Rob M. Moonen

Endotoxin induced chorioamnionitis prevents intestinal development during gestation in fetal sheep.

Chorioamnionitis is the most significant source of prenatal inflammation and preterm delivery. Prematurity and prenatal inflammation are associated with compromised postnatal developmental outcomes, of the intestinal immune defence, gut barrier function and the vascular system. We developed a sheep model to study how the antenatal development of the gut was affected by gestation and/or by endotoxin induced chorioamnionitis. Chorioamnionitis was induced at different gestational ages (GA). Animals were sacrificed at low GA after 2d or 14d exposure to chorioamnionitis. Long term effects of 30d exposure to chorioamnionitis were studied in near term animals after induction of chorioamnionitis. The cellular distribution of tight junction protein ZO-1 was shown to be underdeveloped at low GA whereas endotoxin induced chorioamnionitis prevented the maturation of tight junctions during later gestation. Endotoxin induced chorioamnionitis did not induce an early (2d) inflammatory response in the gut in preterm animals. However, 14d after endotoxin administration preterm animals had increased numbers of T-lymphocytes, myeloperoxidase-positive cells and gammadelta T-cells which lasted till 30d after induction of chorioamnionitis in then near term animals. At early GA, low intestinal TLR-4 and MD-2 mRNA levels were detected which were further down regulated during endotoxin-induced chorioamnionitis. Predisposition to organ injury by ischemia was assessed by the vascular function of third-generation mesenteric arteries. Endotoxin-exposed animals of low GA had increased contractile response to the thromboxane A2 mimetic U46619 and reduced endothelium-dependent relaxation in responses to acetylcholine. The administration of a nitric oxide (NO) donor completely restored endothelial dysfunction suggesting reduced NO bioavailability which was not due to low expression of endothelial nitric oxide synthase. Our results indicate that the distribution of the tight junctional protein ZO-1, the immune defence and vascular function are immature at low GA and are further compromised by endotoxin-induced chorioamnionitis. This study suggests that both prematurity and inflammation in utero disturb fetal gut development, potentially predisposing to postnatal intestinal pathology.

Carbamoyl Phosphate Synthetase Polymorphisms as a Risk Factor for Necrotizing Enterocolitis

A C-to-A nucleotide transversion (T1405N) in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1) has been correlated with low plasma concentrations of l-arginine in neonates. As plasma l-arginine concentrations are decreased in premature infants with necrotizing enterocolitis (NEC), we hypothesized that the CPS1 T1405N polymorphism would correlate with the presence of NEC. We analyzed the CPS1 genotypes for the T1405N polymorphism in 17 preterm infants (≤30 wk and <1500 g) with established NEC, 34 preterm infants without NEC, and 25 healthy term infants. Distribution of genotypes did not differ between the NEC population (CC:AC:AA = 70.6%:23.5%:5.9%) and the preterm control group (CC:AC:AA = 41.2%:35.3%:23.5%; p = 0.110) or the term group (CC:AC:AA = 44%:48%:8%; p = 0.228). The C allele frequency was 82.4% in NEC and 58.8% in preterm control infants (p = 0.018) and analysis for linear trend demonstrated that incidence of NEC increased with the number of C alleles (p = 0.037). The CC genotype was associated with an increased risk of NEC in the preterm infants [odds ratio (OR) = 3.43, 95% confidence interval (CI): 1.01–11.49, p = 0.048), when compared with the grouped together AA/AC genotypes. These data suggest that the CPS1 T1405N polymorphism may be associated with the risk of NEC in preterm infants.

The T1405N Carbamoyl Phosphate Synthetase Polymorphism Does Not Affect Plasma Arginine Concentrations in Preterm Infants

Background A C-to-A nucleotide transversion (T1405N) in the gene that encodes carbamoyl-phosphate synthetase 1 (CPS1) has been associated with changes in plasma concentrations of L-arginine in term and near term infants but not in adults. In preterm infants homozygosity for the CPS1 Thr1405 variant (CC genotype) was associated with an increased risk of having necrotizing enterocolitis (NEC). Plasma L-arginine concentrations are decreased in preterm infants with NEC. Aim To examine the putative association between the CPS1 T1405N polymorphism and plasma arginine concentrations in preterm infants. Methods Prospective multicenter cohort study. Plasma and DNA samples were collected from 128 preterm infants (<30 weeks) between 6 and 12 hours after birth. Plasma amino acid and CPS1 T1405N polymorphism analysis were performed. Results Distribution of genotypes did not differ between the preterm (CC∶CA∶AA = 55.5%∶33.6%∶10.9%, n = 128) and term infants (CC∶CA∶AA = 54.2%∶35.4%∶10.4%, n = 96). There was no association between the CPS1 genotype and plasma L-arginine or L-citrulline concentration, or the ornithine to citrulline ratio, which varies inversely with CPS1 activity. Also the levels of asymmetric dimethylarginine, and symmetric dimethylarginine were not significantly different among the three genotypes. Conclusions The present study in preterm infants did not confirm the earlier reported association between CPS1 genotype and L-arginine levels in term infants.

Plasma Levels of Dimethylarginines in Preterm Very Low Birth Weight Neonates: Its Relation with Perinatal Factors and Short-Term Outcome

Endogenously produced inhibitors of nitric oxide (NO) synthase, in particular asymmetric dimethylarginine (ADMA), are currently considered of importance in various disease states characterized by reduced NO availability. We investigated the association between plasma levels of ADMA, symmetric dimethylarginine (SDMA), l-arginine, and citrulline and perinatal factors and outcome in 130 preterm (gestational age ≤30 weeks) very low birth weight (VLBW, <1500 g) infants. Plasma samples were collected 6–12 h after birth. We did not find significant correlations between ADMA, SDMA, l-arginine, and citrulline levels and gestational age or birth weight. However, the arginine:ADMA ratio (AAR, a better indicator of NO availability than either arginine or ADMA separately) was positively correlated with gestational age. ADMA and arginine levels were not significantly different between males and females but males showed a negative correlation between ADMA levels and gestational age. Perinatal factors such as preeclampsia, chrorioamnionitis, prolonged rupture of membranes, or form of delivery did not significantly alter dimethylarginine levels or AAR. In contrast, the AAR was significantly reduced in the infants with respiratory distress, mechanical ventilation, and systemic hypotension Therefore, our data suggest that altered NO availability may play a role in the respiratory and cardiovascular adaptation in preterm VLBW infants.

Response of Chicken Ductus Arteriosus to Hypercarbic and Normocarbic Acidosis

Background: Changes in pH can have profound effects on vascular tone and reactivity, but their influence on the ductus arteriosus (DA) remains unknown. Objective: To analyzethe effects of hypercarbic and normocarbic acidosis in the reactivity of the chicken DA. Methods: DA rings from 19-day chicken fetuses (total incubation time, 21 days) were mounted in a wire myograph for isometric tension recording. Results: In DA rings (pulmonary side) stimulated with O2, norepinephrine (NE), KCl, or U46619, changes from control conditions (5% CO2, 24 mM NaHCO3, pH 7.4) to 7.5% CO2 (pH 7.25) or 10% CO2 (pH 7.14) induced a concentration-dependent relaxation that reached 43.0% (SD 21.3) of the O2-, 28.6% (SD 23.1) of the NE-, 10.4% (SD 18.7) of the KCl-, and 6.8% (SD 12.6) of the U46619-induced contraction. Hypercarbic-acidosis-induced relaxation was impaired by the non-selective K+ channel blocker tetraethylammonium or the BKCa channel inhibitor iberiotoxin. Normocarbic acidosis (5% CO2, 12 mM NaHCO3, pH 7.13) induced transient relaxation of the DA, which was not affected by the presence of tetraethylammonium or iberiotoxin. Euhydric hypercarbia (10% CO2, 48 mM NaHCO3, pH 7.46) induced a transient contraction of the DA. Conclusions: Our results indicate that the chicken DA is very sensitive to changes in extracellular pH, and that stimulation of BKCa channels may account for the ductal-relaxing effects of hypercarbic acidosis.

Is the C242T Polymorphism of the CYBA Gene Linked with Oxidative Stress-Associated Complications of Prematurity?

The C242T polymorphism of CYBA (cytochrome B-245 alpha chain), the gene encoding the p22phox subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, has been linked to several conditions in which oxidative stress plays a pathogenic role. We investigated in a cohort of 451 preterm infants [gestational age (GA) ≤30 weeks] the association of the polymorphism with the risk of developing neonatal respiratory distress syndrome (RDS), retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), necrotizing enterocolitis, patent ductus arteriosus, or intraventricular hemorrhage. We observed a significant association of the TT/CT genotype with RDS [odds ratio (OR) 2.34, 95% confidence interval (95% CI) 1.28-3.90], ROP (OR 1.72, 95% CI 1.05-2.80), and BPD (OR 1.60, 95% CI 1.05-2.43). When this dominant model was adjusted to account for GA, birth weight, and sex, it remained significant for the three outcomes. This study is the first to address the association of a polymorphism related to the NADPH family with oxidative stress-related complications of prematurity. Since p22phox is essential for reactive oxygen species production by NADPH oxidase, we hypothesize that genetic variations in the protein may lead to differences in susceptibility to oxidative stress-induced damage in preterm infants. Antioxid. Redox Signal. 27, 1432-1438.

Association between the p.Thr1406Asn polymorphism of the carbamoyl-phosphate synthetase 1 gene and necrotizing enterocolitis: A prospective multicenter study.

The p.Thr1406Asn (rs1047891) polymorphism of the carbamoyl-phosphate synthetase 1 (CPS1) gene has been linked to functional consequences affecting the downstream availability of the nitric oxide precursor L-arginine. L-arginine concentrations are decreased in preterm infants with necrotizing enterocolitis (NEC). In this multicenter prospective study, we investigated the association of the p.Thr1406Asn polymorphism with NEC in 477 preterm infants (36 cases of NEC) from 4 European neonatal intensive care units (Maastricht, Las Palmas de Gran Canaria, Mantova, and Milan). Allele and genotype frequencies of the p.Thr1406Asn polymorphism did not significantly differ between the infants with and without NEC. In contrast, the minor A-allele was significantly less frequent in the group of 64 infants with the combined outcome NEC or death before 34 weeks of corrected gestational age than in the infants without the outcome (0.20 vs. 0.31, P = 0.03). In addition, a significant negative association of the A-allele with the combined outcome NEC or death was found using the dominant (adjusted odds ratio, aOR: 0.54, 95% CI 0.29–0.99) and the additive (aOR 0.58, 95% CI 0.36–0.93) genetic models. In conclusion, our study provides further evidence that a functional variant of the CPS1 gene may contribute to NEC susceptibility.

Hypoxia-induced contraction of chicken embryo mesenteric arteries: mechanisms and developmental changes

The fetal cardiovascular responses to acute hypoxia include a redistribution of the cardiac output toward the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-day chicken embryos (E15, E19, E21), and 1- to 45-day-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased toward the first days posthatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.

IL4 Receptor (|[plus]|1902G), IL18 (|[minus]|607A) and VEGF (C-2578A) Single-Nucleotide Polymorphisms were Not Associated with Necrotizing Enterocolitis Risk

Background and Aims: The pathophysiology of necrotizing enterocolitis (NEC) remains elusive and is likely multifactorial. Inadequate response of the inflammatory cytokine cascade and the intestinal circulatory regulation may play a role in the development of NEC. We have examined associations between variants in candidate interleukin (IL) and vascular endothelial growth factor (VEGF) genes with susceptibility to NEC in preterm infants.Methods: DNA samples were collected from 28 preterm infants (gestational age ≤ 30 wk and birth weight < 1500 g) with established NEC (Bells stage ≥ II), 82 controls matched for gestational age and ethnic group and 96 term infants. IL 4 receptor (+1902G), IL18 (-607A) and VEGF (C-2578A) single-nucleotide polymorphisms (SNP) were genotyped and analyzed for their association with NEC.Results: All allele distributions were in Hardy-Weinberg equilibrium. Variant genotypes of IL4 receptor, IL18 and VEGF in cases, controls, and term infants are listed in the table None of the studied SNPs was significantly associated with NEC.TableNone of the studied SNPs was significantly associated with NEC.Conclusions: In contrast to earlier reports, None of the candidate SNPs so far studied was significantly associated with NEC risk in preterm infants. Data from earlier, the present, and future association studies of these SNPs may be added to a meta-analyses to obtain more precise estimates of effects sizes.

383 The T1405N Carbamoyl Phosphate Synthetase Polymorphism Does Not Affect Plasma Arginine Concentrations in Preterm Infants

383 The T1405N Carbamoyl Phosphate Synthetase Polymorphism Does Not Affect Plasma Arginine Concentrations in Preterm Infants