Extreme prematurity-associated alterations of pulmonary inflammatory mediators before and after surfactant administration.

BACKGROUND: The role of prematurity and pulmonary inflammation in the pathogenesis of bronchopulmonary dysplasia (BPD) is very well-defined. However, there is limited knowledge about whether the level of prematurity and surfactant therapy alter the pulmonary cytokines and endothelial growth factor (VEGF). METHODS: This study analyzed the VEGF and cytokines, including interleukin (IL)-1ß, IL-6, IL-8, and IL-10, and tumor necrosis factor α (TNF-α) in the tracheal aspirate (TA) of preterm infants obtained before (within 2 h after birth) and 10-12 h after the administration of the first dose of surfactant. TA was collected from 40 infants of 35 or fewer weeks of gestation, including extremely (Group 1, n = 19), very (Group 2, n = 13), and moderate/late (Group 2, n = 8) preterm neonates. In addition to univariate analysis, controlled regression models estimated the association of perinatal factors with the tested parameters and their role in the development of BPD. RESULT: We recorded significantly lower post-partum levels of VEGF and higher IL-8, IL-1ß, and TNF-α in the TA of Group 1 infants than in Group 2 and 3. Compared to the infants in Group 2 and 3, the post-surfactant increases of pulmonary VEGF, IL-8, IL-10, and TNF-α were more significant in Group 1. All tested parameters in Group 1 and 2 infants, before and after surfactant administration, were comparable. BPD was recorded in nearly 60% of the extremely preterm survivors and was significantly predicted by increased IL-8 before, and elevated TNF-α level after surfactant administration. CONCLUSION: This study indicates the association of birth at extremely preterm gestation with reduction in pulmonary VEGF and exacerbation of pro-inflammatory cytokines followed by greater elevation post-surfactant administration levels of VEGF, IL-8, TNF-α, and IL-10 than in neonates born with gestational age of 28-35 weeks.

Fetal Zone Steroids Show Discrete Effects on Hyperoxia-Induced Attenuation of Migration in Cultured Oligodendrocyte Progenitor Cells.

Cerebral oxygenation disturbances contribute to the pathogenesis of brain lesions in preterm infants with white matter damage. These children are at risk of developing long-term neurodevelopmental disabilities. Preterm birth is associated with sudden hormonal changes along with an untimely increase in oxygen tissue tension. There is a persistent high postnatal production of fetal zone steroids (FZS), which serve in the fetoplacental unit as precursors for placental estrogen synthesis during pregnancy. The role of FZS in events associated with oxygenation differences and their impact on the developing white matter is not well understood. Therefore, we investigated the effect of hyperoxia (80% O2) and subsequent administration of FZS on the protein composition and migration capabilities of immature oligodendrocytes using the OLN93 (rat-derived OPC) cell line as an experimental model. We tested the effect of the FZS, dehydroepiandrosterone (DHEA), 16α-OH-DHEA, and adiol (5-androstene-3ß, 17ß-diol). After 24-hour exposure to hyperoxia, we monitored the changes in the proteome profile following treatment and observed significant alterations in pathways regulating cytoskeletal remodelling, cell migration, and cell survival. Additionally, hyperoxia leads to impaired migration of the OLN93 cells in culture. Administration of the FZS showed positive effects on the migration process under normoxic conditions in general. However, under hyperoxic conditions, the trend was less prominent. The observed effects could be related to changes in levels of cofilin/LIMK pathway-associated proteins. Adiol had a negative effect when administered together with estradiol, and the proteomic data reveal the activation of ephrin receptor signalling that might be responsible for the attenuation of migration. The results suggest that FZS can differentially regulate pathways involved in the migration of OLN93 cells. A deeper insight into the precise role of endogenous FZS would be an essential prerequisite for developing new treatment strategies including supplementation of estradiol and other steroids in preterm infants.

Vascular endothelial growth factor levels and bronchopulmonary dysplasia in preterm infants.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) regulate both vasculogenesis, the development of blood vessels from precursor cells, and angiogenesis, the formation of blood vessels from preexisting vessels. In the fetal lung, high-affinity receptors for VEGF are expressed mainly in alveolar epithelial cells and myocytes, suggesting a paracrine role for VEGF in modulating activities in adjacent vascular endothelium. Previous studies have shown that vascular growth is impaired in bronchopulmonary dysplasia (BPD). OBJECTIVE: The goal of this study was to examine tracheal (T-VEGF) and gastric (G-VEGF) levels in premature infants in the first and third day of life and examine if these levels were associated with the development of BPD. DESIGN/METHODS: Tracheal aspirates from intubated infants and gastric samples from others were obtained on postnatal days 1 (D1) and 3 (D3) from 43 preterm infants (<2000 g birth weight, ≤34 wks gestation). VEGF was quantified by a VEGF Elisa Kit. Demographic, clinical, and pulmonary outcome data were collected including information on respiratory support (oxygenation index (OI) and ventilatory index (VI)) and on the development of BPD, determined at 36 weeks PMA using NICHD criteria. RESULTS: The mean birth weight was 1060 ± 379 g and gestational age 27.5 ± 2.8 wks. BPD was diagnosed in 26 infants who were less mature than the 17 controls without BPD. Day 1 and day 3T-VEGF concentrations did not correlate, but day 3 levels correlated with gestational age (r = 0.75, p < .05). BPD infants, characterized by longer ventilator, CPAP and oxygen days, had day 1T-VEGF levels similar to control infants (126.6 ± 194.7 vs. 149.7 ± 333.2 pg/ml) but day 3 levels were significantly lower (168.9 ± 218.8 vs. 1041.6 ± 676.7 pg/ml). Day 1G-VEGF levels reflected tracheal samples, trending lower in BPD infants. Mode of delivery, race, sex, antenatal steroid administration, chorioamnionitis, sepsis, or growth restriction did not impact VEGF levels. However, lower VEGF levels were associated with a lower VI and lower OI: Day 3 OI correlated with day 3T-VEGF (r = 0.72, p > .05), albeit not significantly. T-VEGF increased from day 1 to day 3 in controls and decreased in BPD infants. There was no relationship between oxygen, CPAP and ventilator days and day 1 or day 3T-VEGF levels. CONCLUSIONS: BPD may be associated with low-serum VEGF levels during the first week of life. This finding is likely related to decreased expression in the lungs of the less mature infants, who are at the highest risk for BPD.

Oxidative Stress and Respiratory Diseases in Preterm Newborns.

Premature infants are exposed to increased generation of reactive oxygen species, and on the other hand, they have a deficient antioxidant defense system. Oxidative insult is a salient part of lung injury that begins as acute inflammatory injury in respiratory distress disease and then evolves into chronic and structural scarring leading to bronchopulmonary dysplasia. Oxidative stress is also involved in the pathogenesis of pulmonary hypertension in newborns through the modulation of the vascular tone and the response to pulmonary vasodilators, with consequent decrease in the density of the pulmonary vessels and thickening of the pulmonary arteriolar walls. Oxidative stress has been recognized as both a trigger and an endpoint for several events, including inflammation, hypoxia, hyperoxia, drugs, transfusions, and mechanical ventilation, with impairment of pulmonary function and prolonged lung damage. Redoxomics is the most fascinating new measure to address lung damage due to oxidative stress. The new challenge is to use omics data to discover a set of biomarkers useful in diagnosis, prognosis, and formulating optimal and individualized neonatal care. The aim of this review was to examine the most recent evidence on the relationship between oxidative stress and lung diseases in preterm newborns. What is currently known regarding oxidative stress-related lung injury pathogenesis and the available preventive and therapeutic strategies are also discussed.

Iron and Neurodevelopment in Preterm Infants: A Narrative Review.

Iron is critical for brain development, playing key roles in synaptogenesis, myelination, energy metabolism and neurotransmitter production. NICU infants are at particular risk for iron deficiency due to high iron needs, preterm birth, disruptions in maternal or placental health and phlebotomy. If deficiency occurs during critical periods of brain development, this may lead to permanent alterations in brain structure and function which is not reversible despite later supplementation. Children with perinatal iron deficiency have been shown to have delayed nerve conduction speeds, disrupted sleep patterns, impaired recognition memory, motor deficits and lower global developmental scores which may be present as early as in the neonatal period and persist into adulthood. Based on this, ensuring brain iron sufficiency during the neonatal period is critical to optimizing neurodevelopmental outcomes and iron supplementation should be targeted to iron measures that correlate with improved outcomes.

Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion.

Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 11ß-hydroxylase activity, sustaining cortisol synthesis. However, the P450c11ß enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11ß-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 11ß-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM-overexpression mice (0.83 vs 0.46 ng/mL, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.

Fetal Zone Steroids and Estrogen Show Sex Specific Effects on Oligodendrocyte Precursor Cells in Response to Oxidative Damage.

Oxygen causes white matter damage in preterm infants and male sex is a major risk factor for poor neurological outcome, which speculates the role of steroid hormones in sex-based differences. Preterm birth is accompanied by a drop in 17ß-estradiol (E2) and progesterone along with increased levels of fetal zone steroids (FZS). We performed a sex-based analysis on the FZS concentration differences in urine samples collected from preterm and term infants. We show that, in preterm urine samples, the total concentration of FZS, and in particular the 16α-OH-DHEA concentration, is significantly higher in ill female infants as compared to males. Since we previously identified Nup133 as a novel target protein affected by hyperoxia, here we studied the effect of FZS, allopregnanolone (Allo) and E2 on differentiation and Nup133 signaling using mouse-derived primary oligodendrocyte progenitor cells (OPCs). We show that the steroids could reverse the effect of hyperoxia-mediated downregulation of Nup133 in cultured male OPCs. The addition of FZS and E2 protected cells from oxidative stress. However, E2, in presence of 16α-OH-DHEA, showed a negative effect on male cells. These results assert the importance of sex-based differences and their potential implications in preterm stress response.

Tear Fluid Angiogenic Factors: Potential Noninvasive Biomarkers for Retinopathy of Prematurity Screening in Preterm Infants.

Purpose: To determine the status of proangiogenic factors in the tear fluid of preterm infants with and without retinopathy of prematurity (ROP). Methods: Preterm infants (n = 36) undergoing routine ROP screening included in the prospective study were categorized as No-ROP (n = 13, no ROP at any visits), ROP (if ROP was present at first visit; n = 18), or No-ROP to ROP (no disease at first visit, but developed ROP subsequently; n = 5). Infants with ROP were also grouped as progressing (n = 7) and regressing (n = 16) based on ROP evolution between the first and subsequent visits. Schirmer's strips were used to collect tear fluid and proangiogenic factors (VEGF, angiogenin, soluble vascular cell adhesion molecule, and fractalkine) levels (in picograms per milliliter) in tear fluid were measured by multiplex ELISA. Results: Lower levels of VEGF (135 ± 69; mean ± standard deviation) and higher levels of angiogenin (6568 ± 4975) were observed in infants with ROP compared with infants without ROP (172.5 ± 54.0; 4139 ± 3909) at the first visit. Significantly lower levels of VEGF were observed in the No-ROP to ROP group compared with the No-ROP and ROP groups. The VEGF and angiogenin levels at the first visit were significantly lower in infants with ROP with progressing disease. Angiogenin levels negatively correlated with birth weight and gestational age in ROP. The area under the curve (AUC) and odds ratio (OR) analysis demonstrated that angiogenin/birth weight (AUC = 0.776; OR, 8.6); angiogenin/gestational age (AUC = 0.706; OR, 7.3) and Angiogenin/VEGF (AUC = 0.806; OR, 14.3) ratios were able to differentiated preterm infants with and without ROP. Conclusions: The association between angiogenin and ROP suggests its possible role in ROP. The ratio of angiogenin level with birth weight, gestational age, and/or VEGF could serve as a potential noninvasive screening biomarker for ROP.

MicroRNA 219-5p inhibits alveolarization by reducing platelet derived growth factor receptor-alpha.

BACKGROUND: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD). METHODS: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants. RESULTS: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation. CONCLUSIONS: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Intestinal SALSA/dmbt1 levels are decreased in prematurely born infants.

The first months of life represent a crucial time period for an infant. Alongside establishing the early microbiome, the mucosal immunological homeostasis is being developed. Both processes may be perturbed in prematurely born infants. The glycoprotein SALSA plays a role in mucosal inflammation and microbial clearance. It is one of the most abundant molecules on the intestinal mucosal surfaces in early life. SALSA binds to many types of microbes and host defence molecules like IgA, C1q and collectin molecules. We here describe the development in faecal SALSA levels during the first three months of life. During these 90 days, the median SALSA level in full-term babies decreased from 1100 µg/mL (range 49-17 000 µg/mL) to 450 µg/mL (range 33-1000 µg/mL). Lower levels of SALSA were observed in prematurely born infants in the same time period. Our novel observation thus indicates an impact of prematurity on an important component of the infant intestinal immune system. Changes in SALSA in early life may have an effect on the early establishment of the human microbiome.

Utility of volatile organic compounds as a diagnostic tool in preterm infants.

BACKGROUND: Volatile organic compounds (VOCs) are hydrocarbons that originate within different healthy and diseased tissues. VOCs can be secreted into the circulation and then excreted in the urine and faeces. In the lungs, VOCs are locally produced and can be detected in exhaled breath. VOCs can be identified using non-invasive techniques, which make their use in preterm infants safe and desirable. METHODS: A systematic search of the literature in PubMed, Embase and Web of Science was conducted looking for VOCs techniques and diagnostic performance in preterm infants. A total of 50 articles identified with only seven papers were included in the final analysis in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). RESULTS: VOCs could diagnose necrotising enterocolitis up to 4 days before a clinical diagnosis; for late onset sepsis, up to 3 days before; and for bronchopulmonary dysplasia, up to 2 weeks before. In addition to these diagnostic uses, VOCs analysis could also distinguish breastfed from formula-fed preterm neonates in the first month of life. CONCLUSION: VOCs analysis is a non-invasive tool that makes the use in preterm infants of preference. VOCs analytic techniques require more research and consensus between researchers to overcome their limitations. IMPACT: Volatile organic compounds are hydrocarbons that can separate between healthy and diseased states in preterm infants. Biomarker panels developed from volatile organic compounds are potential diagnostic tools. The non-invasive nature of acquiring volatile organic compounds markers make it desirable in the paediatric patients. Research into exact chemical components of the volatile organic compounds can inform about the pathophysiology of disease in preterm infants. More robust longitudinal studies with repeated experiments are required before volatile organic compounds can be applied in clinical practice.

Characterisation of fasted state gastric and intestinal fluids collected from children.

Fundamental knowledge about the composition of intestinal fluids in paediatric populations is currently unavailable. This study aimed to characterise gastric and intestinal fluid from paediatric populations. Gastric and intestinal fluid samples were obtained during routine clinical endoscopy from paediatric patients at a large teaching hospital. These fluids were characterised to measure the pH; buffer capacity; osmolality; bile acid concentration and composition. A total of 55 children were recruited to the study aged from 11 months to 15 years of age where 53 gastric fluid samples and 40 intestinal fluid samples were obtained. pH values recorded ranged from pH 0.57 to 11.05 (median: 2.50) in gastric fluids and from 0.89 to 8.97 (median: 3.27) in intestinal fluids. The buffer capacity did not change significantly between gastric and intestinal fluids with median values of 12 mM/L/ΔpH for both fluids. Gastric fluid osmolality values ranged from 1 to 615 mOsm/kg, while intestinal fluid values ranged from 35 to 631 mOsm/kg. Gastric fluid bile acid concentrations ranged from 0.002 to 2.3 mM with a median value of 0.017 mM whilst intestinal fluid bile acid concentrations ranged from 0.0008 to 3.3 mM with a median value of 0.178 mM. Glycocholate; taurocholic acid; glycochenodeoxycholate and taurochenodeoxycholate were the most commonly identified bile acids within paediatric intestinal fluids. All compositional components were associated with large inter-individual variability. Further work is required to develop simulated paediatric media and to explore the impact of these media on drug solubility and dissolution.

Differences in human milk peptide release along the gastrointestinal tract between preterm and term infants.

BACKGROUND & AIMS: Preterm infants are born with a gastrointestinal tract insufficiently developed to digesting large quantities of human milk proteins. Peptides released from the digestion of human milk proteins have been identified with bioactivities that may be beneficial to the developing infant. However, it is unknown how prematurity affects total and bioactive peptide release along the gastrointestinal tract. The aim of this study was to compare milk peptide release from milk to stomach to stool between preterm and term infants. METHODS: Milk, gastric, and stool samples were collected from preterm infants as early collection (days 8 and 9 of life) and late collection (days 21 and 22 of life), and from term infants as early collection. Milk peptides were extracted from the samples and identified using Orbitrap mass spectrometry. Peptide abundance and count were compared across digestion and between the three infant groups at each stage of digestion. RESULTS: Total milk peptide count and abundance increased from milk to stomach then decreased in stool. Total peptide release was similar among the three infant groups for milk and stool samples. In the stomach, preterm early collection had significantly higher peptide abundance and count than the other two groups. Patterns for peptide release from individual milk proteins were distinct from total peptide release both across digestion and among the infant groups. When analyzing single peptides, term early collection gastric samples had significantly higher peptide abundance than preterm early collection for a known antimicrobial peptide, QELLLNPTHQIYPVTQPLAPVHNPISV. CONCLUSIONS: Preterm and term infants digest milk proteins differently along their gastrointestinal tracts. While preterm infants released more total peptides in the stomach, term infants released specific bioactive peptides at higher abundance. We identified a region at the C-terminus of ß-casein that is conserved from milk through stool and from which are released known and potential antimicrobial peptides.

Composition and origin of lung fluid proteome in premature infants and relationship to respiratory outcome.

BACKGROUND: Infants born at extremely low gestational age are at high risk for bronchopulmonary dysplasia and continuing lung disease. There are no early clinical biomarkers for pulmonary outcome and limited therapeutic interventions. OBJECTIVES: We performed global proteomics of premature infant tracheal aspirate (TA) and plasma to determine the composition and source of lung fluid proteins and to identify potential biomarkers of respiratory outcome. METHODS: TA samples were collected from intubated infants in the TOLSURF cohort before and after nitric oxide treatment, and plasma was collected from NO CLD infants. Protein abundance was assayed by HPLC/tandem mass spectrometry and Protein Prospector software. mRNA abundance in mid-gestation fetal lung was assessed by RNA sequencing. Pulmonary morbidity was defined as a need for ventilatory support at term and during the first year. RESULTS: Abundant TA proteins included albumin, hemoglobin, and actin-related proteins. 96 of 137 detected plasma proteins were present in TA (r = 0.69, p<0.00001). Based on lung RNAseq data, ~88% of detected TA proteins in injured infant lung are derived at least in part from lung epithelium with overrepresentation in categories of cell membrane/secretion and stress/inflammation. Comparing 37 infants at study enrollment (7-14 days) who did or did not develop persistent pulmonary morbidity, candidate biomarkers of both lung (eg., annexin A5) and plasma (eg., vitamin D-binding protein) origin were identified. Notably, levels of free hemoglobin were 2.9-fold (p = 0.03) higher in infants with pulmonary morbidity. In time course studies, hemoglobin decreased markedly in most infants after enrollment coincident with initiation of inhaled nitric oxide treatment. CONCLUSIONS: We conclude that both lung epithelium and plasma contribute to the lung fluid proteome in premature infants with lung injury. Early postnatal elevation of free hemoglobin and heme, which are both pro-oxidants, may contribute to persistent lung disease by depleting nitric oxide and increasing oxidative/nitrative stress.

Serious neonatal morbidities are associated with differences in DNA methylation among very preterm infants.

BACKGROUND: Infants born very preterm are more likely to experience neonatal morbidities compared to their term peers. Variations in DNA methylation (DNAm) associated with these morbidities may yield novel information about the processes impacted by these morbidities. METHODS: This study included 532 infants born < 30 weeks gestation, participating in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants study. We used a neonatal morbidity risk score, which was an additive index of the number of morbidities experienced during the NICU stay, including bronchopulmonary dysplasia (BPD), severe brain injury, serious neonatal infections, and severe retinopathy of prematurity. DNA was collected from buccal cells at discharge from the NICU, and DNAm was measured using the Illumina MethylationEPIC. We tested for differential methylation in association with the neonatal morbidity risk score then tested for differentially methylated regions (DMRs) and overrepresentation of biological pathways. RESULTS: We identified ten differentially methylated CpGs (α Bonferroni-adjusted for 706,278 tests) that were associated with increasing neonatal morbidity risk scores at three intergenic regions and at HPS4, SRRD, FGFR1OP, TNS3, TMEM266, LRRC3B, ZNF780A, and TENM2. These mostly followed dose-response patterns, for 8 CpGs increasing DNAm associated with increased numbers of morbidities, while for 2 CpGs the risk score was associated with decreasing DNAm. BPD was the most substantial contributor to differential methylation. We also identified seven potential DMRs and over-representation of genes involved in Wnt signaling; however, these results were not significant after Bonferroni adjustment for multiple testing. CONCLUSIONS: Neonatal DNAm, within genes involved in fibroblast growth factor activities, cellular invasion and migration, and neuronal signaling and development, are sensitive to the neonatal health complications of prematurity. We hypothesize that these epigenetic features may be representative of an integrated marker of neonatal health and development and are promising candidates to integrate with clinical information for studying developmental impairments in childhood.

Cytokine Expression by Human Macrophage-Like Cells Derived from the Monocytic Cell Line THP-1 Differs Between Treatment With Milk from Preterm- and Term-Delivering Mothers and Pasteurized Donor Milk.

Immunomodulatory proteins from human milk may enhance the protection and development of the infant's gut. This study compared the immunomodulatory effects of treatment with milk from preterm-(PM) and term-delivering (TM) mothers and pasteurized donor milk (DM) on cytokine gene expression in human macrophage-like cells derived from the monocytic cell line THP-1. The gene expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-12 (p40), IL-10 and GAPDH in macrophages treated with PM, TM and DM at steady and activated (inflammatory) states were measured using real-time reverse transcription-polymerase chain reaction. TNF-α and IL-6 in macrophages (both states) with DM were higher than PM or TM. IL-10 in steady state macrophages with DM was higher than PM whereas DM increased IL-10 in activated macrophages compared with TM. TM increased IL-6 and IL-12 (p40) in steady state macrophages compared with PM. IL-12 (p40) in activated macrophages with TM was higher than PM. IL-10 in steady state macrophages with TM was higher than PM. These results suggest that DM induces higher gene expression of pro-inflammatory and anti-inflammatory cytokines in macrophages compared with PM or TM. PM reduced gene expression of pro-inflammatory cytokines compared with TM, which may decrease the development of necrotizing enterocolitis and systematic inflammation.

Metabolic changes in early neonatal life: NMR analysis of the neonatal metabolic profile to monitor postnatal metabolic adaptations.

BACKGROUND: A major challenge from the moment a child is delivered is the adaptation to the extrauterine life, where rapid metabolic changes take place. The study of these changes during the first days of human life may assist in the understanding of the metabolic processes that occur at this critical period, which is likely to provide significant clinical insights. To date, metabolomics has become a powerful field, ideal for the monitoring of such dynamic variations, since it offers the possibility to identify alterations in metabolic profiles, even on daily basis. METHODS: The study included 253 healthy newborns (GA 35 to 40 weeks) from the region of Western Greece. Urine samples were collected immediately after birth and at the third day of life. NMR-based metabolomics was used to compare the metabolic urinary profiles of newborns from the first and third day of their life, assessing the impact of six perinatal factors; delivery mode, prematurity, maternal smoking, gender, nutrition and neonatal jaundice. RESULTS: Analysis of urine metabolic fingerprint from the first and third day of life, coupled with multivariate statistics, provides insights into the details of early life metabolic profile differentiation. Αt the third day of life metabolic adaptations are evident, as many differences were noted in urine of healthy neonates within the first 72 h postpartum. Trends in differentiation of metabolites levels between the two groups, late preterm and term newborns, have been also observed. CONCLUSIONS: Newborn's urine metabolic profiles confirmed the rapid changes in their metabolism after birth. Further, ongoing research will enable us to develop one reference model of urinary metabolomics in healthy newborns during the period of adaptation to the extra-uterine life.

Oxygen in the neonatal period: Oxidative stress, oxygen load and epigenetic changes.

Preterm infants frequently require positive pressure ventilation and oxygen supplementation in the first minutes after birth. It has been shown that the amount of oxygen provided during stabilization, the oxygen load, if excessive may cause hyperoxia, and oxidative damage to DNA. Epidemiologic studies have associated supplementation with pure oxygen in the first minutes after birth with childhood cancer. Recent studies have shown that the amount of oxygen supplemented to preterm infants after birth modifies the epigenome. Of note, the degree of DNA hyper-or hypomethylation correlates with the oxygen load provided upon stabilization. If these epigenetic modifications would persist, oxygen supplied in the first minutes after birth could have long term consequences. Further studies with a robust power calculation and long-term follow up are needed to bear out the long-term consequences of oxygen supplementation during postnatal stabilization of preterm infants.

Milk Peptides Survive In Vivo Gastrointestinal Digestion and Are Excreted in the Stool of Infants.

BACKGROUND: Human milk peptides released by gastrointestinal proteases have been identified with bioactivities that can benefit the infant but must first reach their respective sites of activity. Peptides in the stool either survived to or were released inside the intestinal tract, and thus had the opportunity to exert bioactivity there. However, it is unknown whether any milk peptides, bioactive or not, can survive in the stool of infants. OBJECTIVE: The aim of this study was primarily to identify milk peptides in infant stool samples and secondarily test the hypotheses that the milk peptide profiles of stools are different between preterm infants at different days of life and between preterm and term infants. METHODS: Infant stool samples were collected from 16 preterm infants (<34 weeks gestational age) at 8 or 9 and 21 or 22 days of life (DOL), and from 10 term infants (>34 weeks gestational age) at 8 or 9 DOL. Milk peptides were isolated from the stool samples and identified using tandem MS. The peptide counts and abundances were compared between infant groups. RESULTS: In total, 118 exclusively milk-derived peptides from the caseins and α-lactalbumin were present in the stool samples, including some peptides with known or potential bioactivity. The remaining 8014 identified peptides could be derived either from milk or endogenous proteins. Although many individual milk peptides were significantly different between preterm infants at 8/9 and 21/22 DOL and between preterm and term infants, total peptide abundance and count were similar for all 3 groups. CONCLUSIONS: This is the first study to confirm the survival of milk peptides in the stool of infants. Some of the peptides had potential bioactivities that could influence infant gut development. These results are important to understand the physiological relevance of human milk peptides to the infant.

Quality improvement measures for early detection of severe intravenous infiltration in infants.

Intravenous infiltration is one of the most commonly seen morbidity in infants admitted to the neonatal intensive care unit (NICU). The risk of intravenous infiltration in preterm infants is probably due to prolonged peripheral intravenous access requirement for nutritional support and usage of other intravenous medications to support their growth. Infants are more likely to develop intravenous infiltrations due to the increased fragility of their blood vessels, deficient subcutaneous tissue and inability to express pain. As a result, the intravenous infiltrates in infants can rapidly progress to severe stage 3 and stage 4 infiltrates with necrosis if timely intervention is not provided. Also, factors obscuring to identify stage 1 and stage 2 infiltrates, may lead their progression to severe infiltration. Root cause analysis was performed following two severe intravenous infiltrates that required plastic surgery intervention in our level III NICU. Quality improvement measures were implemented. We developed a unique intravenous securing method, conducted educational programmes for NICU staff, increased intravenous site surveillance and ascertained to maintain the intravenous pump pressures in the reference range. The hospital NICU intravenous care policy was updated with quality improvement measures. Data were collected preintervention and postintervention. The incidence of intravenous infiltration in preterm infants varies widely in different places. This may be due to under-reporting of these relatively rare adverse events, but may also be due to the fact that the preterm infants represent a small portion of the patient population. The present study has shown that severe infiltration was associated with an increase in intravenous days. Following the quality improvement measures, there were no reported cases of severe intravenous infiltration. In conclusion, the awareness of the problem with evidence-based quality improvement measures may help in early detection of intravenous infiltrates and decrease the severe intravenous infiltration in infants.