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Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of prematurity. Postulated mechanisms leading to inflammatory necrosis of the ileum and colon include activation of the pathogen recognition receptor Toll-like receptor 4 (TLR4) and decreased levels of transforming growth factor beta (TGFβ). Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a novel damage-associated molecular pattern (DAMP), is a TLR4 ligand and plays a role in a number of inflammatory disease processes. To test the hypothesis that eNAMPT is involved in NEC, an eNAMPT-neutralizing monoclonal antibody, ALT-100, was used in a well-established animal model of NEC. Preterm Sprague–Dawley pups delivered prematurely from timed-pregnant dams were exposed to hypoxia/hypothermia and randomized to control—foster mother dam-fed rats, injected IP with saline (vehicle) 48 h after delivery; control + mAB—foster dam-fed rats, injected IP with 10 µg of ALT-100 at 48 h post-delivery; NEC—orally gavaged, formula-fed rats injected with saline; and NEC + mAb—formula-fed rats, injected IP with 10 µg of ALT-100 at 48 h. The distal ileum was processed 96 h after C-section delivery for histological, biochemical, molecular, and RNA sequencing studies. Saline-treated NEC pups exhibited markedly increased fecal blood and histologic ileal damage compared to controls (q < 0.0001), and findings significantly reduced in ALT-100 mAb-treated NEC pups (q < 0.01). Real-time PCR in ileal tissues revealed increased NAMPT in NEC pups compared to pups that received the ALT-100 mAb (p < 0.01). Elevated serum levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), interleukin-8 (IL-8), and NAMPT were observed in NEC pups compared to NEC + mAb pups (p < 0.01). Finally, RNA-Seq confirmed dysregulated TGFβ and TLR4 signaling pathways in NEC pups that were attenuated by ALT-100 mAb treatment. These data strongly support the involvement of eNAMPT in NEC pathobiology and eNAMPT neutralization as a strategy to address the unmet need for NEC therapeutics. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin [E.sub.2] ([PGE.sub.2]). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum). Intestinal Toll-like receptor-2 (TLR-2), COX-2, [PGE.sub.2], and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and [PGE.sub.2] and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum-treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum. Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of [PGE.sub.2] in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity. epithelial homeostasis; enteral nutrition; mucosal inflammation; probiotics doi: 10.1152/ajpgi.00131.2010.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. Previously, we showed that luminal bile acids (BAs) are increased and correlated with disease development and that the apical sodium-dependent BA transporter (ASBT), which transports BAs from the ileal lumen into enterocytes, is upregulated in rats with NEC. We hypothesized that intraenterocyte, rather than luminal, BAs are associated with NEC and that upregulation of ASBT may be a mechanism by which this occurs. Neonatal rats with or without the ASBT inhibitor SC-435, mice in which ASBT was knocked out, and mice that overproduce BAs were subjected to the NEC protocol. Disease development, ASBT, and the farnesoid X receptor protein, along with luminal and intraenterocyte BA levels, were assessed. In addition, ileal sections from premature infants with and without NEC were examined for ASBT via immunohistology and real-time PCR. When BAs were not transported into enterocytes (rats given SC-435 and ASBT knockout mice), severity and incidence of NEC were reduced. In contrast, in mice that overproduce BAs, ASBT was elevated, intraenterocyte BAs were increased, and disease development was increased. ASBT staining was more intense on the apical membrane of ileal enterocytes from premature infants with NEC than premature infants with non-NEC diagnoses. In addition, ASBT mRNA levels were significantly higher in infants with NEC. These data show that accumulation of intraenterocyte BAs contributes to disease development, elevated ASBT increases disease severity in experimental models of NEC, and ASBT is elevated in human NEC. These data confirm that BAs and upregulation of ASBT play a crucial role in NEC pathogenesis and suggest that inhibition of ASBT could be utilized as a therapeutic modality against this disease. epithelial transport; SC-435; bile salt export pump doi: 10.1152/ajpgi.00242.2010.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis. Autophagy regulates cell homeostasis, but uncontrolled activation of autophagy may lead to cellular injury. The aim was to evaluate the effects of EGF on intestinal autophagy in epithelial cells and in the rat NEC model and measure autophagy in NEC patients. Intestinal epithelial cells (IEC-6) and the rat NEC model were used to study the effect of EGF on intestinal autophagy. Protein levels of Beclin 1 and LC3II were measured in the intestinal epithelium in both in vivo and in vitro models. Ultrastrucrural changes in intestinal epithelium were studied by electron microscopy. Expression of Beclin 1, LC3II, and p62 protein was evaluated in biopsies from NEC patients. Autophagy was induced in IEC-6 cells and inhibited by adding EGF into the culture. In the rat NEC model, EGF treatment of NEC reduced expression of Beclin 1 and LC3II in ileal epithelium. Morphologically, typical signs of autophagy were observed in the epithelium of the NEC group, but not in the EGF group. A strong signal for Beclin 1 and LC3II was detected in the intestine from patients with NEC. Autophagy is activated in the intestinal epithelium of NEC patients and in the ileum of NEC rats. Supplementation of EGF blocks intestinal autophagy in both in vivo and in vitro conditions. Results from this study indicate that EGFmediated protection against NEC injury is associated with regulation of intestinal autophagy. epithelial homeostasis; enteral nutrition; premature; small intestine doi: 10.1152/ajpgi.00076.2010.

Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x [10.sup.6] CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity. adherens junctions; intestinal barrier function; mucins; tight junctions doi: 10.1152/ajpgi.00141.2009.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2-II [18.sup.tm1Aki]/J (NEC IL-[18.sup.-/-]) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-[alpha]-, IL-12-, and IL-1[beta]-positive cells and macrophages were determined in both ileum and liver via immunohistology. I[kappa]B-[alpha] and I[kappa]B-[beta] were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-[18.sup.-/-] mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1[beta] were significantly reduced in IL-[18.sup.-/-] mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-[alpha], ileal IL-1[beta], or IL-12. I[kappa]B-[alpha] and I[kappa]B-[beta] were significantly increased in NEC IL-[18.sup.-/-] mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis. interleukin-18; inflammation

Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function. intestinal permeability; tight junctions; rat; epidermal growth factor

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. However, despite significant morbidity and mortality, the etiology and pathogenesis of NEC are poorly understood. Evidence suggests that ileal proinflammatory mediators such as IL-18 contribute to the pathology associated with this disease. In addition, we have previously shown that upregulation of TNF-[alpha] in the liver is correlated with ileal disease severity in a neonatal rat model of NEC. With the use of a neonatal rat model of NEC, we evaluated the incidence and severity of ileal damage along with the production of both hepatic and ileal proinflammatory cytokines in animals injected with (anti-TNF-[alpha]; n = 23) or without (NEC; n = 25) a monoclonal anti-TNF-[alpha] antibody. In addition, we assessed changes in apoptosis and ileal permeability in the NEC and anti-TNF-[alpha] groups. Ileal damage was significantly decreased, and the incidence of NEC was reduced from 80% to 17% in animals receiving anti-TNF-[alpha]. Hepatic TNF-[alpha] and hepatic and ileal IL-18 were significantly decreased in pups given anti-TNF-[alpha] compared with those sham injected. In addition, ileal luminal levels of both TNF-[alpha] and IL-18 were significantly decreased in the anti-TNF-[alpha]-injected group. Ileal paracellular permeability and the proapoptotic markers Bax and cleaved caspase-3 were significantly decreased in the anti-TNF-[alpha] group. These data show that hepatic TNF-[alpha] is an important component for the development of NEC in the neonatal rat model and suggest that anti-TNF-[alpha] could be used as a potential therapy for human NEC. tumor necrosis factor-[alpha]; anti-tumor necrosis factor-[alpha]; proinflammatory cytokines; liver/gut axis

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC +EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury. epithelial injury; ileum; rat; Bcl-2; Bax

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by mutisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-[alpha], and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-[alpha] levels were also measured. Production of hepatic IL-18 and TNF-[alpha] and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-[alpha] in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis. gastrointestinal system; inflammation; neonatal; gut-liver axis

Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model. Am J Physiol Gastrointest Liver Physiol 282: G156-G164, 2002. First published October 10, 2001; 10.1152/ajpgi.00196.2001.--Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-[alpha] mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC. intestinal injury; inflammation; neonatal intestine; artificial feeding

Background: Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17. Objective: We sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ−/− mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls. Results: In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αβ IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A. Conclusion: Complimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is the most common cause of gastrointestinal-related morbidity and mortality in the neonatal intensive care unit (NICU). Its onset is sudden and the smallest, most premature infants are the most vulnerable. Necrotizing enterocolitis is a costly disease, accounting for nearly 20% of NICU costs annually. Necrotizing enterocolitis survivors requiring surgery often stay in the NICU more than 90 days and are among those most likely to stay more than 6 months. Significant variations exist in the incidence across regions and units. Although the only consistent independent predictors for NEC remain prematurity and formula feeding, others exist that could increase risk when combined. Awareness of NEC risk factors and adopting practices to reduce NEC risk, including human milk feeding, the use of feeding guidelines, and probiotics, have been shown to reduce the incidence of NEC. The purpose of this review is to examine the state of the science on NEC risk factors and make recommendations for practice and research. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E2 (PGE2). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE2, and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE2 and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum-treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum. Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE2 in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity. [ABSTRACT FROM AUTHOR]