A first-in-human phase I/IIa gene transfer clinical trial for Duchenne muscular dystrophy using rAAVrh74.MCK.GALGT2.

In a phase 1/2, open-label dose escalation trial, we delivered rAAVrh74.MCK.GALGT2 (also B4GALNT2) bilaterally to the legs of two boys with Duchenne muscular dystrophy using intravascular limb infusion. Subject 1 (age 8.9 years at dosing) received 2.5 × 1013 vector genome (vg)/kg per leg (5 × 1013 vg/kg total) and subject 2 (age 6.9 years at dosing) received 5 × 1013 vg/kg per leg (1 × 1014 vg/kg total). No serious adverse events were observed. Muscle biopsy evaluated 3 or 4 months post treatment versus baseline showed evidence of GALGT2 gene expression and GALGT2-induced muscle cell glycosylation. Functionally, subject 1 showed a decline in 6-min walk test (6MWT) distance; an increase in time to run 100 m, and a decline in North Star Ambulatory Assessment (NSAA) score until ambulation was lost at 24 months. Subject 2, treated at a younger age and at a higher dose, demonstrated an improvement over 24 months in NSAA score (from 20 to 23 points), an increase in 6MWT distance (from 405 to 478 m), and only a minimal increase in 100 m time (45.6-48.4 s). These data suggest preliminary safety at a dose of 1 × 1014 vg/kg and functional stabilization in one patient.

"Current concepts in assisted mechanical ventilation in the neonate" - Part 2: Understanding various modes of mechanical ventilation and recommendations for individualized disease-based approach in neonates.

Mechanical ventilation is a lifesaving intervention in critically ill preterm and term neonates. However, it has the potential to cause significant damage to the lungs resulting in long-term complications. Understanding the pathophysiological process and having a good grasp of the basic concepts of conventional and high-frequency ventilation is essential for any medical or allied healthcare practitioner involved in the neonates' respiratory management. This review aims to describe the various types and modes of ventilation usually available in neonatal units. It also describes recommendations of an individualized disease-based approach to mechanical ventilation strategies implemented in the authors' institutions.

"Current concepts of mechanical ventilation in neonates" - Part 1: Basics.

Mechanical ventilation is potentially live saving in neonatal patients with respiratory failure. The main purpose of mechanical ventilation is to ensure adequate gas exchange, including delivery of adequate oxygenation and enough ventilation for excretion of CO2. The possibility to measure and deliver small flows and tidal volumes have allowed to develop very sophisticated modes of assisted mechanical ventilation for the most immature neonates, such as volume targeted ventilation, which is used more and more by many clinicians. Use of mechanical ventilation requires a basic understanding of respiratory physiology and pathophysiology of the disease leading to respiratory failure. Understanding pulmonary mechanics, elastic and resistive forces (compliance and resistance), and its influence on the inspiratory and expiratory time constant, and the mechanisms of gas exchange are necessary to choose the best mode of ventilation and adequate ventilator settings to minimize lung injury. Considering the pathophysiology of the disease allows a physiology-based approach and application of these concepts in daily practice for decision making regarding the use of modes and settings of mechanical ventilation, with the ultimate aim of providing adequate gas exchange and minimising lung injury.

Gene Delivery for Limb-Girdle Muscular Dystrophy Type 2D by Isolated Limb Infusion.

In a previous limb-girdle muscular dystrophy type 2D (LGMD2D) clinical trial, robust alpha-sarcoglycan gene expression was confirmed following intramuscular gene (SGCA) transfer. This paved the way for first-in-human isolated limb infusion (ILI) gene transfer trial to the lower limbs. Delivery of scAAVrh74.tMCK.hSGCA via an intravascular route through the femoral artery predicted improved ambulation. This method was initially chosen to avoid safety concerns required for large systemic vascular delivery viral loads. ILI methods were adopted from the extensive chemotherapy experience for treatment of malignancies confined to the extremities. Six LGMD2D subjects were enrolled in a dose-ascending open-label clinical trial. Safety of the procedure was initially assessed in the single limb of a non-ambulant affected adult at a dose of 1 × 1012 vg/kg. Subsequently, ambulatory children (aged 8-13 years) were enrolled and dosed bilaterally with either 1 × 1012 vg/kg/limb or 3 × 1012 vg/kg/limb. The six-minute walk test (6MWT) served as the primary clinical outcome; secondary outcomes included muscle strength (maximum voluntary isometric force testing) and SGCA expression at 6 months. All ambulatory participants except one had pre- and post-treatment muscle biopsies. All four subjects biopsied had confirmed SGCA gene delivery by immunofluorescence, Western blot analysis (14-25% of normal), and vector genome copies (5.4 × 103-7.7 × 104 vg/µg). Muscle strength in the knee extensors (assessed by force generation in kilograms) showed improvement in two subjects that correlated with an increase in fiber diameter post gene delivery. Six-minute walk times decreased or remained the same. Vascular delivery of AAVrh74.tMCK.hSGCA was effective at producing SGCA protein at low doses that correlated with vector copies and local functional improvement restricted to targeted muscles. Future trials will focus on systemic administration to enable targeting of proximal muscles to maximize clinical benefit.

Bridging from Intramuscular to Limb Perfusion Delivery of rAAV: Optimization in a Non-human Primate Study.

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development program to regional limb perfusion, comparing two methods previously established for gene therapy, peripheral venous limb perfusion (VLP) and an intra-arterial push and dwell (IAPD) using rAAV1 and rAAV8 in a non-human primate (rhesus macaque) study. The rhesus AAT transgene was used with a c-myc tag to enable quantification of transgene expression. 5 cohorts of animals were treated with rAAV1-IM, rAAV1-VLP, rAAV1-IAPD, rAAV8-VLP, and rAAV8-IAPD (n = 2-3), with a dose of 6 × 1012 vg/kg. All methods were well tolerated clinically. Potency, as determined by serum levels of AAT, of rAAV1 by the VLP method was twice that observed with direct IM injection; 90 µg/mL with VLP versus 38 µg/mL with direct IM injection. There was an approximately 25-fold advantage in estimated vector genomes retained within the muscle tissue with VLP and a 5-fold improvement in the ratio of total vector genomes retained within muscle as compared with liver. The other methods were intermediate in the potency and retention of vector genomes. Examination of muscle enzyme (CK) levels indicated rAAV1-VLP to be equally safe as compared with IM injection, while the IAPD method showed significant CK elevation. Overall, rAAV1-VLP demonstrates higher potency per vector genome injected and a greater total vector retention within the muscle, as compared to IM injection, while enabling a much greater total dose to be delivered, with equivalent safety. These data provide the basis for continuation of the dose escalation of the rAAV1-AAT program in patients and bode well for rAAV-VLP as a platform for replacement of secreted proteins.

Predicting Risk of Infection in Infants with Congenital Diaphragmatic Hernia.

OBJECTIVE: To predict incident bloodstream infection and urinary tract infection (UTI) in infants with congenital diaphragmatic hernia (CDH). STUDY DESIGN: We conducted a retrospective analysis using the Children's Hospital Neonatal Database during 2010-2016. Infants with CDH admitted at 22 participating regional neonatal intensive care units were included; patients repaired or discharged to home prior to admission/referral were excluded. The primary outcome was death or the occurrence of bloodstream infection or UTI prior to discharge. Factors associated with this outcome were used to develop a multivariable equation using 80% of the cohort. Validation was performed in the remaining 20% of infants. RESULTS: Median gestation and postnatal age at referral in this cohort (n = 1085) were 38 weeks and 3.1 hours, respectively. The primary outcome occurred in 395 patients (36%); and was associated with low birth weight, low Apgar, low admission pH, renal and associated anomalies, patch repair, and extracorporeal membrane oxygenation (P < .001 for all; area under receiver operating curve = 0.824; goodness of fit χ2 = 0.52). After omitting death from the outcome measure, admission pH, patch repair of CDH, and duration of central line placement were significantly associated with incident bloodstream infection or UTI. CONCLUSIONS: Infants with CDH are at high risk of infection which was predicted by clinical factors. Early identification and low threshold for sepsis evaluations in high-risk infants may attenuate acquisition and the consequences of these infections.

An Isolated Limb Infusion Method Allows for Broad Distribution of rAAVrh74.MCK.GALGT2 to Leg Skeletal Muscles in the Rhesus Macaque.

Recombinant adeno-associated virus (rAAV)rh74.MCK.GALGT2 is a muscle-specific gene therapy that is being developed to treat forms of muscular dystrophy. Here we report on an isolated limb infusion technique in a non-human primate model, where hindlimb blood flow is transiently isolated using balloon catheters to concentrate vector in targeted leg muscles. A bilateral dose of 2.5 × 1013 vector genomes (vg)/kg/limb was sufficient to induce GALGT2-induced glycosylation in 10%-60% of skeletal myofibers in all leg muscles examined. There was a 19-fold ± 6-fold average limb-wide increase in vector genomes per microgram genomic DNA at a bilateral dose of 2.5 × 1013 vg/kg/limb compared with a bilateral dose of 6 × 1012 vg/kg/limb. A unilateral dose of 6 × 1013 vg/kg/limb showed a 12- ± 3-fold increase in treated limb muscles compared to contralateral untreated limb muscles, which received vector only after release into the systemic circulation from the treated limb. Variability in AAV biodistribution between different segments of the same muscle was 125% ± 18% for any given dose, while variability between the same muscle for any given treatment dose was 45% ± 7%. These experiments demonstrate that treatment of muscles throughout the leg with rAAVrh74.MCK.GALGT2 can be accomplished safely using an isolated limb infusion technique, where balloon catheters transiently isolate the limb vasculature, but that intra- and inter-muscle transduction variability is a significant issue.

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Acquired Infection and Antimicrobial Utilization During Initial NICU Hospitalization in Infants With Congenital Diaphragmatic Hernia.

BACKGROUND: In addition to substantial medical and surgical intervention, neonates with congenital diaphragmatic hernia often have concurrent concerns for acquired infection. However, few studies focus on infection and corresponding antimicrobial utilization in this population. METHODS: The Children's Hospital Neonatal Database was queried for congenital diaphragmatic hernia infants hospitalized from January 2010 to February 2016. Patient charts were linked to the Pediatric Health Information Systems database. Descriptive clinical data including delivery history, cultures sent, diagnosed infection, antimicrobial use and outcomes were reported. RESULTS: A total of 1085 unique patients were identified after data linkages; 275 (25.3%) were born at <37 weeks' gestation. Bacteremia at delivery (2/1085) and in the first 7 days of life (8/1085) was less common than later infection, but 976 patients (89.9%) were treated with antibiotics. Median number of days on antibiotics was 6 [3,11] for those without a documented infection and 21 [13,36] for those with positive cultures. Incidence of urinary tract infection, bacteremia and pneumonia increased significantly over time and was most common after 28 days. Antibiotic use, conversely, decreased over time (92% of infants in week 1 to 44% in week 4 and beyond). CONCLUSIONS: Although culture positivity increased with age, risk of these selected infections was relatively low for a population in neonatal intensive care unit. An important mismatch is observed between culture negativity and high rates of antibiotic utilization. These data identify opportunities for antibiotic stewardship quality improvement programs.

Induction of T-Cell Infiltration and Programmed Death Ligand 2 Expression by Adeno-Associated Virus in Rhesus Macaque Skeletal Muscle and Modulation by Prednisone.

Use of adeno-associated virus (AAV) to transduce genes into skeletal muscles can be associated with T-cell responses to viral capsid and/or to transgenic protein. Intramuscular mononuclear cell infiltrates primarily consisting of CD8+ T cells and also containing FOXP3+ regulatory T cells were present in rhesus macaque skeletal muscle treated with rAAVrh74.MCK.GALGT2 by vascular delivery. Administration of oral prednisone prior to AAV gene delivery and throughout the study reduced such infiltrates by 60% at 24 weeks post AAV delivery compared with AAV-treated animals not receiving prednisone, regardless of the presence of pre-existing AAV serum antibodies at the time of treatment. The majority of CD8+ T cells in AAV-treated muscles expressed activated caspase 3 and programmed cell death protein 1 (PD1), suggesting ongoing programmed cell death. AAV-transduced skeletal muscles also had elevated expression of programmed death ligand 2 (PDL2) on skeletal myofibers, and this increase in expression extended to muscles where transgene was not overexpressed. These data demonstrate that prednisone can reduce the extent of intramuscular T-cell infiltrates in AAV-treated muscles, which may aid in achieving long-term transgene expression, as may the induction of PDL2 expression on skeletal myofibers to promote PD1-mediated programmed T-cell death.

Short-term weight gain velocity in infants with congenital diaphragmatic hernia (CDH).

BACKGROUND: Appropriate post-natal growth remains a mainstay of therapeutic goals for infants with CDH, with the hypothesis that optimizing linear growth will improve survival through functional improvements in pulmonary hypoplasia. However, descriptions of growth and the effect on survival are limited in affected infants. OBJECTIVE: Describe in-hospital weight gain related to survival among infants with CDH. DESIGN/METHODS: Children's Hospitals Neonatal Database (CHND) identified infants with CDH born ≥34weeks' gestation (2010-14). Exclusion criteria were: admission age>7days, death/discharge age<14days, or surgical CDH repair prior to admission. Weight gain velocity (WGV: g/kg/day) was calculated using an established exponential approximation and the cohort stratified by Q1: <25%ile, Q2-3: 25-75%ile, and Q4: >75%ile. Descriptive measures and unadjusted Kaplan-Meier analyses describe the implications of WGV on mortality/discharge. RESULTS: In 630 eligible infants, median WGV was 4.6g/kg/day. After stratification by WGV [Q1: (n=156; <3.1g/kg/day); Q2-3 (n=316; 3.1-5.9g/kg/day), and Q4 (n=158, >5.9g/kg/day)] infants in Q1 had shortest median length of stay, less time on TPN and intervention for gastro-esophageal reflux relative to the other WGV strata (p<0.01 for all). Unadjusted survival estimates revealed that Q1 [hazard ratio (HR)=9.5, 95% CI: 5.7, 15.8] and Q4 [HR=2.9, 95% CI: 1.7, 5.1, p<0.001 for both] WGV were strongly associated with NICU mortality relative to Q2-3 WGV. CONCLUSION: Variable WGV is evident in infants with CDH. Highest and lowest WGV appear to be related to adverse outcomes. Efforts are needed to develop nutritional strategies targeting optimal growth.

Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells.

We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.

An arginase-1 SNP that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia enhances NO-mediated apoptosis in lymphocytes.

Arginase and nitric oxide synthase (NOS) share a common substrate, l-arginine, and have opposing effects on vascular remodeling. Arginase is the first step in polyamine and proline synthesis necessary for cellular proliferation, while NO produced from NOS promotes apoptosis. Previously, we identified a single nucleotide polymorphism (SNP) in the arginase-1 (ARG1) gene, rs2781666 (T-allele) that was associated with a decreased risk for developing pulmonary hypertension (PH) in a cohort of infants with bronchopulmonary dysplasia (BPD). In this study, we utilized lymphocytes from neonates (the only readily available cells from these patients expressing the two genotypes of interest) with either the rs2781666 SNP (TT) or wild type (GG) to test the hypothesis that the protection of the ARG1 SNP against the development of PH in BPD would involve augmented NO production leading to more apoptosis. Lymphocytes were stimulated with IL-4, IL-13, and phorbol myristate acetate (PMA). We found that TT lymphocytes had similar levels of arginase I and arginase II expression, but there was a tendency for lower urea production (a surrogate marker of arginase activity), than in the GG lymphocytes. The TT lymphocytes also had significantly greater NO production than did GG lymphocytes despite no differences in iNOS expression between genotypes. Furthermore, the TT lymphocytes had lower numbers of viable cells, and higher levels of cleaved caspase-3 than did GG lymphocytes. Inhibiting NOS activity using Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) significantly decreased cleaved caspase-3 levels in the TT lymphocytes. These data demonstrate that the TT genotype results in greater levels of NO production leading to more apoptosis, which is consistent with the concept that BPD patients with the TT genotype are protected against the development of PH by producing greater basal levels of endogenous NO.

A single nucleotide polymorphism in the dimethylarginine dimethylaminohydrolase gene is associated with lower risk of pulmonary hypertension in bronchopulmonary dysplasia.

AIM: Pulmonary hypertension (PH) develops in 25-40% of bronchopulmonary dysplasia (BPD) patients, substantially increasing mortality. We have previously found that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) production, is elevated in patients with BPD-associated PH. ADMA is metabolised by N(á´³) ,N(á´³) -dimethylarginine dimethylaminohydrolase (DDAH). Presently, we test the hypothesis that there are single nucleotide polymorphisms (SNPs) in DDAH1 and/or DDAH2 associated with the development of PH in BPD patients. METHODS: BPD patients were enrolled (n = 98) at Nationwide Children's Hospital. Clinical characteristics and 36 SNPs in DDAH1 and DDAH2 were compared between BPD-associated PH patients (cases) and BPD-alone patients (controls). RESULTS: In BPD patients, 25 (26%) had echocardiographic evidence of PH (cases). In this cohort, DDAH1 wild-type rs480414 was 92% sensitive and 53% specific for PH in BPD, and the DDAH1 SNP rs480414 decreased the risk of PH in an additive model of inheritance (OR = 0.39; 95% CI [0.18-0.88], p = 0.01). CONCLUSION: The rs480414 SNP in DDAH1 may be protective against the development of PH in patients with BPD. Furthermore, the DDAH1 rs480414 may be a useful biomarker in developing predictive models for PH in patients with BPD.

Short-term outcomes and medical and surgical interventions in infants with congenital diaphragmatic hernia.

OBJECTIVE: The aim of this study is to characterize medical and surgical therapies and short-term outcomes in infants with congenital diaphragmatic hernia (CDH). STUDY DESIGN: Retrospective analysis of CDH infants admitted to 27 children's hospitals submitting data to Children's Hospital Neonatal Database (CHND) from 2010 to 2013, stratified by gestational age, birth weight, and survival. RESULTS: A total of 572 infants were identified, 508 (89%) born ≥ 34 weeks' gestation and ≥ 2 kg. More mature infants had higher APGAR scores, shorter duration of mechanical ventilation, and were more likely to receive extracorporeal membrane oxygenation (ECMO). Overall, mortality for the cohort was 29%, with mortality lower in infants born ≥ 34 weeks' gestation and ≥ 2 kg (26 vs. 50%, p < 0.01). Nonsurvivors were more likely to receive treatment with high-frequency oscillatory ventilation (HFOV), vasopressors, pulmonary vasodilators, and ECMO, and to have associated major congenital anomalies than survivors. In hospital morbidity and complications were relatively uncommon among survivors. CONCLUSION: Infants with CDH have a high risk of morbidity and mortality, and for preterm infants with CDH those risks are amplified. Patterns of respiratory and circulatory support appeared to be different for survivors. In addition to established data registries, this consortium of regional neonatal intensive care units provides a new collaborative effort to describe short-term outcomes for infants referred with CDH.

Resveratrol prevents hypoxia-induced arginase II expression and proliferation of human pulmonary artery smooth muscle cells via Akt-dependent signaling.

Pulmonary artery smooth muscle cell (PASMC) proliferation plays a fundamental role in the vascular remodeling seen in pulmonary hypertensive diseases associated with hypoxia. Arginase II, an enzyme regulating the first step in polyamine and proline synthesis, has been shown to play a critical role in hypoxia-induced proliferation of human PASMC (hPASMC). In addition, there is evidence that patients with pulmonary hypertension have elevated levels of arginase in the vascular wall. Resveratrol, a natural polyphenol found in red wine and grape skins, has diverse biochemical and physiological actions including antiproliferative properties. Furthermore, resveratrol has been shown to attenuate right ventricular and pulmonary artery remodeling, both pathological components of pulmonary hypertension. The present studies tested the hypothesis that resveratrol would prevent hypoxia-induced pulmonary artery smooth muscle cell proliferation by inhibiting hypoxia-induced arginase II expression. Our data indicate that hypoxia-induced hPASMC proliferation is abrogated following treatment with resveratrol. In addition, the hypoxic induction of arginase II was directly attenuated by resveratrol treatment. Furthermore, we found that the inhibitory effect of resveratrol on arginase II in hPASMC was mediated through the PI3K-Akt signaling pathway. Supporting these in vitro findings, resveratrol normalized right ventricular hypertrophy in an in vivo neonatal rat model of chronic hypoxia-induced pulmonary hypertension. These novel data support the notion that resveratrol may be a potential therapeutic agent in pulmonary hypertension by preventing PASMC arginase II induction and proliferation.

Comparison of routine laboratory measures of heparin anticoagulation for neonates on extracorporeal membrane oxygenation.

Our objective was to determine the best measure of heparin anticoagulation in neonatal patients on extracorporeal membrane oxygenation. Activated clotting time (ACT), activated partial thromboplastin time (aPTT), and antifactor Xa levels, along with corresponding heparin infusion rates and heparin bolus volumes, were collected from neonates receiving ECMO at our institution from 2008 to 2013. After natural log transformation of antifactor Xa, ACT, and aPTT, overall correlations between antifactor Xa levels and either ACT or aPTT and correlations between these tests and heparin infusion rates were evaluated using linear mixed models that accounted for both within- and between-patient correlations. Twenty-six neonates with an average weight of 3.4 kg (standard deviation .7) had a total of 27 separate ECMO runs during the study period. Within each patient, ACT (r = .40, p < .0001) and aPTT (r = .48, p < .0001) were both directly correlated with antifactor Xa levels. In contrast, between patients, only aPTT maintained a direct correlation with antifactor Xa (r = .61, p = .07), whereas ACT showed a statistically significant inverse correlation with antifactor Xa (r = -.48, p = .04). Compared with ACT, aPTT is more consistently reflective of the anticoagulation status both within each patient on ECMO and between patients treated with ECMO. Future efforts to develop standardized heparin infusion algorithms for patients on ECMO should consider using aPTT levels to monitor anticoagulation.

Asymmetric dimethylarginine does not inhibit arginase activity and is pro-proliferative in pulmonary endothelial cells.

Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. L-Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater L-arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (L-arginine, ADMA, N(G) -monomethyl-L-arginine (L-NMMA) and N(G) -nitro-L-arginine methyl ester (L-NAME)). Only L-arginine resulted in measurable urea production (Km = 6.9 ± 0.8 mmol/L; Vmax = 6.6 ± 0.3 µmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, L-NMMA and L-NAME in the presence of 1 mmol/L l-arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.

Lung development alterations in newborn mice after recovery from exposure to sublethal hyperoxia.

Exposure of newborn mice to hyperoxia arrests lung development, with resultant pathological characteristics similar to bronchopulmonary dysplasia in infants born prematurely. We tested the hypothesis that aberrations in lung development caused by 14 days of sublethal hyperoxia would be reversed during 14 days of recovery to room air (RA) when the concentration of oxygen exposure was weaned gradually. Newborn FVB mice were exposed to 85% oxygen or RA for 14 days. Weaning from hyperoxia was by either transfer directly into RA or a decrease in the concentration of oxygen by 10% per days. At 28 days, pups were euthanized, and the lungs were inflation fixed and assessed. At postnatal day 28, lungs of mice weaned abruptly from hyperoxia had fewer (6 ± 0.6 versus 10 ± 0.7; P < 0.001) alveoli per high-powered field and larger alveoli (4050 ± 207 versus 2305 ± 182 µm(2)) than animals weaned gradually; both hyperoxia-exposed groups were different from lungs obtained from air-breathing controls (20 ± 0.5 alveoli per high-powered field; P < 0.001). The results are consistent with the absence of catch-up alveolarization in this model and indicate that the long-term consequences of early exposures to hyperoxia merit closer examination. The effects of abrupt weaning to RA observed further suggest that weaning should be considered in experimental models of newborn exposure to hyperoxia.

Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin α2 surrogates.

Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P < 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.