Paneth cell ontogeny in term and preterm ovine models.

Introduction: Paneth cells are critically important to intestinal health, including protecting intestinal stem cells, shaping the intestinal microbiome, and regulating host immunity. Understanding Paneth cell biology in the immature intestine is often modeled in rodents with little information in larger mammals such as sheep. Previous studies have only established the distribution pattern of Paneth cells in healthy adult sheep. Our study aimed to examine the ontogeny, quantification, and localization of Paneth cells in fetal and newborn lambs at different gestational ages and with perinatal transient asphyxia. We hypothesized that ovine Paneth cell distribution at birth resembles the pattern seen in humans (highest concentrations in the ileum) and that ovine Paneth cell density is gestation-dependent. Methods: Intestinal samples were obtained from 126-127 (preterm, with and without perinatal transient asphyxia) and 140-141 (term) days gestation sheep. Samples were quantified per crypt in at least 100 crypts per animal and confirmed as Paneth cells through in immunohistochemistry. Results: Paneth cells had significantly higher density in the ileum compared to the jejunum and were absent in the colon. Discussion: Exposure to perinatal transient asphyxia acutely decreased Paneth cell numbers. These novel data support the possibility of utilizing ovine models for understanding Paneth cell biology in the fetus and neonate.

State of the art review on machine learning and artificial intelligence in the study of neonatal necrotizing enterocolitis.

Necrotizing Enterocolitis (NEC) is one of the leading causes of gastrointestinal emergency in preterm infants. Although NEC was formally described in the 1960's, there is still difficulty in diagnosis and ultimately treatment for NEC due in part to the multifactorial nature of the disease. Artificial intelligence (AI) and machine learning (ML) techniques have been applied by healthcare researchers over the past 30 years to better understand various diseases. Specifically, NEC researchers have used AI and ML to predict NEC diagnosis, NEC prognosis, discover biomarkers, and evaluate treatment strategies. In this review, we discuss AI and ML techniques, the current literature that has applied AI and ML to NEC, and some of the limitations in the field.

The fetal response to maternal inflammation is dependent upon maternal IL-6 in a murine model.

BACKGROUND: The induction of maternal inflammation in mice leads to fetal injury that is believed to be IL-6 dependent. The fetal inflammatory response, defined by elevated fetal or amniotic fluid IL-6, has been described as a potential mechanism for subsequent fetal injury. The role of maternal IL-6 production and signaling in the fetal IL-6 response is currently unclear. METHODS: Genetic and anti-IL-6 antibody strategies were used to systematically block the maternal IL-6 response during inflammation. Chorioamnionitis was induced using intraperitoneal injection of lipopolysaccharide (LPS) at mid gestation (E14.5) and late gestation (E18.5). This model was used in pregnant C57Bl/6 dams, IL6-/- dams, C57Bl/6 dams treated with anti-IL-6 (blocks both classical and trans-signaling) or anti-gp130 antibodies (blocks trans-signaling only) and IL6+/- dams. Six hours following LPS injection, maternal serum, placental tissue, amniotic fluid and fetal tissue or serum were collected. A bead-based multiplex assay was used to evaluate levels of IL-6, KC, IL-1ß, TNF, IL-10, IL-22, IFN-γ, IL-13 and IL-17A. RESULTS: Chorioamnionitis in C57Bl/6 dams was characterized by elevated maternal serum levels of IL-6, KC and IL-22 with litter loss during mid gestation. The fetal response to maternal inflammation in C57Bl/6 mice was primarily characterized by elevated levels of IL-6, KC and IL-22 in the placenta, amniotic fluid and fetus during both mid and late gestation. A global IL-6 knockout (IL6-/-) eradicated the maternal, placental, amniotic fluid and fetal IL-6 response to LPS during mid and late gestation and improved litter survival, while minimally influencing the KC or IL-22 responses. Blocking maternal classical IL-6 signaling in C57Bl/6 dams at the time of LPS exposure diminished the maternal, placental, amniotic fluid and fetal IL-6 response during mid and late gestation, while blocking maternal IL-6 trans-signaling only affected fetal IL-6 expression. To evaluate whether maternal IL-6 was crossing the placenta and reaching the fetus, IL-6+/- dams were utilized in the chorioamnionitis model. IL-6+/- dams mounted a systemic inflammatory response following injection with LPS, characterized by elevated IL-6, KC and IL-22. IL-6-/- pups born to IL6+/- dams had decreased amniotic fluid levels of IL-6 and undetectable levels of fetal IL-6 compared to IL-6+/+ littermate controls. CONCLUSION: The fetal response to systemic maternal inflammation is dependent upon maternal IL-6 signaling, but maternal IL-6 is not crossing the placenta and reaching the fetus at detectable levels.

State-of-the-art review and update of in vivo models of necrotizing enterocolitis.

NEC remains one of the most common causes of mortality and morbidity in preterm infants. Animal models of necrotizing enterocolitis (NEC) have been crucial in improving our understanding of this devastating disease and identifying biochemical pathways with therapeutic potential. The pathogenesis of NEC remains incompletely understood, with no specific entity that unifies all infants that develop NEC. Therefore, investigators rely on animal models to manipulate variables and provide a means to test interventions, making them valuable tools to enhance our understanding and prevent and treat NEC. The advancements in molecular analytic tools, genetic manipulation, and imaging modalities and the emergence of scientific collaborations have given rise to unique perspectives and disease correlates, creating novel pathways of investigation. A critical review and understanding of the current phenotypic considerations of the highly relevant animal models of NEC are crucial to developing novel therapeutic and preventative strategies for NEC.

Hyaluronic Acid 35 kDa Protects against a Hyperosmotic, Formula Feeding Model of Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC), an inflammatory disease of the intestine, is a common gastrointestinal emergency among preterm infants. Intestinal barrier dysfunction, hyperactivation of the premature immune system, and dysbiosis are thought to play major roles in the disease. Human milk (HM) is protective, but the mechanisms underpinning formula feeding as a risk factor in the development of NEC are incompletely understood. Hyaluronic acid 35 kDa (HA35), a bioactive glycosaminoglycan of HM, accelerates intestinal development in murine pups during homeostasis. In addition, HA35 prevents inflammation-induced tissue damage in pups subjected to murine NEC, incorporating Paneth cell dysfunction and dysbiosis. We hypothesized HA35 treatment would reduce histological injury and mortality in a secondary mouse model of NEC incorporating formula feeding. NEC-like injury was induced in 14-day mice by dithizone-induced disruption of Paneth cells and oral gavage of rodent milk substitute. Mortality and histological injury, serum and tissue cytokine levels, stool bacterial sequencing, and bulk RNA-Seq comparisons were analyzed. HA35 significantly reduced the severity of illness in this model, with a trend toward reduced mortality, while RNA-Seq analysis demonstrated HA35 upregulated genes associated with goblet cell function and innate immunity. Activation of these critical protective and reparative mechanisms of the small intestine likely play a role in the reduced pathology and enhanced survival trends of HA-treated pups subjected to intestinal inflammation in this secondary model of NEC, providing potentially interesting translational targets for the human preterm disease.

Early Antibiotic Exposure Alters Intestinal Development and Increases Susceptibility to Necrotizing Enterocolitis: A Mechanistic Study.

Increasing evidence suggests that prolonged antibiotic therapy in preterm infants is associated with increased mortality and morbidities, such as necrotizing enterocolitis (NEC), a devastating gastrointestinal pathology characterized by intestinal inflammation and necrosis. While a clinical correlation exists between antibiotic use and the development of NEC, the potential causality of antibiotics in NEC development has not yet been demonstrated. Here, we tested the effects of systemic standard-of-care antibiotic therapy for ten days on intestinal development in neonatal mice. Systemic antibiotic treatment impaired the intestinal development by reducing intestinal cell proliferation, villi height, crypt depth, and goblet and Paneth cell numbers. Oral bacterial challenge in pups who received antibiotics resulted in NEC-like intestinal injury in more than half the pups, likely due to a reduction in mucous-producing cells affecting microbial-epithelial interactions. These data support a novel mechanism that could explain why preterm infants exposed to prolonged antibiotics after birth have a higher incidence of NEC and other gastrointestinal disorders.

Bifidobacterium longum Subspecies infantis Strain EVC001 Decreases Neonatal Murine Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a disease mainly of preterm infants with a 30-50% mortality rate and long-term morbidities for survivors. Treatment strategies are limited and have not improved in decades, prompting research into prevention strategies, particularly with probiotics. Recent work with the probiotic B. infantis EVC001 suggests that this organism may generate a more appropriate microbiome for preterm infants who generally have inappropriate gut colonization and inflammation, both risk factors for NEC. Experimental NEC involving Paneth cell disruption in combination with bacterial dysbiosis or formula feeding was induced in P14-16 C57Bl/6 mice with or without gavaged B. infantis. Following completion of the model, serum, small intestinal tissue, the cecum, and colon were harvested to examine inflammatory cytokines, injury, and the microbiome, respectively. EVC001 treatment significantly decreased NEC in a bacterial dysbiosis dependent model, but this decrease was model-dependent. In the NEC model dependent on formula feeding, no difference in injury was observed, but trending to significant differences was observed in serum cytokines. EVC001 also improved wound closure at six and twelve hours compared to the sham control in intestinal epithelial monolayers. These findings suggest that B. infantis EVC001 can prevent experimental NEC through anti-inflammatory and epithelial barrier restoration properties.

A critical evaluation of current definitions of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants, with significant mortality and long-term morbidity among survivors. Multiple NEC definitions exist, but no formal head-to-head evaluation has been performed. We hypothesized that contemporary definitions would perform better in evaluation metrics than Bell's and range features would be more frequently identified as important than yes/no features. METHODS: Two hundred and nineteen patients from the University of Iowa hospital with NEC, intestinal perforation, or NEC concern were identified from a 10-year retrospective cohort. NEC presence was confirmed by a blinded investigator. Evaluation metrics were calculated using statistics and six supervised machine learning classifiers for current NEC definitions. Feature importance evaluation was performed on each decision tree classifier. RESULTS: Newer definitions outperformed Bell's staging using both standard statistics and most machine learning classifiers. The decision tree classifier had the highest overall machine learning scores, which resulted in Non-Bell definitions having high sensitivity (0.826, INC) and specificity (0.969, ST), while Modified Bell (IIA+) had reasonable sensitivity (0.783), but poor specificity (0.531). Feature importance evaluation identified nine criteria as important for diagnosis. CONCLUSIONS: This preliminary study suggests that Non-Bell NEC definitions may be better at diagnosing NEC and calls for further examination of definitions and important criteria. IMPACT: This article is the first formal head-to-head evaluation of current available definitions of NEC. Non-Bell NEC definitions may be more effective in identifying NEC based on findings from traditional measures of diagnostic performance and machine learning techniques. Nine features were identified as important for diagnosis from the definitions evaluated within the decision tree when performing supervised classification machine learning. This article serves as a preliminary study to formally evaluate the definitions of NEC utilized and should be expounded upon with a larger and more diverse patient cohort.

Routine Administration of a Multispecies Probiotic Containing Bifidobacterium and Lactobacillus to Very Low Birth Weight Infants Had No Significant Impact on the Incidence of Necrotizing Enterocolitis.

Background: Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity in preterm infants, and prevention and treatment strategies have remained largely unchanged over the past several decades. As understanding of the microbiome has increased, probiotics have been hypothesized as a possible strategy for decreasing rates of NEC, and several studies have noted significant decreases in rates of NEC after initiation of probiotics in preterm infants. However, a recent AAP report cited caution on the use of probiotic use in part because studies of probiotic use in ELBW infants are lacking. As our unit began routine use of probiotics for all infants <33 weeks in 2015 and we are a leading institution for intact survival of ELBW infants, we attempted to answer if probiotic use can impact the rate of NEC in VLBW and ELBW infants. Methods: We conducted a single-center retrospective chart review of infants with modified Bell's stage ≥2a NEC for the 4 years prior to and 5 years after initiation of a protocol involving routine supplementation of a multispecies probiotic to premature infants at the University of Iowa, Stead Family Children's Hospital. The primary outcome measures were rates of modified Bell's stage ≥2a NEC and all-cause pre-discharge mortality at our institution before and after initiation of routine probiotic supplementation in 2015. Results: In our institution, neither the rates of modified Bell's stage ≥2a NEC, nor the rates of all-cause mortality were significantly altered in very low birth weight (VLBW) infants by the initiation of routine probiotic use (NEC rates pre-probiotic 2.1% vs. post-probiotic 1.5%; all-cause mortality rates pre-probiotic 8.4% vs. post-probiotic 7.4%). Characteristics of our two cohorts were overall similar except for a significantly lower 5-minute APGAR score in infants in the post-probiotic epoch (pre-probiotic 8 vs. post-probiotic 6 p = 0.0316), and significantly more infants in the post-probiotic epoch received probiotics (pre-probiotics 0% vs. post-probiotics 65%; p < 0.0001). Similarly, probiotic use had no impact on the incidence of NEC when we restricted our data to only extremely low birth weight (ELBW) infants (pre-probiotics 1.6% vs post-probiotics 4.1%). When we restricted our analysis to only inborn infants, probiotics still had no impact on NEC rates in VLBW infants (1.5% pre- and 1.1% post-probiotic, p = 0.61) or ELBW infants (2% pre- and 2.1% post-probiotic, p = 0.99) Conclusions: Contrary to other studies, we found no significant difference in rates of modified Bell's stage ≥2a NEC or all-cause pre-discharge mortality in VLBW infants following routine administration of a multispecies probiotic supplement.

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types.

MicroRNA-mediated regulation is critical for the proper development and function of the small intestinal (SI) epithelium. However, it is not known which microRNAs are expressed in each of the cell types of the SI epithelium. To bridge this important knowledge gap, we performed comprehensive microRNA profiling in all major cell types of the mouse SI epithelium. We used flow cytometry and fluorescence-activated cell sorting with multiple reporter mouse models to isolate intestinal stem cells, enterocytes, goblet cells, Paneth cells, enteroendocrine cells, tuft cells, and secretory progenitors. We then subjected these cell populations to small RNA-sequencing. The resulting atlas revealed highly enriched microRNA markers for almost every major cell type (https://sethupathy-lab.shinyapps.io/SI_miRNA/). Several of these lineage-enriched microRNAs (LEMs) were observed to be embedded in annotated host genes. We used chromatin-run-on sequencing to determine which of these LEMs are likely cotranscribed with their host genes. We then performed single-cell RNA-sequencing to define the cell type specificity of the host genes and embedded LEMs. We observed that the two most enriched microRNAs in secretory progenitors are miR-1224 and miR-672, the latter of which we found is deleted in hominin species. Finally, using several in vivo models, we established that miR-152 is a Paneth cell-specific microRNA.NEW & NOTEWORTHY In this study, first, microRNA atlas (and searchable web server) across all major small intestinal epithelial cell types is presented. We have demonstrated microRNAs that uniquely mark several lineages, including enteroendocrine and tuft. Identification of a key marker of mouse secretory progenitor cells, miR-672, which we show is deleted in humans. We have used several in vivo models to establish miR-152 as a specific marker of Paneth cells, which are highly understudied in terms of microRNAs.

Toll-like receptor 4 and myeloid differentiation factor 88 are required for gastric bypass-induced metabolic effects.

BACKGROUND: Toll-like receptor 4 (TLR4) has been suggested as one of the forefront cross-communicators between the intestinal bacteria and the host to regulate inflammatory signals and energy homeostasis. High-fat diet-induced inflammation is mediated by changes in gut microbiota and requires a functional TLR-4, the deficiency of which renders mice resistant to diet-induced obesity and its associated metabolic dysfunction. Furthermore, gut microbiota was suggested to play a key role in the beneficial effects of Roux-en-Y gastric bypass (RYGB), a commonly performed bariatric procedure. OBJECTIVES: To explore whether TLR4, myeloid differentiation factor 8 (MyD88; 1 of its key downstream signaling regulators) and gut microbiota play an integrative role in RYGB-induced metabolic outcomes. SETTING: Animal- based study. METHOD: We performed RYGB in TLR4 and MyD88 knock-out (KO) mice and used fecal microbiota transplant (FMT) from RYGB-operated animals to these genetic mouse models to address our questions. RESULTS: We demonstrate that RYGB reduces TLR4 expression explicitly in the small and large intestine of C57Blc/6J mice. We also show that TLR4 KO mice have an attenuated glucoregulatory response to RYGB. In addition, we reveal that MyD88 KO mice fail to respond to all RYGB-induced metabolic effects. Finally, fecal microbiota transplant from RYGB-operated mice into TLR4 KO and MyD88 KO naïve recipients fails to induce a metabolic phenotype similar to that of the donors, as it does in wild-type recipients. CONCLUSION: TLR4 and MyD88 are required for RYGB-induced metabolic response that is likely mediated by gut microbiome.

Acceleration of Small Intestine Development and Remodeling of the Microbiome Following Hyaluronan 35 kDa Treatment in Neonatal Mice.

The beneficial effects of human milk suppressing the development of intestinal pathologies such as necrotizing enterocolitis in preterm infants are widely known. Human milk (HM) is rich in a multitude of bioactive factors that play major roles in promoting postnatal maturation, differentiation, and the development of the microbiome. Previous studies showed that HM is rich in hyaluronan (HA) especially in colostrum and early milk. This study aims to determine the role of HA 35 KDa, a HM HA mimic, on intestinal proliferation, differentiation, and the development of the intestinal microbiome. We show that oral HA 35 KDa supplementation for 7 days in mouse pups leads to increased villus length and crypt depth, and increased goblet and Paneth cells, compared to controls. We also show that HA 35 KDa leads to an increased predominance of Clostridiales Ruminococcaceae, Lactobacillales Lactobacillaceae, and Clostridiales Lachnospiraceae. In seeking the mechanisms involved in the changes, bulk RNA seq was performed on samples from the terminal ileum and identified upregulation in several genes essential for cellular growth, proliferation, and survival. Taken together, this study shows that HA 35 KDa supplemented to mouse pups promotes intestinal epithelial cell proliferation, as well as the development of Paneth cells and goblet cell subsets. HA 35 KDa also impacted the intestinal microbiota; the implications of these responses need to be determined.

Potential Prenatal Origins of Necrotizing Enterocolitis.

Necrotizing enterocolitis is a serious and yet incompletely understood gastrointestinal disease of infancy that predominately impacts premature neonates. Prevention is a key strategy for the management of necrotizing enterocolitis. Although postnatal risk factors have been the focus of prevention efforts, obstetric complications, including intrauterine inflammation and infection, growth restriction, preeclampsia, and prenatal medications, have been associated with an increased risk of necrotizing enterocolitis. This article reviews the evidence behind the prenatal risk factors for necrotizing enterocolitis, and discusses how these risk factors may elucidate the pathogenesis of necrotizing enterocolitis and provide insight into prevention and treatment.

Defining necrotizing enterocolitis: current difficulties and future opportunities.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in hospitalized infants. First classified through Bell staging in 1978, a number of additional definitions of NEC have been proposed in the subsequent decades. In this review, we summarize eight current definitions of NEC, and explore similarities and differences in clinical signs and radiographic features included within these definitions, as well as their limitations. We highlight the importance of a global consensus on defining NEC to improve NEC research and outcomes, incorporating input from participants at an international NEC conference. We also highlight the important role of patient-families in helping to redefine NEC.

Hope on the horizon: promising novel therapies for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains among the most common and devastating diseases in neonates. Despite advances in neonatal clinical care, specific treatment strategies and diagnostic modalities remain lacking. As a result, morbidity and mortality remain high. Improved understanding of the pathogenesis of NEC has the potential for improved therapeutics. Some of the areas of research leading to promising discoveries include inhibition of Toll-like receptor signaling, modulation of vascular endothelial growth factor signal pathways, defining metabolomic alterations in NEC to discover potential biomarkers, probing for genetic predispositions to NEC susceptibility, determining mechanistic relations between anemia and NEC, and microflora modulation through the use of probiotics. All of these areas may represent novel promising approaches to the prevention and treatment of NEC. This review will focus on these current and possible therapeutic perspectives.

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine.

Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.

Feeding Formula Eliminates the Necessity of Bacterial Dysbiosis and Induces Inflammation and Injury in the Paneth Cell Disruption Murine NEC Model in an Osmolality-Dependent Manner.

Necrotizing enterocolitis (NEC) remains a significant cause of morbidity and mortality in preterm infants. Formula feeding is a risk factor for NEC and osmolality, which is increased by the fortification that is required for adequate growth of the infant, has been suggested as a potential cause. Our laboratory has shown that Paneth cell disruption followed by induction of dysbiosis can induce NEC-like pathology in the absence of feeds. We hypothesized adding formula feeds to the model would exacerbate intestinal injury and inflammation in an osmolality-dependent manner. NEC-like injury was induced in 14-16 day-old C57Bl/6J mice by Paneth cell disruption with dithizone or diphtheria toxin, followed by feeding rodent milk substitute with varying osmolality (250-1491 mOsm/kg H2O). Animal weight, serum cytokines and osmolality, small intestinal injury, and cecal microbial composition were quantified. Paneth cell-disrupted mice fed formula had significant NEC scores compared to controls and no longer required induction of bacterial dysbiosis. Significant increases in serum inflammatory markers, small intestinal damage, and overall mortality were osmolality-dependent and not related to microbial changes. Overall, formula feeding in combination with Paneth cell disruption induced NEC-like injury in an osmolality-dependent manner, emphasizing the importance of vigilance in designing preterm infant feeds.