK activation. Disruption of ET-1 ROCK interactions may increase vascular growth in PPHN.
Kawasaki Disease (KD) can result in fatal coronary artery aneurysms especially in untreated patients. Our recent studies of KD vascular pathology revealed subacute/chronic vasculitis that began early in the illness with proliferation of smooth muscle cell derived-myofibroblasts in a complex extracellular matrix (ECM). We hypothesized that there is dysregulation of specific ECM and adhesion molecules in KD coronary arteries.
Gene expression profiling for ECM and adhesion molecules was performed on 6 acute KD and 8 control coronary arteries using a targeted real-time PCR array approach.
Integrins alpha4 and alphaM (ITGA4, ITGAM), collagen 1A1 (COL1A1), and matrix metalloproteinase 7 (MMP-7) were significantly upregulated in KD coronary arteries compared with controls. Immunohistochemistry with anti- ITGAM antibodies revealed expression on inflammatory cells within the coronary artery wall in KD patients but not controls.
Integrins ITGA4 and ITGAM are upregulated in KD vasculopathy, likely promoting inflammatory recruitment that stimulates smooth muscle cell transition to myofibroblasts and their proliferation. MMP-7 likely enhances myofibroblast proliferation and luminal lesion expansion, and overexpression of COL1A1 may lead to coronary artery stenosis. Identification of the molecular pathogenesis of KD vasculopathy may lead to the development of circulating biomarkers and to directed therapeutic interventions.
Sertraline, a selective serotonin reuptake inhibitor (SSRI), is the most commonly prescribed therapy for maternal depression. Epidemiologic studies have linked SSRI exposure with decreased fetal growth, altered autonomic regulation, and cardiac malformations. We hypothesized SSRI exposure decreases left ventricular volumes and increases adult sympathetic nervous system activation, resulting in increased adult heart rates.
C57BL/6 mice received saline or sertraline (5 or 15 mg/kg/day i.p.) on postnatal days 1–14. Adult phenotypes were assessed at 5 months.
Sertraline-exposed mice had smaller left ventricular internal diameters in diastole (control 4.0 ± 0.1 mm, SSRI 3.7 ± 0.1 mm, p < 0.05), decreased stroke volumes (control 46 ± 2.6 μL, SSRI 37 ± 2.3 μL, p < 0.05), higher heart rates (control 530 ± 13 beats per minute (bpm), SSRI 567 ± 6 bpm, p <0.05) and increased urinary excretion of noradrenaline (control 174 ± 29.4 ng/mL, SSRI 276 ± 35.1 ng/mL, p<0.05). These changes were associated with increased cerebral serotonin transporter (5-HTT) expression.
Neonatal sertraline exposure causes long term changes in cardiac morphology and physiology. We speculate that early life SSRI exposure impairs cardiomyocyte growth and central serotonin signaling, leading to a small left heart syndrome in adult mice.
Acute otitis media (AOM) is a frequent complication of viral upper respiratory tract infection (URI). We hypothesized that severity of nasopharyngeal cellular injury during URI, as measured by lactate dehydrogenase (LDH) concentrations in nasopharyngeal secretions (NPS), is related to AOM complication.
LDH concentrations were determined in NPS samples (n=594) which were collected at the initial visit for URI from 183 children who were followed for development of AOM. A subset of NPS samples (n= 134) were analyzed for interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) α concentrations.
AOM complication was independently predicted by LDH concentrations (median mU/ml with AOM = 2438 vs. without AOM = 1573, estimate=0.276; P=0.02). LDH effect on AOM development was highest during the first 4 days of URI. LDH concentrations were higher in URIs due to adenoviruses, bocaviruses, and rhinoviruses when compared to virus-negative samples (P <0.05). There was a positive correlation between concentrations of LDH and all cytokines (P< 0.001).
LDH concentrations in NPS are positively associated with AOM risk, suggesting that the severity of nasopharyngeal inflammatory injury during URI contributes to the development of AOM, and that reduction of inflammatory injury may reduce the risk for AOM.
We sought to disentangle the contributions of perinatal systemic inflammation and small for gestational age (SGA) to the occurrence of low Bayley Mental Development Indices (MDIs) at age 2 years.
We measured the concentration of 25 inflammation-related proteins in blood obtained during the first 2 postnatal weeks from 805 infants who were born before the 28th week of gestation and who had MDI measurements at age 2 years and were able to walk independently.
SGA newborns who did not have systemic inflammation (a concentration of an inflammation-related protein in the top quartile for gestational age on 2 days a week apart) were at greater risk of an MDI < 55, but not 55–69, than their peers who had neither SGA nor systemic inflammation. SGA infants who had elevated blood concentrations of IL-1beta, TNF-alpha, or IL-8 during the first two postnatal weeks were at even higher risk of an MDI < 55 than their SGA peers without systemic inflammation and of their non-SGA peers with systemic inflammation.
SGA appears to place very preterm newborns at increased risk of a very low MDI. Systemic inflammation adds considerably to the increased risk.
Fetal growth restriction is reported to be associated with impaired placental iron transport. Transferrin receptor (TfR) is a major placental iron transporter in humans, but is unstudied in sheep. TfR is regulated by both iron and nitric oxide (NO), the molecule produced by endothelial NOS (eNOS). We hypothesized that limited placental development downregulates both placental TfR and eNOS expression, thereby lowering fetal tissue iron.
An ovine surgical uterine space restriction (USR) model, combined with multifetal gestation, tested the extremes of uterine and placental adaptation. Blood, tissues, and placentomes from non-space restricted (NSR) singletons were compared to USR fetuses at 120 or 130 days of gestation (GD).
When expressed proportionate to fetal weight, liver iron content did not differ while renal iron was higher in USR vs. NSR fetuses. Renal TfR protein expression did not differ, but placental TfR expression was lower in USR fetuses at GD130. Placental levels of TfR correlated to eNOS. TfR was localized throughout the placentome, including the hemophagous zone, implicating a role for TfR in ovine placental iron transport.
In conclusion, fetal iron was regulated in an organ-specific fashion. In USR fetuses, NO-mediated placental adaptations may prevent the normal upregulation of placental TfR at GD130.
We have previously demonstrated that enterally administered heparin-binding EGF-like growth factor (HB-EGF) produced in Escherichia coli decreases the incidence and severity of intestinal injury in a neonatal rat model of necrotizing enterocolitis (NEC). In preparation for upcoming human clinical trials, large-scale production of HB-EGF according to Good Manufacturing Practice (GMP) has been successfully accomplished using a Pichia pastoris yeast system. The current studies used a neonatal rat model of NEC to elucidate several important preclinical characteristics of HB-EGF therapy. We found that enteral administration of HB-EGF (800 μg/kg/dose) four times a day effectively reduced the incidence and severity of NEC, that Pichia-derived HB-EGF was not significantly different from E. coli-derived HB-EGF in preventing NEC, that EGF was not superior to HB-EGF in preventing NEC, and that prophylactic administration of HB-EGF added to formula starting with the first feed or 12 h later significantly reduced the incidence of NEC, with no change in the incidence of NEC noted if HB-EGF was added to the formula starting 24, 48, or 72 h after birth. Thus, large-scale production of GMP-grade HB-EGF in Pichia pastoris yeast produces a biologically active molecule suitable for human clinical trials.
Prematurely born infants are often treated with supraphysiologic amounts of oxygen, which is associated with lung injury and the development of diseases such as bronchopulmonary dysplasia. Complimentary responses between the lung and liver during the course of hyperoxic lung injury have been studied in adult animals, but little is known about this relationship in neonates. These studies tested the hypothesis that oxidant stress occurs in the livers of newborn mice in response to continuous hyperoxia exposure. Greater levels of glutathione disulfide and nitrotyrosine were detected in lung tissues but not liver tissues from newborn mice exposed to hyperoxia than in room air-exposed controls. However, early increases in 5-lipoxygenase and cyclooxygenases-2 protein levels and increases in total hydroxyeicosatetraenoic acid and prostaglandin levels were observed in the liver tissues of hyperoxia-exposed pups. These studies indicate that free radical oxidation occurs in the lungs of newborn pups exposed to hyperoxia, and alterations in lipid metabolism could be a primary response in the liver tissues. The findings of this study identify possible new mechanisms associated with hyperoxic lung injury in a newborn model of bronchopulmonary dysplasia and thus open opportunities for research.
We investigated the hypothesis that oxidative stress in persistent pulmonary hypertension of the newborn (PPHN) impairs voltage gated potassium (Kv) channel function. We induced PPHN in fetal lambs by prenatal ligation of ductus arteriosus; controls had sham ligation. We studied changes in the tone of pulmonary artery rings and Kv channel current of freshly isolated pulmonary artery smooth muscle cells (PASMC) using standard techniques. 4-Aminopyridine (4-AP), a Kv channel antagonist, induced dose dependent constriction of control PA rings; this response was attenuated in PPHN pulmonary arteries. Exogenous superoxide and peroxynitrite inhibited the response to 4-AP in control rings. Tiron, a superoxide scavenger, improved the response to 4-AP in PPHN rings. 4-AP inhibited the NOS- independent relaxation response to ATP in control PA rings. Relaxation response to ATP was blunted in PPHN rings and was improved by NOS antagonist, n-nitro-l- arginine methyl ester (L-NAME). 4-AP attenuated this response in L-NAME treated PPHN rings. Exogenous superoxide suppressed 4-AP sensitive Kv current in control PASMC. Kv channel current was attenuated in cells from PPHN lambs and was restored by tiron. Oxidative stress impairs Kv channel function in PPHN. Superoxide scavengers may improve pulmonary vasodilation in PPHN in part by restoring Kv channel function.