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Introduction: The neonatal sequential organ failure assessment (nSOFA) score is a tool for calculating mortality risk of infants in the neonatal intensive care unit. The utility of the nSOFA in determining the risk of mortality or the association with surgical intervention among infants with necrotizing enterocolitis (NEC) has not been investigated. Methods: We performed a retrospective, cohort study of preterm (Results: Of the 259 infants, nSOFA scores for infants who died (n = 39) or had the composite outcome of surgery or death (n = 114) were significantly higher (p < 0.05) early in the NEC course compared to nSOFA scores for infants who survived medical NEC. Twelve hours after evaluation, the area under the receiver operating characteristic curve was 0.87 (95% confidence interval [CI], 0.80–0.93) to discriminate for mortality and 0.84 (95% CI, 0.79–0.90) for surgery or death (p < 0.001). A maximum nSOFA score of ≥4 at −6, 0, 6, or 12 h following evaluation was associated with a 20-fold increase in mortality and 19-fold increase in surgery or death compared with a score of p < 0.001). Conclusion: In this multicenter cohort, the nSOFA score was able to discriminate well for death as well as surgery or death among infants with NEC. The nSOFA is a clinical research tool that may be used in infants with NEC to improve classification by objective quantification of organ dysfunction.

Due to the changing complex healthcare environment, educational innovation is essential to meet the needs of current and future neonatal-perinatal medicine (NPM) leaders. Greater clinical demands, decreased academic funding, and expanded graduate medical education program requirements have negatively impacted time for teaching and educational scholarship potentially limiting innovation in the field. By focusing on adult learning principles, embracing technology, and promoting collaboration, today's educators are preparing the next generation of neonatologists. Current innovations include regionalizing simulation boot camps, leveraging virtual learning to increase accessibility, developing niche training opportunities, and incorporating population health principles within existing quality initiatives. Areas in need of additional innovation include faculty and fellow development for teaching skills, expansion of educational networks, and dissemination and financial support of educational scholarship. These efforts and future innovations will require medical institutions and national NPM organizations to further invest in the medical educator as part of their missions.

Necrotizing enterocolitis (NEC) causes significant morbidity and mortality in premature infants; therefore, the identification of therapeutic and preventative strategies against NEC remains a high priority. The ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) is well known to contribute to the regulation of intestinal microbial communities and amelioration of intestinal inflammation. However, the role of AhR signaling in NEC is unclear. Experimental NEC was induced in 4-d-old wild-type mice or mice lacking AhR expression in the intestinal epithelial cells or AhR expression in CD11c+ cells (AhRΔCD11c) by subjecting animals to twice daily hypoxic stress and gavage feeding with formula supplemented with LPS and enteric bacteria. During NEC, compared with wild-type mice treated with vehicle, littermates treated with an AhR proligand, indole-3-carbinol, had reduced expression of Il1b and Marco, a scavenger receptor that mediates dendritic cell activation and the recognition and clearance of bacterial pathogens by macrophages. Furthermore, indole-3-carbinol treatment led to the downregulation of genes involved in cytokine and chemokine, as revealed by pathway enrichment analysis. AhR expression in the intestinal epithelial cells and their cre-negative mouse littermates were similarly susceptible to experimental NEC, whereas AhRΔCD11c mice with NEC exhibited heightened inflammatory responses compared with their cre-negative mouse littermates. In seeking to determine the mechanisms involved in this increased inflammatory response, we identified the Tim-4− monocyte–dependent subset of macrophages as increased in AhRΔCD11c mice compared with their cre-negative littermates. Taken together, these findings demonstrate the potential for AhR ligands as a novel immunotherapeutic approach to the management of this devastating disease.

Neonatal-perinatal medicine fellows must achieve a meaningful accomplishment in scholarly activity as part of their training. Despite the requirement for scholarly training in fellowship, there is a vanishingly small number of MD-only physician-scientists pursuing a research-oriented career. Recent neonatal trainees have identified several factors that preclude their careers in research-focused academic neonatology, including lower pay in academic positions, inadequate training in research techniques, and the perception that individuals in research careers have a poor work-life balance. High competition for limited pediatric research funds also contributes to a diminishing pool of physician-scientists in neonatology. This small number of physician-scientists is threatened by a high rate of attrition among physicians who enter this career path. In order to prevent further declines in the number of neonatal physician-scientists, we need improvements in funding and strong intra- and cross-institutional mentorship to foster individuals interested in a career as a physician-scientist.

With over one-third of pregnancies in the United States being delivered by cesarean and the growing knowledge of morbidities associated with repeat cesarean deliveries, the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the Society for Maternal-Fetal Medicine, and the American College of Obstetricians and Gynecologists convened a workshop to address the concept of preventing the first cesarean. The available information on maternal and fetal factors, labor management and induction, and non-medical factors leading to the first cesarean were reviewed as well as the implications of the first cesarean on future reproductive health. Key points were identified to assist with reduction in cesarean rates including that labor induction should be performed primarily for medical indication; if done for non-medical indications, the gestational age should be at least 39 weeks or more and the cervix should be favorable, especially in the nulliparous patient. Review of the current literature demonstrates the importance of adhering to appropriate definitions for failed induction and arrest of labor progress. The diagnosis of “failed induction” should only be made after an adequate attempt. Adequate time for normal latent and active phases of the first stage, and for the second stage, should be allowed, as long as the maternal and fetal conditions permit. The adequate time for each of these stages appears to be longer than traditionally estimated. Operative vaginal delivery is an acceptable birth method when indicated, and can safely prevent cesarean delivery. Given the progressively declining use, it is critical that training and experience in operative vaginal delivery is facilitated and encouraged. When discussing the first cesarean with a patient, counseling should include its effect on future reproductive health.

Background Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in the neonatal ICU with minimal progress in the research. Methods Federal webpages were queried to look for funding opportunity announcements (FOAs) and to develop lists of funded projects on NEC to identify gaps in NEC-related research topics. Results Over the past 30 years, the National Institutes of Health (NIH) issued two FOAs to stimulate research on NEC with $4.1 million set aside for the first year of respective funding. We identified 23 recently funded studies of which 18 were research projects, 4 training grants, and 1 conference grant support. Only one grant focused on parent and family engagement in the NICU. Conclusion There are significant research gaps that can be addressed with adequate funding from the federal government on the prevention and treatment of NEC.

Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients’ intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli , which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.

BACKGROUND: Surgery residents complete their research training early in residency. Non-surgical trainees typically have research incorporated toward the last two years of their fellowship, conferring an advantage to apply for grants with recent research experience and preliminary data. METHODS: The NIH RePORTER database was queried for K08 awardees trained in medicine, pediatrics, and surgery from 2013–2017. 406 K08 recipients were identified and time from completion of clinical training to achieving a K08 award was measured. Data were compared using ANOVA and expressed as mean. P

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors., Cliff Luke et al. report that lysoptosis is a eukaryotic stand-alone regulated cell death pathway. They identify that this new cell death modality predominates in the absence of neutralizing endogenous inhibitors.

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.

Objective Necrotizing enterocolitis (NEC) is the most common and lethal gastrointestinal disease affecting preterm infants. NEC develops suddenly and is characterized by gut barrier destruction, an inflammatory response, intestinal necrosis and multi-system organ failure. There is currently no method for early NEC detection, and the pathogenesis of NEC remains unclear. Design To further understand the molecular mechanisms that support NEC, we used solution phase hybridization and next-generation DNA sequencing of bisulfite converted DNA to perform targeted genome-wide analysis of DNA methylation at high read depth. Results We found that ileal samples from surgical NEC infants (n = 5) exist in a broadly hypermethylated state relative to their non-NEC counterparts (n = 9). These trends were not uniform, with hypermethylation being most consistently observed outside CpG islands and promoters. We further identified several biologically interesting gene promoters that displayed differential methylation in NEC and a number of biological pathways that appear dysregulated in NEC. We also found that DNA methylation patterns identified in ileal NEC tissue were correlated with those found and published previously in stool samples from NEC-affected infants. Conclusion We confirmed that surgical NEC is associated with broad DNA hypermethylation in the ileum, and this may be detectable in stool samples of affected individuals. Thus, an epigenomic liquid biopsy of stool may have significant potential as a biomarker with respect to the diagnostic/predictive detection of NEC. Our findings, along with recent similar observations in colon, suggest that epigenomic dysregulation is a significant feature of surgical NEC. These findings motivate future studies which will involve the longitudinal screening of samples obtained prior to the onset of NEC. Our long-term goal is the development of novel screening, diagnostic and phenotyping methods for NEC.

Summary Recapitulating human NEC using animal models has been insightful in dissecting the signaling pathways, immune-mediated mechanisms, genetic signatures, and the intestinal architecture of NEC. This protocol describes an in vivo murine NEC model, using hypoxia and formula containing lipopolysaccharide and enteric bacteria derived from an infant with NEC. With this mouse model, we aim to further dissect NEC pathogenesis and develop new therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Mihi et al. (2021)., Graphical abstract, Highlights • This protocol describes the induction of murine necrotizing enterocolitis (NEC) • This mouse model is based on the use of hypoxia and formula supplemented with LPS • This mouse model can be used to study NEC pathogenesis and therapeutic targets, Recapitulating human NEC using animal models has been insightful in dissecting the signaling pathways, immune-mediated mechanisms, genetic signatures, and the intestinal architecture of NEC. This protocol describes an in vivo murine NEC model, using hypoxia and formula containing lipopolysaccharide and enteric bacteria derived from an infant with NEC. With this mouse model, we aim to further dissect NEC pathogenesis and develop new therapeutic strategies.

PurposeNecrotizing enterocolitis is associated with decreased intestinal perfusion and ischemia. Paneth cells, specialized epithelial cells, have been shown to regulate the intestinal vasculature and disruption of these cells has been associated with NEC. We hypothesized that Paneth cell disruption in immature mice intestine would decrease the perfusion of the intestinal microvasculature.MethodsPaneth cells were disrupted in P14-16 mice using chemical (dithizone) and transgenic (diphtheria toxin) methodology. Six hours after Paneth cell disruption, Dylight 488 was injected directly into the left ventricle and allowed to perfuse for 5 minutes prior to intestinal harvesting. Tissue samples were evaluated with confocal fluorescence microscopy to quantify intestinal perfusion and samples were quantified by real time RT-PCR for gene expression.ResultsDithizone treatment significantly decreased intestinal perfusion compared to controls (p 0.21). Intestines from all treatment groups had similar PECAM staining, but intestines treated with dithizone had significantly decreased nNOS and iNOS gene expression compared to controls (p

Hemodynamic instability of the preterm infant has challenged neonatologists since the inception of the field. The inherent abnormal condition and dynamic physiology of the preterm infant make it difficult to define and subsequently treat neonatal hypotension. Since neonatal hypotension contributes to the several complications of prematurity, significant research efforts have been carried out to learn how to define and treat it. We review the obstacles encountered in defining and treating neonatal hypotension and apply this knowledge to the special consideration of preterm infants with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a life-threatening disease of the gastrointestinal tract that primarily affects premature neonates. Infants with NEC experience a high risk for increased morbidity and mortality, thus indicating a need to implement early medical management to minimize the progression to advanced stages of disease. The objective of this chapter is to discuss the initial clinical approach to an infant with NEC, including strategies for bowel rest and the use of serial abdominal radiographs to monitor for pathognomonic features of NEC. Additionally, this chapter will provide an overview of the evidence for antibiotic choice in the treatment of NEC. We then discuss the evaluation and management of common hematologic abnormalities seen during NEC, such as anemia, thrombocytopenia, and coagulopathy. Lastly, we describe complications that can occur despite appropriate NEC management, including multiorgan compromise, short bowel syndrome, intestinal strictures, and NEC recurrence.

Necrotizing enterocolitis (NEC) is an acute inflammatory disease that unforeseeably develops in very low birth weight premature infants. NEC is characterized by impairment of the intestinal barrier resulting in intestinal necrosis and multisystem organ failure. Animal models of NEC have contributed significantly to a better understanding of the underlying molecular mechanisms of the disease and facilitated the exploration of potential new therapeutic strategies. Here, we provide a detailed protocol that recapitulates some of the main histological and transcriptional features of human NEC in newborn mice.

A successful Neonatal–Perinatal Medicine fellowship (NPM-F) program requires presence and insight of national and institutional supervisory organizations as well as effective program-specific leaders: program director (PD), associate program director (APD), program coordinator (PC), and core faculty. It is becoming more common for PDs and APDs to have advanced training in medical education and conduct medical education research. While NPM-F program leaders benefit from a strong national NPM educator community, they face challenges of increased regulatory burden and unclear national guidelines with variable local interpretation for protected time. National and local organizations can support program leaders and promote their academic success while reducing burnout and turnover by providing leadership training, academic mentoring, and adequate protected time for research and program-specific tasks.

Neonatal necrotizing enterocolitis (NEC) is a devastating and unpredictable gastrointestinal disease with a high mortality rate in premature infants. Currently, no predictive or diagnostic biomarkers exist for NEC. Clinical intervention is reactive to the overt manifestations of disease resulting in high levels of morbidity and mortality. To better understand the molecular mechanisms that underpin NEC, we have undertaken a high resolution genome wide epigenomic analysis using solution phase hybridization and next generation DNA sequencing of bisulfite converted DNA. Our data reveal a broad and significant genomic hypermethylation in surgical NEC tissues compared to non-NEC controls. These changes were found to be far more pronounced in regions outside CpG islands and gene regulatory elements, which suggests that NEC-specific hypermethylation is not a non-specific global phenomenon. We identified a number of important biological pathways that are dysregulated in NEC and observed a clear association between NEC methylation changes and gene expression. Significantly, we found that the same patterns of global methylation identified in surgical NEC tissue are also detectable in stool samples from affected infants. To our knowledge, this is the first evidence of a methylomic signature that is both associated with NEC and detectable non-invasively. These findings point towards a new opportunity for the development of novel screening, diagnostic and phenotyping methods for NEC that could be deployed in the NICU for improved detection of this devastating disease.

Preterm birth affects more than 10% of all births worldwide. Such infants are much more prone to Growth Faltering (GF), an issue that has been unsolved despite the implementation of numerous interventions aimed at optimizing preterm infant nutrition. To improve the ability for early prediction of GF risk for preterm infants we collected a comprehensive, large, and unique clinical and microbiome dataset from 3 different sites in the US and the UK. We use and extend machine learning methods for GF prediction from clinical data. We next extend graphical models to integrate time series clinical and microbiome data. A model that integrates clinical and microbiome data improves on the ability to predict GF when compared to models using clinical data only. Information on a small subset of the taxa is enough to help improve model accuracy and to predict interventions that can improve outcome. We show that a hierarchical classifier that only uses a subset of the taxa for a subset of the infants is both the most accurate and cost-effective method for GF prediction. Further analysis of the best classifiers enables the prediction of interventions that can improve outcome.

Background Necrotizing enterocolitis (NEC) remains one of the overall leading causes of death in premature infants, and the pathogenesis is unpredictable and not well characterized. The aim of our study was to determine the molecular phenotype of NEC via transcriptomic and epithelial cell-specific epigenomic analysis, with a specific focus on DNA methylation. Methods Using laser capture microdissection, epithelial cell-specific methylation signatures were characterized by whole-genome bisulfite sequencing of ileal and colonic samples at the time of surgery for NEC and after NEC had healed at reanastomosis (n = 40). RNA sequencing was also performed to determine the transcriptomic profile of these samples, and a comparison was made to the methylome data. Results We found that surgical NEC has a considerable impact on the epigenome by broadly increasing DNA methylation levels, although these effects are less pronounced in genomic regions associated with the regulation of gene expression. Furthermore, NEC-related DNA methylation signatures were influenced by tissue of origin, with significant differences being noted between colon and ileum. We also identified numerous transcriptional changes in NEC and clear associations between gene expression and DNA methylation. Conclusions We have defined the intestinal epigenomic and transcriptomic signatures during surgical NEC, which will advance our understanding of disease pathogenesis and may enable the development of novel precision medicine approaches for NEC prediction, diagnosis and phenotyping.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in the neonatal ICU with minimal progress in the research.Federal webpages were queried to look for funding opportunity announcements (FOAs) and to develop lists of funded projects on NEC to identify gaps in NEC-related research topics.Over the past 30 years, the National Institutes of Health (NIH) issued two FOAs to stimulate research on NEC with $4.1 million set aside for the first year of respective funding. We identified 23 recently funded studies of which 18 were research projects, 4 training grants, and 1 conference grant support. Only one grant focused on parent and family engagement in the NICU.There are significant research gaps that can be addressed with adequate funding from the federal government on the prevention and treatment of NEC.

Preterm infants are a vulnerable population at risk of intestinal dysbiosis. The newborn microbiome is dominated by Bifidobacterium species, though abnormal microbial colonization can occur by exogenous factors such as mode of delivery, formula feeding, and exposure to antibiotics. Therefore, preterm infants are predisposed to sepsis and necrotizing enterocolitis (NEC), a fatal gastrointestinal disorder, due to an impaired intestinal barrier, immature immunity, and a dysbiotic gut microbiome. Properties of human milk serve as protection in the prevention of NEC. Human milk oligosaccharides (HMOs) and the microbiome of breast milk are immunomodulatory components that provide intestinal homeostasis through regulation of the microbiome and protection of the intestinal barrier. Enteral probiotic supplements have been trialed to evaluate their impact on establishing intestinal homeostasis. Here, we review the protective role of HMOs, probiotics, and synbiotic combinations in protecting a vulnerable population from the pathogenic features associated with necrotizing enterocolitis.

Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.

Author(s): Stanford, Amy H; Gong, Huiyu; Noonan, Mackenzie; Lewis, Angela N; Gong, Qingqing; Lanik, Wyatt E; Hsieh, Jonathan J; Lueschow, Shiloh R; Frey, Mark R; Good, Misty; McElroy, Steven J | Abstract: BackgroundPreterm infants are susceptible to unique pathology due to their immaturity. Mouse models are commonly used to study immature intestinal disease, including necrotizing enterocolitis (NEC). Current NEC models are performed at a variety of ages, but data directly comparing intestinal developmental stage equivalency between mice and humans are lacking.MethodsSmall intestines were harvested from C57BL/6 mice at 3-4 days intervals from birth to P28 (n = 8 at each age). Preterm human small intestine samples representing 17-23 weeks of completed gestation were obtained from the University of Pittsburgh Health Sciences Tissue Bank, and at term gestation during reanastamoses after resection for NEC (n = 4-7 at each age). Quantification of intestinal epithelial cell types and messenger RNA for marker genes were evaluated on both species.ResultsOverall, murine and human developmental trends over time are markedly similar. Murine intestine prior to P10 is most similar to human fetal intestine prior to viability. Murine intestine at P14 is most similar to human intestine at 22-23 weeks completed gestation, and P28 murine intestine is most similar to human term intestine.ConclusionUse of C57BL/6J mice to model the human immature intestine is reasonable, but the age of mouse chosen is a critical factor in model development.

Type 17 cytokines have been strongly implicated in mucosal immunity in part by regulating the production of antimicrobial peptides. Using a mouse model of C. rodentium infection which causes colitis, we found that intestinal IL-17RA and IL-17RC was partially required for control of infection in the colon and IL-17 regulates the production of luminal hydrogen peroxide as well as expression of Tnsf13. Reduced Tnfsf13 expression was associated with a profound defect in generating C. rodentium specific IgA+ antibody secreting cells. Taken together, intestinal IL-17R signaling plays key roles in controlling invading pathogens in part by regulating luminal hydrogen peroxide as well as regulating the generation of pathogen specific IgA+ antibody secreting cells.

Significance The gut-originating pathogen Escherichia coli has been associated with a portion of cases of late-onset sepsis (LOS), a leading cause of neonatal mortality. While it remains unclear how E. coli may gain access to the systemic circulation from the intestine, breast milk may protect against bacterial translocation and reduces the risk of LOS. Here we show a mechanism whereby gut-residing E. coli gain systemic access and have developed an animal model replicating this mechanism to explore the protective effects of breast milk in LOS., Late-onset sepsis (LOS) is a highly consequential complication of preterm birth and is defined by a positive blood culture obtained after 72 h of age. The causative bacteria can be found in patients’ intestinal tracts days before dissemination, and cohort studies suggest reduced LOS risk in breastfed preterm infants through unknown mechanisms. Reduced concentrations of epidermal growth factor (EGF) of maternal origin within the intestinal tract of mice correlated to the translocation of a gut-resident human pathogen Escherichia coli, which spreads systemically and caused a rapid, fatal disease in pups. Translocation of Escherichia coli was associated with the formation of colonic goblet cell-associated antigen passages (GAPs), which translocate enteric bacteria across the intestinal epithelium. Thus, maternally derived EGF, and potentially other EGFR ligands, prevents dissemination of a gut-resident pathogen by inhibiting goblet cell-mediated bacterial translocation. Through manipulation of maternally derived EGF and alteration of the earliest gut defenses, we have developed an animal model of pathogen dissemination which recapitulates gut-origin neonatal LOS.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using suspension and imaging mass cytometry coupled with single-cell RNA sequencing, we demonstrate severe inflammation in patients with NEC. NEC mucosa could be subtyped by an influx of three distinct neutrophil phenotypes (immature, newly emigrated, and aged). Furthermore, CD16+CD163+ monocytes/Mϕ, correlated with newly emigrated neutrophils, were specifically enriched in NEC mucosa, found adjacent to the blood vessels, and increased in circulation of infants with surgical NEC, suggesting trafficking from the periphery to areas of inflammation. NEC-specific monocytes/Mϕ transcribed inflammatory genes, including TREM1, IL1A, IL1B, and calprotectin, and neutrophil recruitment genes IL8, CXCL1, CXCL2, CXCL5 and had enrichment of gene sets in pathways involved in chemotaxis, migration, phagocytosis, and reactive oxygen species generation. In summary, we identify a novel subtype of inflammatory monocytes/Mϕ associated with NEC that should be further evaluated as a potential biomarker of surgical NEC and a target for the development of NEC-specific therapeutics.

Necrotizing enterocolitis (NEC) is a severe intestinal disease of premature infants with high mortality. Studies suggest a causative relationship between red blood cell (RBC) transfusion and NEC; however, whether RBC transfusion leads to worse outcomes in NEC is unknown. We sought to determine whether RBC transfusion was associated with an increased risk of surgical NEC and mortality.In this retrospective study, 115 patients were enrolled with NEC Bell's stage 2A or greater from 2010-2015. Patients were classified based on the timing of RBC transfusion before NEC: ≤72 h,72 h, and no transfusion. Variables including gestational age (GA), birth weight (BW), feedings, and hematocrit levels were analyzed. Outcomes were surgical intervention for NEC following RBC transfusion and mortality.Twenty-three (20%) infants developed NEC ≤ 72 h after RBC transfusion, 16 (69.6%) required surgery with a mortality rate of 21.7% (n = 5). Seventeen (15%) infants developed NEC 72 h after RBC transfusion, 12 (70.6%) required surgery with a mortality rate of 23.5% (n = 4). 75 (65%) patients developed NEC without RBC transfusion, 17 (22.7%) required surgery with a mortality rate of 4% (n = 3). Lower GA and BW were significantly associated with RBC transfusion and the need for surgical intervention. RBC transfusion ≤72 h before NEC was associated with surgical NEC (pairwise adjusted P 0.001) and mortality (pairwise adjusted P = 0.048). However, multivariable logistic regression analysis revealed RBC transfusion is not an independent risk factor for surgical NEC.Infants of lower GA and BW were more likely to receive an RBC transfusion before NEC, which was significantly associated with surgical intervention and an increasing risk of mortality. Judicious use of transfusions in premature infants may improve NEC outcomes.

Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal disease of the premature infant. We have recently shown that NEC development occurs after an increase in proinflammatory CD4+Th17 (Th17) cells and reduced anti-inflammatory Foxp3+ regulatory T cells (Tregs) to the premature small intestine of mice and humans, which can be experimentally reversed in mice by administration of all-trans retinoic acid (ATRA). We have also shown that NEC is characterized by apoptosis of Lgr5-positive intestinal stem cells (ISCs–Lgr5+cells) within the crypts of Lieberkühn, which are subsequently essential for intestinal homeostasis. We now hypothesize that the normal lymphocyte balance within the lamina propria of the intestine can be achieved via administration of ATRA which restores mucosal integrity by preventing the loss of intestinal stem cells. Utilizing both in vivo and in vitro strategies we now demonstrate that Th17 recruitment and Treg depletion lead to increased apoptosis within ISC niches, significantly impairing proliferative capacity and mucosal healing. ATRA exerted its protective effects by preventing T cell imbalance, ultimately leading to the protection of the ISC pool preventing the development of NEC in mice. These findings raise the exciting possibility that dietary manipulations could prevent and treat NEC by modulating lymphocyte balance and the ISC pool within the newborn small intestine.

Key Points Question Unlike candidate biomarkers inclusive for all forms of systemic inflammation, can dysfunction in host management of microbiota have a high positive predictive value as a biomarker for necrotizing enterocolitis? Findings In this diagnostic study of 136 premature infants, high amounts of intestinal alkaline phosphatase protein in stool and low intestinal alkaline phosphatase enzyme activity were associated with diagnosis of necrotizing enterocolitis. There was no association of intestinal alkaline phosphatase measures with non–gastrointestinal tract infections. Meaning Measuring the inability of intestinal alkaline phosphatase to maintain host-microbiota homeostasis can potentially guide decisions for personalized care and treatment when an infant is most susceptible to developing necrotizing enterocolitis., This diagnostic study evaluates whether aberrant intestinal alkaline phosphatase biochemistry in infant stool is a molecular biomarker for necrotizing enterocolitis and not associated with sepsis in the absence of necrotizing enterocolitis., Importance Necrotizing enterocolitis (NEC) in preterm infants is an often-fatal gastrointestinal tract emergency. A robust NEC biomarker that is not confounded by sepsis could improve bedside management, lead to lower morbidity and mortality, and permit patient selection in randomized clinical trials of possible therapeutic approaches. Objective To evaluate whether aberrant intestinal alkaline phosphatase (IAP) biochemistry in infant stool is a molecular biomarker for NEC and not associated with sepsis. Design, Setting, and Participants This multicenter diagnostic study enrolled 136 premature infants (gestational age

IL-17A and IL-22 derived from Th17 cells play a significant role in mucosal immunity and inflammation. TGF-β and IL-6 promote Th17 differentiation; however, these cytokines have multiple targets. The identification and screening of additional molecules that regulate IL-17A and IL-22 responses in certain inflammatory conditions is of great clinical significance. In this study, we show that CDDO-Im, a specific Nrf2 activator, promotes IL-17A and IL-22 responses in murine Th17 cells. In contrast, CDDO-Im inhibits IL-17A response in multiple sclerosis patient-derived PBMCs. However, Nrf2 specifically regulates IL-22 response in vivo. Nrf2 acts through the regulation of antioxidant response element (ARE) binding motifs in target genes to induce or repress transcription. Promoter analysis revealed that Il17a, Rorc, and Ahr genes have several ARE motifs. We showed that Nrf2 bound to ARE repressor (ARE-R2) of Rorc and inhibited Rorc-dependent IL-17A transactivation. The luciferase reporter assay data showed that CDDO-Im regulated Ahr promoter activity. Chromatin immunoprecipitation quantitative PCR data showed that Nrf2 bound to ARE of AhR. Finally, we confirmed that the CDDO-Im–mediated induction of IL-22 production in CD4+ T cells was abrogated in CD4-specific Ahr knockout mice (AhrCD4). CH-223191, a specific AhR antagonist, inhibits CDDO-Im–induced IL-22 production in CD4+ T cells, which further confirmed the AhR-dependent regulation. Collectively, our data showed that Nrf2 via AhR pathways regulated IL-22 response in CD4+ T cells.

Necrotizing enterocolitis (NEC) remains a leading cause of preterm infant mortality. NEC is multifactorial and believed a consequence of intestinal immaturity, microbial dysbiosis, and an exuberant inflammatory response. Over the past decade, exaggerated Toll-like receptor 4 (TLR4) activity in the immature intestine of preterm neonates emerged as an inciting event preceding NEC. Increased TLR4 signaling in epithelial cells results in the initiation of an uncontrolled immune response and destruction of the mucosal barrier. This article discusses the state of the science of the molecular mechanisms involved in TLR4-mediated inflammation during NEC and the development of new therapeutic strategies to prevent NEC.

We seek to define the mechanisms leading to the development of lung disease in the setting of neonatal necrotizing enterocolitis (NEC), a life-threatening gastrointestinal disease of premature infants characterized by the sudden onset of intestinal necrosis. NEC development in mice requires activation of the LPS receptor TLR4 on the intestinal epithelium, through its effects on modulating epithelial injury and repair. Although NEC-associated lung injury is more severe than the lung injury that occurs in premature infants without NEC, the mechanisms leading to its development remain unknown. In this study, we now show that TLR4 expression in the lung gradually increases during postnatal development, and that mice and humans with NEC-associated lung inflammation express higher levels of pulmonary TLR4 than do age-matched controls. NEC in wild-type newborn mice resulted in significant pulmonary injury that was prevented by deletion of TLR4 from the pulmonary epithelium, indicating a role for pulmonary TLR4 in lung injury development. Mechanistically, intestinal epithelial TLR4 activation induced high-mobility group box 1 release from the intestine, which activated pulmonary epithelial TLR4, leading to the induction of the neutrophil recruiting CXCL5 and the influx of proinflammatory neutrophils to the lung. Strikingly, the aerosolized administration of a novel carbohydrate TLR4 inhibitor prevented CXCL5 upregulation and blocked NEC-induced lung injury in mice. These findings illustrate the critical role of pulmonary TLR4 in the development of NEC-associated lung injury, and they suggest that inhibition of this innate immune receptor in the neonatal lung may prevent this devastating complication of NEC.

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout ( Il22Ra1 Hep−/− ) and Stat3 knockout ( Stat3 Hep −/− ) mice were generated and subjected to intra-abdominal infection with Klebsiella pneumoniae , which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3 . Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae , which was dependent on lipocalin 2 (LCN2). However, in vivo , rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti- K. pneumoniae bactericidal activity in vitro . These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections.

The type 17 cytokine receptors are thought to play critical roles in mucosal immunity. We have shown that intestinal epithelial expression of IL–17 receptor control the development of microbiota in the terminal ileum and conditional deletion of these receptors results in overgrowth of segmented filamentous bacteria. To study the roles of these receptors in response to an attaching and effacing pathogen we challenged intestinal specific Il17ra mice and Il17rc mice with Citrobacter rodentium (C. rodentium). Conditional deletion of Il17ra and l17rc Iresulted in increased bacterial growth in the colon as well as enhanced dissemination to the liver suggesting that intestinal epithelial IL–17 receptors expression is required for control of this infection. This was supported by unbiased RNAseq analysis of the colon that showed diminished expression of Pigr, Tnfsf13 and Nox1 in the absence of Il17ra. IgA has crucial role in mucosal immunity to protect from pathogenic bacteria. PIGR binds dimeric IgA and translocation into lumen, while TNFSF13 activates induce differentiation to plasma cells following–crass switch. C. rodentium specific IgA in stool and C. rodentium specific IgA producing cells in lamina propria were reduced in Il17ra mice. These results suggested IL-17 receptor signaling regulates transcytosis of C. rodentium specific IgA as well as secretion of bacteria specific IgA. On the other hands Nox1 can produce apical hydrogen peroxide. Nox1 null mice were more sensitive for C. rodentium infection. Furthermore Nox1 was expressed by IL–17A stimulation in colonic organoid. Taken together, IL–17 receptor signaling control to produce C. rodentium specific IgA through PIGR and TNFSF13 as well as hydrogen peroxide through NOX1.

Author(s): Good, Misty; McElroy, Steven J; Berger, Jennifer N; Moore, Daniel J; Wynn, James L

Breast milk contains immunomodulating components that are beneficial to newborns during maturation of their immune system. Human breast milk composition is influenced by an infant’s gestational and chronological age, lactation stage, and the mother and infant’s health status. Major immunologic components in human milk, such as secretory immunoglobulin A (IgA) and growth factors, have a known role in regulating gut barrier integrity and microbial colonization, which therefore protect against the development of a life-threatening gastrointestinal illness affecting newborn infants called necrotizing enterocolitis (NEC). Breast milk is a known protective factor in the prevention of NEC when compared with feeding with commercial formula. Breast milk supplements infants with human milk oligosaccharides, leukocytes, cytokines, nitric oxide, and growth factors that attenuate inflammatory responses and provide immunological defenses to reduce the incidence of NEC. This article aims to review the variety of immunomodulating components in breast milk that protect the infant from the development of NEC.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

This study describes the number and characteristics of physician-scientists who are funded by National Institutes of Health independent investigator awards.

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.