PROJECT SUMMARY Necrotizing enterocolitis (NEC) is a life-threatening, gastrointestinal emergency affecting 7-10% of preterm infants with mortality as high as 30-50%. "Leaky gut", or intestinal barrier immaturity with elevated intestinal permeability (IP), is the proximate cause of susceptibility to NEC in preterm neonates. Early detection of leaky gut is essential to identify infants at risk for NEC to reduce disease severity. No clinical factor, routine laboratory test or biomarker alone or in combination have been described that identify newborns susceptible to develop NEC. We postulate that a mucosal immune phenotype detectable in fecal extracts, is reflective of the intestinal microenvironment and postnatal barrier maturation and can distinguish infants at high risk for developing NEC. The goal of this study is to investigate the diagnostic value of fecal immune biomarkers to identify “at-risk” neonates with leaky gut prior to NEC onset. When used alone or in combination with other IP-associated neonatal factors (i.e., postmenstrual age, feeding, microbiota), the immune phenotype can provide the basis of a non- invasive, stool-based method to detect neonatal leaky gut. To achieve this goal, we will leverage existing stool samples collected from a cohort of 205 preterms (<33 weeks gestation), from whom both gut microbiome (16S rRNA gene sequencing and metagenome) and IP (urine non-metabolized sugar probes lactulose and rhamnose) are known. Two aims are proposed: 1) to conduct an in-depth characterization of cytokines and chemokines in fecal extracts of preterm infants during the first 7-10 days of life, and to determine the diagnostic value of fecal immune biomarkers in identification of aberrantly elevated IP as a major risk factor for NEC; 2) to define longitudinal changes of fecal immune biomarkers associated with improved or persistently elevated IP. At the completion of this study, we will have identified a discriminatory mucosal immune phenotype representing a novel, non-invasive stool-based diagnostic/screening tool to identify preterm newborn “at risk” for developing NEC. This study will also provide new insights into the interplay between host immunity and gut microbiota dysbiosis that contributes to heightened barrier injury in early life. These results will facilitate clinical studies to evaluate rationally designed interventions for early NEC prevention and to promote healthy intestinal barrier functions and newborn health.