Type 2 immune polarization is associated with cardiopulmonary disease in preterm infants.

Postnatal maturation of the immune system is poorly understood, as is its impact on illnesses afflicting term or preterm infants, such as bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension. These are both cardiopulmonary inflammatory diseases that cause substantial mortality and morbidity with high treatment costs. Here, we characterized blood samples collected from 51 preterm infants longitudinally at five time points, 20 healthy term infants at birth and age 3 to 16 weeks, and 5 healthy adults. We observed strong associations between type 2 immune polarization in circulating CD3+CD4+ T cells and cardiopulmonary illness, with odds ratios up to 24. Maternal magnesium sulfate therapy, delayed hepatitis B vaccination, and increasing fetal, but not maternal, chorioamnionitis severity were associated with attenuated type 2 polarization. Blocking type 2 mediators such as interleukin-4 (IL-4), IL-5, IL-13, or signal transducer and activator of transcription 6 (STAT6) in murine neonatal cardiopulmonary disease in vivo prevented changes in cell type composition, increases in IL-1ß and IL-13, and losses of pulmonary capillaries, but not gains in larger vessels. Thereby, type 2 blockade ameliorated lung inflammation, protected alveolar and vascular integrity, and confirmed the pathological impact of type 2 cytokines and STAT6. In-depth flow cytometry and single-cell transcriptomics of mouse lungs further revealed complex associations between immune polarization and cardiopulmonary disease. Thus, this work advances knowledge on developmental immunology and its impact on early life disease and identifies multiple therapeutic approaches that may relieve inflammation-driven suffering in the youngest patients.

Characterization of the pathoimmunology of necrotizing enterocolitis reveals novel therapeutic opportunities.

Necrotizing enterocolitis (NEC) is a severe, currently untreatable intestinal disease that predominantly affects preterm infants and is driven by poorly characterized inflammatory pathways. Here, human and murine NEC intestines exhibit an unexpected predominance of type 3/TH17 polarization. In murine NEC, pro-inflammatory type 3 NKp46-RORγt+Tbet+ innate lymphoid cells (ILC3) are 5-fold increased, whereas ILC1 and protective NKp46+RORγt+ ILC3 are obliterated. Both species exhibit dysregulation of intestinal TLR repertoires, with TLR4 and TLR8 increased, but TLR5-7 and TLR9-12 reduced. Transgenic IL-37 effectively protects mice from intestinal injury and mortality, whilst exogenous IL-37 is only modestly efficacious. Mechanistically, IL-37 favorably modulates immune homeostasis, TLR repertoires and microbial diversity. Moreover, IL-37 and its receptor IL-1R8 are reduced in human NEC epithelia, and IL-37 is lower in blood monocytes from infants with NEC and/or lower birthweight. Our results on NEC pathomechanisms thus implicate type 3 cytokines, TLRs and IL-37 as potential targets for novel NEC therapies.

X-linked adrenoleukodystrophy: Pathology, pathophysiology, diagnostic testing, newborn screening and therapies.

Adrenoleukodystrophy (ALD) is a rare X-linked disease caused by a mutation of the peroxisomal ABCD1 gene. This review summarizes our current understanding of the pathogenic cell- and tissue-specific roles of lipid species in the context of experimental therapeutic strategies and provides an overview of critical historical developments, therapeutic trials and the advent of newborn screening in the USA. In ALD, very long-chain fatty acid (VLCFA) chain length-dependent dysregulation of endoplasmic reticulum stress and mitochondrial radical generating systems inducing cell death pathways has been shown, providing the rationale for therapeutic moiety-specific VLCFA reduction and antioxidant strategies. The continuing increase in newborn screening programs and promising results from ongoing and recent therapeutic investigations provide hope for ALD.

X-linked Adrenoleukodystrophy: Pathology, Pathophysiology, Diagnostic Testing, Newborn Screening, and Therapies.

Adrenoleukodystrophy (ALD) is a rare X-linked disease caused by a mutation of the peroxisomal ABCD1 gene. This review summarizes our current understanding of the pathogenic cell- and tissue-specific role of lipid species in the context of experimental therapeutic strategies and provides an overview of critical historical developments, therapeutic trials, and the advent of newborn screening in the United States. In ALD, very long chain fatty acid (VLCFA) chain-length-dependent dysregulation of endoplasmic reticulum stress and mitochondrial radical generating systems inducing cell death pathways has been shown, providing the rationale for therapeutic moiety-specific VLCFA reduction and antioxidant strategies. The continuing increase in newborn screening programs and promising results from ongoing and recent therapeutic investigations provide hope for ALD.

Attitudes of Australian health professionals towards rapid genomic testing in neonatal and paediatric intensive care.

We investigated the attitudes of intensive care physicians and genetics professionals towards rapid genomic testing in neonatal and paediatric intensive care units (NICU/PICU). A mixed-methods study (surveys and interviews) was conducted at 13 Australian hospitals and three laboratories involved in multi-center implementation of rapid genomic testing. We investigated experience and confidence with genomic tests among intensivists; perceived usefulness of genomic diagnostic results; preferences for service delivery models; and implementation readiness among genetic services. The overall survey response rate was 59%, 47% for intensivists (80/170), and 75% (91/121) for genetics professionals. Intensivists reported moderate confidence with microarray tests and lower confidence with genomic tests. The majority of intensivists (77%), clinical geneticists (87%) and genetic counsellors (82%) favoured a clinical genetics-led service delivery model of genomic testing. Perceived clinical utility of genomic results was lower in the intensivist group compared to the genetics professionals group (20 v 50%, p < 0.001). Interviews (n = 6 intensivists; n = 11 genetic counselors) demonstrated support for implementation, with concerns relating to implementation environment and organizational readiness. Overall, our findings support initial implementation of genomic testing in NICU/PICU as part of an interdisciplinary service delivery model that promotes gradual adoption of genomics by the intensive care workforce while ensuring safety, sustainability, and efficiency.

Accuracy of five formulae to determine the insertion length of umbilical venous catheters.

INTRODUCTION: Umbilical venous catheter (UVC) placement is a common neonatal procedure. It is important to position the UVC tip accurately at the first attempt to prevent complications and minimise handling. Catheters positioned too low need to be removed, but catheters positioned too high may be withdrawn in a sterile fashion to a safe position. We aimed to determine the precision and accuracy of five published formulae developed to guide UVC placement. METHODS: This was a prospective observational study. Following UVC insertion, anteroposterior and lateral X-rays were performed to identify catheter tip position. Parameters required to apply the five formulae were recorded. Insertion lengths were then calculated and compared with the gold standard (UVC tip at the level of the diaphragm on the lateral X-ray). They were also used to classify predicted UVC tip position as either correct (UVC tip at or up to 1 cm above the diaphragm), too high or too low. RESULTS: Of 118 eligible infants, 70 had the UVC tip in a position where measurements could be used. Their median (IQR) gestational age and weight were 28.5 (26-36) weeks and 1035 (745-2788) g, respectively. The predicted success rate for each formula ranged from 44.9% to 55.7%. A formula based on birth weight had the highest rate of either correct or high position (95.8%). CONCLUSIONS: Inserting a UVC into a safe position on first attempt is difficult and low tip placement is common. Around half of UVCs need to be manipulated to achieve the desired position.

Epigenetic Influences on Neurodevelopment at 11 Years of Age: Protocol for the Longitudinal Peri/Postnatal Epigenetic Twins Study at 11 Years of Age (PETS@11).

Neurodevelopment is sensitive to genetic and pre/postnatal environmental influences. These effects are likely mediated by epigenetic factors, yet current knowledge is limited. Longitudinal twin studies can delineate the link between genetic and environmental factors, epigenetic state at birth and neurodevelopment later in childhood. Building upon our study of the Peri/postnatal Epigenetic Twin Study (PETS) from gestation to 6 years of age, here we describe the PETS 11-year follow-up in which we will use neuroimaging and cognitive testing to examine the relationship between early-life environment, epigenetics and neurocognitive outcomes in mid-childhood. Using a within-pair twin model, the primary aims are to (1) identify early-life epigenetic correlates of neurocognitive outcomes; (2) determine the developmental stability of epigenetic effects and (3) identify modifiable environmental risk factors. Secondary aims are to identify factors influencing gut microbiota between 6 and 11 years of age to investigate links between gut microbiota and neurodevelopmental outcomes in mid-childhood. Approximately 210 twin pairs will undergo an assessment at 11 years of age. This includes a direct child cognitive assessment, multimodal magnetic resonance imaging, biological sampling, anthropometric measurements and a range of questionnaires on health and development, behavior, dietary habits and sleeping patterns. Data from complementary data sources, including the National Assessment Program - Literacy and Numeracy and the Australian Early Development Census, will also be sought. Following on from our previous focus on relationships between growth, cardiovascular health and oral health, this next phase of PETS will significantly advance our understanding of the environmental interactions that shape the developing brain.

Quantitation of the cellular content of saliva and buccal swab samples.

Buccal swabs and saliva are the two most common oral sampling methods used for medical research. Often, these samples are used interchangeably, despite previous evidence that both contain buccal cells and blood leukocytes in different proportions. For some research, such as epigenetic studies, the cell types contributing to the analysis are highly relevant. We collected such samples from twelve children and twenty adults and, using Papanicolaou staining, measured the proportions of epithelial cells and leukocytes through microscopy. To our knowledge, no studies have compared cellular heterogeneity in buccal swab and saliva samples from adults and children. We confirmed that buccal swabs contained a higher proportion of epithelial cells than saliva and that children have a greater proportion of such cells in saliva compared to adults. At this level of resolution, buccal swabs and saliva contained similar epithelial cell subtypes. Gingivitis in children was associated with a higher proportion of leukocytes in saliva samples but not in buccal swabs. Compared to more detailed and costly methods such as flow cytometry or deconvolution methods used in epigenomic analysis, the procedure described here can serve as a simple and low-cost method to characterize buccal and saliva samples. Microscopy provides a low-cost tool to alert researchers to the presence of oral inflammation which may affect a subset of their samples. This knowledge might be highly relevant to their specific research questions, may assist with sample selection and thus might be crucial information despite the ability of data deconvolution methods to correct for cellular heterogeneity.

Relationship between Epigenetic Maturity and Respiratory Morbidity in Preterm Infants.

OBJECTIVE: To assess associations between epigenetic maturity of extremely preterm babies (born at less than 28 weeks of gestation), neonatal interventions, and respiratory outcomes, including the administration of surfactant and postnatal corticosteroids, duration of assisted ventilation, and development of bronchopulmonary dysplasia (BPD). STUDY DESIGN: DNA was extracted from neonatal blood spots collected after birth from 143 extremely preterm infants born 1991-1992 in Victoria, Australia and used to determined DNA methylation (DNAm). A DNAm based gestational age was determined using our previously published method. The residual of DNAm gestational age and clinically estimated gestational age (referred to as "gestational age acceleration") was used as a measure to assess developmental maturity. Associations between gestational age acceleration and respiratory interventions and morbidities were determined. RESULTS: Infants with higher gestational age acceleration were less likely to receive surfactant (P = .009) or postnatal corticosteroids (P = .008), had fewer days of assisted ventilation (P = .01), and had less BPD (P = .02). Respiratory measures are known to correlate with gestational age; however, models comparing each with clinically estimated gestational age were improved by the addition of the gestational age acceleration measure in the model. CONCLUSIONS: Gestational age acceleration correlates with respiratory interventions and outcomes of extremely preterm babies. Surfactant and postnatal corticosteroid use, assisted ventilation days, and BPD rates were all lower in babies who were epigenetically more mature than their obstetrically estimated gestational age. This suggests that gestational age acceleration is a clinically relevant metric of developmental maturity.

Accuracy of 11 formulae to guide umbilical arterial catheter tip placement in newborn infants.

BACKGROUND: Umbilical arterial catheter (UAC) insertion is a common procedure in the neonatal intensive care unit (NICU). Correct placement of the tip of the UAC at first attempt minimises handling of the infant and reduces the risk of infection and complications. We aimed to determine the accuracy of 11 published formulae to guide UAC placement. METHODS: This was a one-year prospective observational study in a tertiary NICU. Clinicians used their preferred formula for UAC insertion, with X-rays performed immediately post-procedure to check the tip position. Birth weight and measurements included in the 11 formulae were recorded within 48 hours. The gold standard insertion distance was defined as the distance from the abdominal wall to the mid-descending aorta, at T8 level on X-ray (range T6-T10). Insertion length using the 11 formulae was calculated and compared with this gold standard distance. RESULTS: One hundred and three infants were included, with median (IQR) gestational age and weight of 28 (26-33.5) weeks and 980 (780-2045) g, respectively. The predicted value of the 11 formulae to place the UAC in correct position ranged from 51.0% to 73.8%. Formulae that involved direct body part measurements showed the highest predicted success rates, smallest mean difference from T8 and narrowest limits of agreement using the Bland-Altman method. CONCLUSION: Success rates for accurate UAC placement are highest when formulae that involve body measurements are used. However, even the most accurate method would result in more than 25% of UACs needing manipulation to achieve an optimal position.

Pulmonary hypertension associated with bronchopulmonary dysplasia in preterm infants.

Bronchopulmonary dysplasia (BPD) and BPD-associated pulmonary hypertension (BPD-PH) are chronic inflammatory cardiopulmonary diseases with devastating short- and long-term consequences for infants born prematurely. The immature lungs of preterm infants are ill-prepared to achieve sufficient gas exchange, thus usually necessitating immediate commencement of respiratory support and oxygen supplementation. These therapies are life-saving, but they exacerbate the tissue damage that is inevitably inflicted on a preterm lung forced to perform gas exchange. Together, air-breathing and necessary therapeutic interventions disrupt normal lung development by aggravating pulmonary inflammation and vascular remodelling, thus frequently precipitating BPD and PH via an incompletely understood pathogenic cascade. BPD and BPD-PH share common risk factors, such as low gestational age at birth, fetal growth restriction and perinatal maternal inflammation; however, these risk factors are not unique to BPD or BPD-PH. Occurring in 17-24% of BPD patients, BPD-PH substantially worsens the morbidity and mortality attributable to BPD alone, thus darkening their outlook; for example, BPD-PH entails a mortality of up to 50%. The absence of a safe and effective therapy for BPD and BPD-PH renders neonatal cardiopulmonary disease an area of urgent unmet medical need. Besides the need to develop new therapeutic strategies, a major challenge for clinicians is the lack of a reliable method for identifying babies at risk of developing BPD and BPD-PH. In addition to discussing current knowledge on pathophysiology, diagnosis and treatment of BPD-PH, we highlight emerging biomarkers that could enable clinicians to predict disease-risk and also optimise treatment of BPD-PH in our tiniest patients.

An epigenetic clock for gestational age at birth based on blood methylation data.

BACKGROUND: Gestational age is often used as a proxy for developmental maturity by clinicians and researchers alike. DNA methylation has previously been shown to be associated with age and has been used to accurately estimate chronological age in children and adults. In the current study, we examine whether DNA methylation in cord blood can be used to estimate gestational age at birth. RESULTS: We find that gestational age can be accurately estimated from DNA methylation of neonatal cord blood and blood spot samples. We calculate a DNA methylation gestational age using 148 CpG sites selected through elastic net regression in six training datasets. We evaluate predictive accuracy in nine testing datasets and find that the accuracy of the DNA methylation gestational age is consistent with that of gestational age estimates based on established methods, such as ultrasound. We also find that an increased DNA methylation gestational age relative to clinical gestational age is associated with birthweight independent of gestational age, sex, and ancestry. CONCLUSIONS: DNA methylation can be used to accurately estimate gestational age at or near birth and may provide additional information relevant to developmental stage. Further studies of this predictor are warranted to determine its utility in clinical settings and for research purposes. When clinical estimates are available this measure may increase accuracy in the testing of hypotheses related to developmental age and other early life circumstances.

A prospective evaluation of whole-exome sequencing as a first-tier molecular test in infants with suspected monogenic disorders.

PURPOSE: To prospectively evaluate the diagnostic and clinical utility of singleton whole-exome sequencing (WES) as a first-tier test in infants with suspected monogenic disease. METHODS: Singleton WES was performed as a first-tier sequencing test in infants recruited from a single pediatric tertiary center. This occurred in parallel with standard investigations, including single- or multigene panel sequencing when clinically indicated. The diagnosis rate, clinical utility, and impact on management of singleton WES were evaluated. RESULTS: Of 80 enrolled infants, 46 received a molecular genetic diagnosis through singleton WES (57.5%) compared with 11 (13.75%) who underwent standard investigations in the same patient group. Clinical management changed following exome diagnosis in 15 of 46 diagnosed participants (32.6%). Twelve relatives received a genetic diagnosis following cascade testing, and 28 couples were identified as being at high risk of recurrence in future pregnancies. CONCLUSIONS: This prospective study provides strong evidence for increased diagnostic and clinical utility of singleton WES as a first-tier sequencing test for infants with a suspected monogenic disorder. Singleton WES outperformed standard care in terms of diagnosis rate and the benefits of a diagnosis, namely, impact on management of the child and clarification of reproductive risks for the extended family in a timely manner.Genet Med 18 11, 1090-1096.

Predictors of unfavorable thermal outcome during newborn emergency retrievals.

OBJECTIVE: Maintenance of normal body temperature is a challenge during transports. We aimed to identify predisposing factors for unfavorable thermal outcome during emergency retrievals of neonates. METHODS: Demographic data and clinical variables for transports performed over a 2-year period were extracted from the Newborn Emergency Transport Service (Victoria, Australia) database. Arrival temperatures outside normothermia (36.5°-37.5°C) were defined as an unfavorable outcome. RESULTS: Normothermia on arrival at the receiving hospital was achieved in 78% of 1,261 transports. The strongest predictor of unfavorable thermal outcome was an abnormal temperature at the start of the retrieval (odds ratio [OR] = 8.04; 95% confidence interval [CI], 5.91-10.95; P < .001) followed by very low weight on transport (< 1,500 g; OR = 2.49; 95% CI, 1.63-3.80; P < .001) and respiratory support (OR = 1.81; 95% CI, 1.29-2.54; P = .001). Medications (eg, inotropes and sedation/muscle relaxation) or central/peripheral venous/arterial lines were not significant predictors of outcome when temperature at retrieval start, weight at transport, and respiratory support were adjusted as cofactors. Mode of transport (road, fixed wing, or rotary wing aircraft) and outside temperature were not associated with thermal outcome. CONCLUSION: Abnormal temperature at the start of the retrieval, very low transport weight, and respiratory support were strong predictors of unfavorable thermal outcome during neonatal emergency transports.

Newborn screening for X-linked adrenoleukodystrophy: further evidence high throughput screening is feasible.

X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible.

Analysis of epigenetic changes in survivors of preterm birth reveals the effect of gestational age and evidence for a long term legacy.

BACKGROUND: Preterm birth confers a high risk of adverse long term health outcomes for survivors, yet the underlying molecular mechanisms are unclear. We hypothesized that effects of preterm birth can be mediated through measurable epigenomic changes throughout development. We therefore used a longitudinal birth cohort to measure the epigenetic mark of DNA methylation at birth and 18 years comparing survivors of extremely preterm birth with infants born at term. METHODS: Using 12 extreme preterm birth cases and 12 matched, term controls, we extracted DNA from archived neonatal blood spots and blood collected in a similar way at 18 years of age. DNA methylation was measured at 347,789 autosomal locations throughout the genome using Infinium HM450 arrays. Representative methylation differences were confirmed by Sequenom MassArray EpiTYPER. RESULTS: At birth we found 1,555 sites with significant differences in methylation between term and preterm babies. At 18 years of age, these differences had largely resolved, suggesting that DNA methylation differences at birth are mainly driven by factors relating to gestational age, such as cell composition and/or maturity. Using matched longitudinal samples, we found evidence for an epigenetic legacy associated with preterm birth, identifying persistent methylation differences at ten genomic loci. Longitudinal comparisons of DNA methylation at birth and 18 years uncovered a significant overlap between sites that were differentially-methylated at birth and those that changed with age. However, we note that overlapping sites may either differ in the same (300/1,555) or opposite (431/1,555) direction during gestation and aging respectively. CONCLUSIONS: We present evidence for widespread methylation differences between extreme preterm and term infants at birth that are largely resolved by 18 years of age. These results are consistent with methylation changes associated with blood cell development, cellular composition, immune induction and age at these time points. Finally, we identified ten probes significantly associated with preterm individuals and with greater than 5% methylation discordance at birth and 18 years that may reflect a long term epigenetic legacy of preterm birth.

Amplitude-integrated electroencephalography in newborns with inborn errors of metabolism.

BACKGROUND: The utility of amplitude-integrated electroencephalography (aEEG) monitoring has been established for patients with neonatal hypoxic-ischemic encephalopathy. OBJECTIVE: To evaluate the role of aEEG in the diagnostic process and treatment of patients with encephalopathy due to inborn errors of metabolism. METHODS: Cases collected through an international registry were divided into 5 groups of metabolic disorders. Common aEEG features were sought for each group. RESULTS: In total, 21/30 (70%) cases had abnormal aEEG background patterns, 18/30 (60%) showed seizure activity. Patients with disorders of energy metabolism, hyperammonemia, and organic/amino acidopathies often showed marked aEEG depression with seizure activity. In contrast, aEEGs of patients with peroxisomal disorders did not show major background abnormalities but seizures were present in 5/6 subjects. We report two features of interest: firstly, two tracings displayed an unusual upward shift of the lower aEEG amplitude margin. Secondly, aEEGs of infants with non-ketotic hyperglycinemia showed a pattern we refer to as 'high-frequency burst-suppression pattern'. CONCLUSIONS: aEEG in patients with inborn errors of metabolism frequently reveals abnormalities and assists clinicians in the clinical assessment, management and monitoring of these patients.

Use of amplitude integrated electroencephalography (aEEG) in patients with inborn errors of metabolism - a new tool for the metabolic geneticist.

Patients with metabolic disorders often, especially as newborns, present with encephalopathy and seizures, frequently requiring intensive care during metabolic crises. Cerebral function monitoring using amplitude integrated electroencephalography (aEEG) can be utilized to supplement clinical assessment and other monitoring already in use in the intensive care setting. In this technique, a one or two-channel EEG tracing is obtained, processed, compressed and displayed. Use of aEEG is well established in evaluation and treatment of newborns with hypoxic ischemic encephalopathy. The basis of aEEG interpretation is the recognition of patterns which have been defined for different degrees of encephalopathy. Seizures are identified on the compressed tracing in combination with analysis of the corresponding raw EEG tracing. This review discusses the experience, although limited at this time, with use of aEEG in infants with inborn errors of metabolism. Through an international collaborative, the International Registry for Cerebral Function Monitoring in Patients with Genetics Disorders and Brain Malformations, aEEG tracings of patients with inborn errors of metabolism were collected. The features of 25 traces are included in this review. This collection includes patients with hyperammonemia (HA, n=4), disorders of energy metabolism (DEM, n=9), disorders of amino and organic acid metabolism (DAOAM, n=7), and peroxisomal disorders (PD, n=5). Fifteen of 25 patients demonstrated encephalopathic changes, including patients with HA, DEM and DAOAM, but not PD. In 15 of 25 patients seizure potentials were identified. In HA, DEM, and DAOAM both encephalopathy and seizures may coincide, while in peroxisomal disorders seizures were seen without background patterns indicating encephalopathy, likely due to neuronal migration defects as the underlying cause. The current experience with the use of aEEG in these patients, while limited, indicates that cerebral function monitoring techniques have a role in treatment of patients with metabolic disorders, especially during metabolic crises. Use of aEEG in this patient group is encouraged if locally available, and collaborative efforts to collect data on aEEG use in patients with metabolic disorders are encouraged to further define the scope and utility of this procedure.