Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of ß-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

Reducing umbilical catheter migration rates by using a novel securement device.

OBJECTIVE: This study evaluates the effectiveness of a novel device, LifeBubble, in reducing umbilical cord catheter (UC) migration and associated complications in neonates. STUDY DESIGN: A retrospective review was performed at Oregon Health & Science University's NICU (2019-2021) to compare standard adhesive securement with LifeBubble. The primary outcomes were UC migration, discontinuation due to malposition, and CLABSI incidence. Differences between groups were statistically analyzed and logistic regression used to adjust for potential confounders. RESULTS: Among 118 neonates (57 LifeBubble, 61 adhesive), LifeBubble significantly reduced migration of any UC > 1 vertebral body (12.3% vs. 55.7%), including UVC migration (5.3% vs. 39.3%) and UAC migration (7.0% vs 23.0%), as well as UVC discontinuation due to malposition (5.6% vs 37.7%). The number needed to treat (NNT) to prevent one instance of UVC discontinuation is 4. CONCLUSION: LifeBubble effectively reduces UC migration and premature discontinuation, indicating its potential to enhance neonatal care and safety.

Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid.

Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of ß-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

Oxygenation Fluctuations Associated with Severe Retinopathy of Prematurity: Insights from a Multimodal Deep Learning Approach.

Purpose: Retinopathy of prematurity (ROP) is one of the leading causes of blindness in children. Although the role of oxygen in the pathophysiology of ROP is well established, a precise understanding of the dynamic relationship between oxygen exposure ROP incidence and severity is lacking. The purpose of this study was to evaluate the correlation between time-dependent oxygen variables and the onset of ROP. Design: Retrospective cohort study. Participants: Two hundred thirty infants who were born at a single academic center and met the inclusion criteria were included. Infants are mainly born between January 2011 and October 2022. Methods: Patient data were extracted from electronic health records (EHRs), with sufficient time-dependent oxygen data. Clinical outcomes for ROP were recorded as none/mild or moderate/severe (defined as type II or worse). Mixed-effects linear models were used to compare the 2 groups in terms of dynamic oxygen variables, such as daily average and the coefficient of variation (COV) fraction of inspired oxygen (FiO2). Support vector machine (SVM) and long-short-term memory (LSTM)-based multimodal models were trained with fivefold cross-validation to predict which infants would develop moderate/severe ROP. Gestational age (GA), birth weight, and time-dependent oxygen variables were used to develop predictive models. Main Outcome Measures: Model cross-validation performance was evaluated by computing the mean area under the receiver operating characteristic (AUROC) curve, precision, recall, and F1 score. Results: We found that both daily average and COV of FiO2 were associated with more severe ROP (adjusted P < 0.001). With fivefold cross-validation, the multimodal LSTM models had higher performance than the best static models (SVM using GA and 3 average FiO2 features) and SVM models trained on GA alone (mean AUROC = 0.89 ± 0.04 vs. 0.86 ± 0.05 vs. 0.83 ± 0.04). Conclusions: The development of severe ROP might not only be influenced by oxygen exposure but also by its fluctuation, which provides direction for future study of pathophysiological factors associated with severe ROP development. Additionally, we demonstrated that multimodal neural networks can be a method to extract useful information from time-series data, which may be a valuable methodology for the investigation of other diseases using EHR data. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

The amniotic fluid proteome changes across gestation in humans and rhesus macaques.

Amniotic fluid is a complex biological medium that offers protection to the fetus and plays a key role in normal fetal nutrition, organogenesis, and potentially fetal programming. Amniotic fluid is also critically involved in longitudinally shaping the in utero milieu during pregnancy. Yet, the molecular mechanism(s) of action by which amniotic fluid regulates fetal development is ill-defined partly due to an incomplete understanding of the evolving composition of the amniotic fluid proteome. Prior research consisting of cross-sectional studies suggests that the amniotic fluid proteome changes as pregnancy advances, yet longitudinal alterations have not been confirmed because repeated sampling is prohibitive in humans. We therefore performed serial amniocenteses at early, mid, and late gestational time-points within the same pregnancies in a rhesus macaque model. Longitudinally-collected rhesus amniotic fluid samples were paired with gestational-age matched cross-sectional human samples. Utilizing LC-MS/MS isobaric labeling quantitative proteomics, we demonstrate considerable cross-species similarity between the amniotic fluid proteomes and large scale gestational-age associated changes in protein content throughout pregnancy. This is the first study to compare human and rhesus amniotic fluid proteomic profiles across gestation and establishes a reference amniotic fluid proteome. The non-human primate model holds promise as a translational platform for amniotic fluid studies.

Building better barriers: how nutrition and undernutrition impact pediatric intestinal health.

Chronic undernutrition is a major cause of death for children under five, leaving survivors at risk for adverse long-term consequences. This review focuses on the role of nutrients in normal intestinal development and function, from the intestinal epithelium, to the closely-associated mucosal immune system and intestinal microbiota. We examine what is known about the impacts of undernutrition on intestinal physiology, with focus again on the same systems. We provide a discussion of existing animal models of undernutrition, and review the evidence demonstrating that correcting undernutrition alone does not fully ameliorate effects on intestinal function, the microbiome, or growth. We review efforts to treat undernutrition that incorporate data indicating that improved recovery is possible with interventions focused not only on delivery of sufficient energy, macronutrients, and micronutrients, but also on efforts to correct the abnormal intestinal microbiome that is a consequence of undernutrition. Understanding of the role of the intestinal microbiome in the undernourished state and correction of the phenotype is both complex and a subject that holds great potential to improve recovery. We conclude with critical unanswered questions in the field, including the need for greater mechanistic research, improved models for the impacts of undernourishment, and new interventions that incorporate recent research gains. This review highlights the importance of understanding the mechanistic effects of undernutrition on the intestinal ecosystem to better treat and improve long-term outcomes for survivors.

Dilemmas in establishing preterm enteral feeding: where do we start and how fast do we go?

Beginning and achieving full enteral nutrition is a key step in the care of preterm infants, particularly very low birth weight (VLBW) infants. As is true for many organ system-specific complications of prematurity, the gastrointestinal tract must complete in utero development ex utero while concurrently serving a physiologic role reserved for after completion of full term development. The preterm gut must assume the placental function of the interface between a source of energy, precursors for anabolism, and micronutrients, and the developing infant-through digestion and absorption of milk, instead of directly from the mother via the uteroplacental interface. The benefits of enteral nourishment in preterm infants are counterbalanced by gastrointestinal complications of prematurity: dysmotility leading to difficulty establishing and advancing feeds, and the risk of necrotizing enterocolitis (NEC). Concern for these complications can prolong the need for parenteral nutrition with an associated increase in risk for central line-associated bloodstream infection (CLABSI) and parenteral nutrition (PN)-associated cholestasis or liver disease (PNALD). Thus, a daily issue facing neonatologists caring for preterm infants is how to optimally begin, advance, and reach full enteral nutrition sufficient to satisfy the nutrient, energy, and fluid requirements of VLBW infants while minimizing risk. In this perspective, we provide an overview of the approaches and supporting data for starting and advancing enteral feeds in preterm infants, particularly very low birth weight infants, and we discuss the significant gaps in knowledge that accompany current approaches. This framework recognizes the dilemmas of preterm feeding initiation and advancement and identifies areas of opportunity for further investigation.

Carnitine-Acylcarnitine Translocase Deficiency Masked by Extreme Prematurity.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare disorder of long chain fatty acid oxidation with a very high mortality rate due to cardiomyopathy or multiorgan failure. We present the course of a very premature infant with early onset CACT deficiency complicated by multiple episodes of necrotizing enterocolitis, sepsis, and liver insufficiency, followed by eventual demise. The complications of prematurity, potentiated by the overlay of CACT deficiency, contributed to the difficulty of reaching the ultimate diagnosis of CACT deficiency.

Impaired placental hemodynamics and function in a non-human primate model of gestational protein restriction.

Maternal malnutrition increases fetal and neonatal morbidity, partly by affecting placental function and morphology, but its impact on placental hemodynamics are unknown. Our objective was to define the impact of maternal malnutrition on placental oxygen reserve and perfusion in vivo in a rhesus macaque model of protein restriction (PR) using advanced imaging. Animals were fed control (CON, 26% protein), 33% PR diet (17% protein), or a 50% PR diet (13% protein, n = 8/group) preconception and throughout pregnancy. Animals underwent Doppler ultrasound and fetal biometry followed by MRI at gestational days 85 (G85) and 135 (G135; term is G168). Pregnancy loss rates were 0/8 in CON, 1/8 in 33% PR, and 3/8 in 50% PR animals. Fetuses of animals fed a 50% PR diet had a smaller abdominal circumference (G135, p < 0.01). On MRI, placental blood flow was decreased at G135 (p < 0.05) and placental oxygen reserve was reduced (G85, p = 0.05; G135, p = 0.01) in animals fed a 50% PR diet vs. CON. These data demonstrate that a 50% PR diet reduces maternal placental perfusion, decreases fetal oxygen availability, and increases fetal mortality. These alterations in placental hemodynamics may partly explain human growth restriction and stillbirth seen with severe PR diets in the developing world.

Dilemmas in human milk fortification.

Fortification of human milk is the standard of care for very low birth weight (VLBW) infants and is required to support adequate postnatal growth and development. Achieving adequate growth velocity and preventing growth faltering is critical for the developing neonatal brain and optimizing long-term neurodevelopmental outcomes. Mother's milk is the gold standard nutrition to feed preterm infants, however, it does not provide the nutrients needed to support the growth of VLBW infants. After the decision is made to use mother's milk (if available) or alternatively, donor human milk, many dilemmas exist with regards to additional treatment decisions surrounding the type of fortification to use, when to fortify, and the duration of fortification. In this article, we will review the differences in mother's milk compared to donor milk, the different types of human milk fortifiers, the optimal timing of fortification, and discuss when to discontinue human milk fortification.

Perioperative Improvement in Pulmonary Function in Infants with Congenital Diaphragmatic Hernia.

OBJECTIVE: The objective of this study was to compare serial changes in pulmonary function in contemporary infants with congenital diaphragmatic hernia managed with a gentle ventilation approach. STUDY DESIGN: Observational cohort, single-center study of infants ≥350/7 weeks gestation at delivery with congenital diaphragmatic hernia. Functional residual capacity (FRC), passive respiratory compliance, and passive respiratory resistance were measured presurgical and postsurgical repair and within 2 weeks of discharge. A 1-way analysis of variance for repeated measures was used to evaluate the change in FRC, passive respiratory compliance, and passive respiratory resistance over these repeated measures. RESULTS: Twenty-eight infants were included in the analysis with a mean gestational age of 38.3 weeks and birth weight of 3139 g. We found a significant increase in FRC across the 3 time points (mean in mL/kg [SD]: 10.9 [3.6] to 18.5 [5.2] to 24.2 [4.4]; P < .0001). There was also a significant increase in passive respiratory compliance and decrease in passive respiratory resistance. In contrast to a previous report, there were survivors in the current cohort with a preoperative FRC of <9 mL/kg. The mean FRC measured at discharge was in the range considered within normal limits. Sixteen infants had prenatal measurements of the lung-to-head ratio, but there was no relationship between the lung-to-head ratio and preoperative or postoperative FRC measurements. CONCLUSIONS: Infants with congenital diaphragmatic hernia demonstrate significant increases in FRC and improvements in respiratory mechanics measured preoperatively and postoperatively and at discharge. We speculate these improvements are due to the surgical resolution of the mechanical obstruction to lung recruitment and that after achieving preoperative stability, repair should not be delayed given these demonstrable postoperative improvements.

Shaping infant development from the inside out: Bioactive factors in human milk.

Human breast milk is the optimal nutrition for all infants and is comprised of many bioactive and immunomodulatory components. The components in human milk, such as probiotics, human milk oligosaccharides (HMOs), extracellular vesicles, peptides, immunoglobulins, growth factors, cytokines, and vitamins, play a critical role in guiding neonatal development beyond somatic growth. In this review, we will describe the bioactive factors in human milk and discuss how these factors shape neonatal immunity, the intestinal microbiome, intestinal development, and more from the inside out.

Body composition measurement for the preterm neonate: using a clinical utility framework to translate research tools into clinical care.

Body composition analysis to distinguish between fat mass and fat-free mass is an established research approach to assess nutritional status. Within neonatal medicine, preterm infant body composition is linked with later health outcomes including neurodevelopment and cardiometabolic health. Mounting evidence establishing fat-free mass as an indicator of nutritional status, coupled with the availability of testing approaches that are feasible to use in preterm infants, have enhanced interest in measuring body composition in the neonatal intensive care unit (NICU) setting. In this paper, we use the concept of clinical utility-the added value of a new methodology over current standard care-as a framework for assessing several existing body composition methodologies with potential for clinical application to preterm neonates. We also use this framework to identify remaining knowledge gaps and prioritize efforts to advance our understanding of clinically-oriented body composition testing in the NICU.

Bifidobacteriumlongum subsp. infantis EVC001 Administration Is Associated with a Significant Reduction in the Incidence of Necrotizing Enterocolitis in Very Low Birth Weight Infants.

OBJECTIVE: To assess the effects of Bifidobacteriumlongum subsp. infantis EVC001 (Binfantis EVC001) administration on the incidence of necrotizing enterocolitis (NEC) in preterm infants in a single level IV neonatal intensive care unit (NICU). STUDY DESIGN: Nonconcurrent retrospective analysis of 2 cohorts of very low birth weight (VLBW) infants not exposed and exposed to Binfantis EVC001 probiotic at Oregon Health & Science University from 2014 to 2020. Outcomes included NEC incidence and NEC-associated mortality, including subgroup analysis of extremely low birth weight (ELBW) infants. Log-binomial regression models were used to compare the incidence and risk of NEC-associated outcomes between the unexposed and exposed cohorts. RESULTS: The cumulative incidence of NEC diagnoses decreased from 11.0% (n = 301) in the no EVC001 (unexposed) cohort to 2.7% (n = 182) in the EVC001 (exposed) cohort (P < .01). The EVC001 cohort had a 73% risk reduction of NEC compared with the no EVC001 cohort (adjusted risk ratio, 0.27; 95% CI, 0.094-0.614; P < .01) resulting in an adjusted number needed to treat of 13 (95% CI, 10.0-23.5) for Binfantis EVC001. NEC-associated mortality decreased from 2.7% in the no EVC001 cohort to 0% in the EVC001 cohort (P = .03). There were similar reductions in NEC incidence and risk for ELBW infants (19.2% vs 5.3% [P < .01]; adjusted risk ratio, 0.28; 95% CI, 0.085-0.698 [P = .02]) and mortality (5.6% vs 0%; P < .05) in the 2 cohorts. CONCLUSIONS: In this observational study of 483 VLBW infants, Binfantis EVC001 administration was associated with significant reductions in the risk of NEC and NEC-related mortality. Binfantis EVC001 supplementation may be considered safe and effective for reducing morbidity and mortality in the NICU.

Acute right ventricular failure associated with pulmonary hypertension in pediatrics: understanding the hemodynamic profiles.

Pulmonary hypertension (PHTN) is a common pathology in pediatrics, arising from a diverse array of etiologies and manifesting in equally diverse patient populations. The inpatient management of these infants and children may be complicated by dynamic and at times severe increases in pulmonary vascular resistance (PVR) and right ventricular (RV) afterload. Yet absent are cognitively accessible heuristics in the field whereby providers can reconcile the various clinical manifestations they observe with an understanding of the cardiac physiology at play, and therefore, appropriate physiology-driven interventions. Described herein is a framework for understanding the pathophysiology of four clinical phenotypes which are driven by two echocardiographic patient characteristics: the presence or absence of an atrial communication and the capacity of the right ventricle to maintain ventricular-vascular coupling. Application of this paradigm may facilitate accurate interpretation of observed clinical data, and alignment of treatment strategies with the underlying pathophysiology.

Peptides Derived from In Vitro and In Vivo Digestion of Human Milk Are Immunomodulatory in THP-1 Human Macrophages.

BACKGROUND: Milk proteins contain many encrypted bioactive peptides. Whether these bioactive peptides are released in the infant intestine and exert immunomodulatory activity remains unknown. OBJECTIVE: This study examined in vitro immunomodulatory activities of peptides from in vitro- and in vivo-digested human milk. METHODS: Peptides were extracted from in vitro-digested human milk and pooled intestinal samples from 8 infants fed human milk. Peptides extracted from in vitro-digested samples were fractionated. The in vitro effects of these peptides and fractions on the secretion of TNF-α and IL-8 in LPS-treated human immune THP-1 macrophages were evaluated. The significance of differences between in vitro peptide fraction treatment and control on cytokine production was analyzed by t test. LC-MS/MS-based peptidomics was conducted to identify the peptides. The peptides were screened for potential bioactivity using a sequence homology search using the Milk Bioactive Peptide Database (MBPDB). RESULTS: Six fractions of the peptide mixture extracted from the in vitro-digested human milk significantly inhibited TNF-α production by LPS-challenged THP-1 macrophages. Fractions F4, F8, F11, F14, and F17 attenuated IL-8 secretion, and F6/7 and F18 increased IL-8 secretion. Peptides extracted from the pooled in vivo intestinal samples attenuated both TNF-α and IL-8 secretion. There were 266 and 418 peptides identified in the in vitro and in vivo samples, respectively. Among the peptides, 34 and 50 in the in vitro and in vivo samples, respectively, had >80% sequence similarity to bioactive peptides in the MBPDB. CONCLUSIONS: Peptides released by in vitro and in vivo infant digestion of human milk were immunomodulatory in human immune cells; fractions F4, F8, and F11 were anti-inflammatory; and F6/7 and F18 were proinflammatory. Thirteen peptides were present in all fractions with anti-inflammatory activity, and 38 peptides were present in all fractions with proinflammatory activity. These peptides potentially contributed to the observed immunomodulatory activity of the peptide mixtures.

Microfluidic photoreactor to treat neonatal jaundice.

While in most cases, jaundice can be effectively treated using phototherapy, severe cases require exchange transfusion, a relatively risky procedure in which the neonate's bilirubin-rich blood is replaced with donor blood. Here, we examine extracorporeal blood treatment in a microfluidic photoreactor as an alternative to exchange transfusion. This new treatment approach relies on the same principle as phototherapy but leverages microfluidics to speed up bilirubin removal. Our results demonstrate that high-intensity light at 470 nm can be used to rapidly reduce bilirubin levels without causing appreciable damage to DNA in blood cells. Light at 470 nm was more effective than light at 505 nm. Studies in Gunn rats show that photoreactor treatment for 4 h significantly reduces bilirubin levels, similar to the bilirubin reduction observed for exchange transfusion and on a similar time scale. Predictions for human neonates demonstrate that this new treatment approach is expected to exceed the performance of exchange transfusion using a low blood flow rate and priming volume, which will facilitate vascular access and improve safety.

Pulmonary Function Tests in Very Low Birth Weight Infants Screened for Pulmonary Hypertension: A Pilot Study.

OBJECTIVE: To compare pulmonary function tests (PFTs), specifically respiratory system resistance (Rrs) and compliance (Crs), in very low birth weight (VLBW) infants with and without pulmonary hypertension. STUDY DESIGN: Infants were included who underwent PFTs at 34-38 weeks postmenstrual age (PMA) as part of our pulmonary hypertension screening guidelines for infants born at ≤1500 g requiring respiratory support at ≥34 weeks PMA. One pediatric cardiologist reviewed and estimated right ventricular or pulmonary arterial pressure and defined pulmonary hypertension as an estimated pulmonary arterial pressure or right ventricular pressure greater than one-half the systemic pressure. Rrs and Crs were measured with the single breath occlusion technique and functional residual capacity with the nitrogen washout method according to standardized criteria. RESULTS: Twelve VLBW infants with pulmonary hypertension and 39 without pulmonary hypertension were studied. Those with pulmonary hypertension had significantly lower birth weight and a trend toward a lower gestational age. There were no other demographic differences between the groups. The infants with pulmonary hypertension had significantly higher Rrs (119 vs 78 cmH2O/L/s; adjusted P = .012) and significantly lower Crs/kg (0.71 vs 0.92 mL/cmH2O/kg; P = .04). CONCLUSIONS: In this pilot study of VLBW infants screened for pulmonary hypertension at 34-38 weeks PMA, those with pulmonary hypertension had significantly increased Rrs and decreased Crs compared with those without pulmonary hypertension. Additional studies are needed to further phenotype infants with evolving BPD and pulmonary hypertension.

Congenital Diaphragmatic Hernia Management: A Systematic Review and Care Pathway Description Including Volume-Targeted Ventilation.

BACKGROUND: Although it is well established that standardized treatment protocols improve outcomes for infants with congenital diaphragmatic hernia (CDH), there remains variance between existing protocols. PURPOSE: The purpose of this article was to review current literature on protocols for CDH management in the preoperative period and to describe a care pathway integrating best practice elements from existing literature with volume-targeted ventilation strategies previously in place at a major tertiary care center in the Pacific Northwestern United States. METHODS/SEARCH STRATEGY: A systematic review of literature was performed according to PRISMA guidelines to identify current publications on CDH protocols and examine them for similarities and differences, particularly regarding ventilation strategies. FINDINGS/RESULTS: Although existing protocols from multiple regions worldwide shared common goals of reducing barotrauma and delaying surgery until a period of clinical stabilization was achieved, their strategies varied. None included volume-targeted ventilation with pressure limitation as a method of avoiding ventilation-induced lung injury (VILI). IMPLICATIONS FOR PRACTICE: Institutions that routinely manage infants with CDH should have a standardized treatment protocol in place, as this is shown to improve outcomes. This may include volume-targeted ventilation with pressure limitation as a successful VILI-limiting strategy. IMPLICATIONS FOR RESEARCH: While standardized protocols have been shown to increase survival rate for infants with CDH, more research is needed to determine what these protocols should include. Specifically, there is a need for future study on the most appropriate ventilation mode for this population.