S-Nitrosylated fetal hemoglobin in neonatal human blood.

BACKGROUND: Nitric oxide (NO) and its derivatives play important roles in the cardiopulmonary transition upon birth and in other oxygen-sensitive developmental milestones. One mechanism for the coupling of oxygen sensing and signaling by NO species is via the formation of an S-nitrosothiol (SNO) moiety on hemoglobin (Hb, forming SNO-Hb) and its release from the red blood cell in hypoxia. Although SNO-Hb formed on adult-type Hb (HbA, forming SNO-HbA) has been documented in physiological and pathophysiological human states, the fetal variant, SNO-HbF, has thus far not been isolated or characterized in human blood. METHODS AND RESULTS: We developed a technique capable of separating Hbs A and F under conditions that preserve SNO. We then measured SNO-HbF in the blood of healthy and premature or otherwise ill neonates using the gold standard for SNO measurement, mercury-coupled photolysis-chemiluminescence. SNO-HbF levels were in the range of those previously reported for HbA in adults. We found that SNO-HbF was more abundant at earlier gestational age (<30 weeks), even when accounting for the absolute HbF level. CONCLUSIONS: The ability to monitor SNO-HbF could provide new insights into fetal development and the perinatal transition, and has potential as a biomarker relevant to the management of neonatal diseases.

Right ventricular echocardiographic indices predict poor outcomes in infants with persistent pulmonary hypertension of the newborn.

AIMS: Infants with persistent pulmonary hypertension of the newborn (PPHN) have elevated pulmonary vascular resistance that can lead to right ventricular (RV) failure and death. Clinicians must decide which infants will fail conventional therapy and require transfer to extra corporeal membrane oxygenation (ECMO) centres, but accurate echocardiographic predictors have not been identified. We assessed echocardiographic measurements of RV pressure and function in predicting progression to death or ECMO in infants with PPHN. METHODS AND RESULTS: Echocardiograms for infants ≥35-week gestation with a clinical diagnosis of PPHN were retrospectively reviewed. Traditional and strain echocardiographic measures were compared for those with or without the primary outcome of ECMO/cardiovascular death. Receiver operator curves identified cut points for measures that were significantly different. Of the 86 subjects analysed, 25 (29%) of the patients had the primary outcome of ECMO/death. The ECMO/death group had diminished tricuspid annular plane systolic excursion (TAPSE; P = 0.002) and RV global longitudinal peak strain (GLPS; P = 0.03), a predominant right-to-left shunt across the patent ductus arteriosus (PDA; P = 0.05), and an elevated oxygenation index (OI; P < 0.001). Sensitivity/specificity for TAPSE <4 mm was 56 and 85%, and for GLPS greater than or equal to -9% was 52 and 77%. CONCLUSION: TAPSE, GLPS, and right-to-left PDA shunting were associated with progression to death/ECMO. RV free wall strain was not associated with the outcome, suggesting that diminished global strain better reflects clinical outcomes in this group. These thresholds may assist in the decision-making to transfer high-risk infants to ECMO centres.

Management Practice and Mortality for Infants with Congenital Diaphragmatic Hernia.

OBJECTIVE: Congenital diaphragmatic hernia (CDH) is fatal in 20 to 40% of cases, largely due to pulmonary dysmaturity, lung hypoplasia, and persistent pulmonary hypertension. Evidence for survival benefit of inhaled nitric oxide (iNO), extracorporeal membrane oxygenation (ECMO), and other medical interventions targeting pulmonary hypertension is lacking. We assessed medical interventions and mortality over time in a large multicenter cohort of infants with CDH. STUDY DESIGN: We identified all infants ≥ 34 weeks' gestation with CDH discharged from 29 neonatal intensive care units between 1999 and 2012 with an average of ≥ 2 CDH admissions per year. We examined mortality and the proportion of infants exposed to medical interventions, comparing four periods of time: 1999-2001, 2002-2004, 2005-2007, and 2008-2012. RESULTS: We identified 760 infants with CDH. From 1999-2001 to 2008-2012, use of iNO increased from 20% of infants to 50%, sildenafil use increased from 0 to 14%, and milrinone use increased from 0 to 22% (p < 0.001). Overall mortality (28%) did not significantly change over time compared with the earliest time period. CONCLUSION: Despite changing use of iNO, sildenafil, and milrinone, CDH mortality has not significantly decreased in this population of infants.

Transbilayer phospholipid movements in ABCA1-deficient cells.

Tangier disease is an inherited disorder that results in a deficiency in circulating levels of HDL. Although the disease is known to be caused by mutations in the ABCA1 gene, the mechanism by which lesions in the ABCA1 ATPase effect this outcome is not known. The inability of ABCA1 knockout mice (ABCA1-/-) to load cholesterol and phospholipids onto apoA1 led to a proposal that ABCA1 mediates the transbilayer externalization of phospholipids, an activity integral not only to the formation of HDL particles but also to another, distinct process: the recognition and clearance of apoptotic cells by macrophages. Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient engulfment of the apoptotic cells, and it has been proposed that ABCA1 is required for transbilayer externalization of PS to the surface of both cell types. To determine whether ABCA1 is responsible for any of the catalytic activities known to control transbilayer phospholipid movements, these activities were measured in cells from ABCA1-/- mice and from Tangier individuals as well as ABCA1-expressing HeLa cells. Phospholipid movements in either normal or apoptotic lymphocytes or in macrophages were not inhibited when cells from knockout and wildtype mice or immortalized cells from Tangier individuals vs normal individuals were compared. Exposure of PS on the surface of normal thymocytes, apoptotic thymocytes and elicited peritoneal macrophages from wildtype and knockout mice or B lymphocytes from normal and Tangier individuals, as measured by annexin V binding, was also unchanged. No evidence was found of ABCA1-stimulated active PS export, and spontaneous PS movement to the outer leaflet in the presence or absence of apoA1 was unaffected by the presence or absence of ABCA1. Normal or Tangier B lymphocytes and macrophages were also identical in their ability to serve as targets or phagocytes, respectively, in apoptotic cell clearance assays. No evidence was found to support the suggestion that ABCA1 is involved in transport to the macrophage cell surface of annexins I and II, known to enhance phagocytosis of apoptotic cells. These results show that mutations in ABCA1 do not measurably reduce the rate of transbilayer movements of phospholipids in either the engulfing macrophage or the apoptotic target, thus discounting catalysis of transbilayer movements of phospholipids as the mechanism by which ABCA1 facilitates loading of phospholipids and cholesterol onto apoA1.

Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax.

BACKGROUND: Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined. RESULTS: We found that immortalization occurred earlier and more frequently when the myogenic cells lacked the pro-apoptotic protein Bax. Furthermore, myogenesis was altered by Bax inactivation as Bax-null cells produced muscle colonies with more nuclei than wild-type cells, though a lower percentage of the Bax-null nuclei were incorporated into multinucleate myotubes. In vivo, both the fast and slow myofibers in Bax-null muscles had smaller cross-sectional areas than those in wild-type muscles. After immortalization, both Bax-null and Bax-positive myogenic cells expressed desmin, retained the capacity to form multinucleate myotubes, expressed p19ARF protein, and retained p53 functions. Expression of p16INK4a, however, was found in only about half of the immortalized myogenic cell lines. CONCLUSIONS: Mouse myogenic cells can undergo spontaneous immortalization via a mechanism that can include, but does not require, loss of p16INK4a, and also does not require inactivation of p19ARF or p53. Furthermore, loss of Bax, which appears to be a downstream effector of p53, accelerates immortalization of myogenic cells and alters myogenesis.