Immature Intestine Research Articles

Mo1960 Modulation of the Inflammatory Response by Epidermal Growth Factor in the Immature Human Intestine

Mo1960 Modulation of the Inflammatory Response by Epidermal Growth Factor in the Immature Human Intestine

Anti-inflammatory effects of epidermal growth factor on the immature human intestine

The inflammatory response of the preterm infants' intestine underlines its inability to respond to hemodynamic stress, microbes, and nutrients. Recent evidence suggests that exogenous epidermal growth factor (EGF) exerts a therapeutic influence on neonatal enteropathies. However, the molecular mechanisms underlying the beneficial effects of EGF remain to be clarified. The purpose of this study was to evaluate the impact of EGF on the gene expression profiles of the developing human small and large intestine at midgestation in serum-free organ cultures using microarrays. The gene expression profiles of cultured human fetal ileal and colonic explants were investigated in the absence or presence of a physiological concentration of 50 ng/ml EGF for 48 h. Data were analyzed with the Ingenuity Pathway Analysis (IPA) software and confirmed by qPCR. We found a total of 6,474 differentially expressed genes in the two segments in response to EGF. IPA functional analysis revealed that in addition to differentially modulating distinct cellular, molecular, and physiological functions in the small and large intestine, EGF regulated the inflammatory response in both intestinal segments in a distinct manner. For instance, several intestinal-derived chemokines such as CCL2, CCL25, CXCL5, and CXCL10 were found to be differentially regulated by EGF in the immature ileum and colon. The findings showing the anti-inflammatory influence of exogenous EGF suggests a mechanistic basis for the beneficial effects of EGF on neonatal enteropathies. These results reinforce growing evidence that by midgestation, the human small intestine and colon rely on specific and distinct regulatory pathways.

Paneth cell ablation in the presence of Klebsiella pneumoniae induces necrotizing enterocolitis (NEC)-like injury in immature murine small intestine

SUMMARYNecrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. During NEC pathogenesis, bacteria are able to penetrate innate immune defenses and invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Normally, Paneth cells appear in the intestinal crypts during the first trimester of human pregnancy. Paneth cells constitute a major component of the innate immune system by producing multiple antimicrobial peptides and proinflammatory mediators. To better understand the possible role of Paneth cell disruption in NEC, we quantified the number of Paneth cells present in infants with NEC and found that they were significantly decreased compared with age-matched controls. We were able to model this loss in the intestine of postnatal day (P)14-P16 (immature) mice by treating them with the zinc chelator dithizone. Intestines from dithizone-treated animals retained approximately half the number of Paneth cells compared with controls. Furthermore, by combining dithizone treatment with exposure to Klebsiella pneumoniae, we were able to induce intestinal injury and inflammatory induction that resembles human NEC. Additionally, this novel Paneth cell ablation model produces NEC-like pathology that is consistent with other currently used animal models, but this technique is simpler to use, can be used in older animals that have been dam fed, and represents a novel line of investigation to study NEC pathogenesis and treatment.

Human foetal intestinal fibroblasts are hyper-responsive to lipopolysaccharide stimulation

Background Intestinal myofibroblasts contribute to immune regulation in adults with inflammatory bowel disease but have not been characterised in neonatal intestinal inflammatory diseases. Aims To compare lipopolysaccharide (LPS)-stimulated interleukin-8 (IL-8) production between human foetal and mature intestinal myofibroblasts in vitro. Methods Foetal, neonatal and adult cells were stimulated with increasing concentrations of E. coli LPS. In LPS stimulated foetal myofibroblasts, Toll-like receptor 4 mRNA expression was assessed by real-time PCR whilst Toll-like receptor 4 receptor activity was determined using anti-Toll-like receptor 4 antibody. Mitogen activated protein kinase pathway activity was assessed using chemical inhibitors and Western blotting. IL-8 production was measured by quantitative ELISA. Results IL-8 production by LPS stimulated foetal myofibroblasts occurred in a dose dependent manner. Toll-like receptor 4 expression was constitutive and Toll-like receptor 4 receptor blockade reduced IL-8 production by 42% ( P = 0.0262). C-Jun N-terminal kinase, p38 and NF-κB inhibitors significantly attenuated LPS stimulated IL-8 production by 42%, 33% and 2%, respectively. Mitogen activated protein kinase activity was confirmed by the presence of phosphorylated proteins on Western blots. Conclusion These data demonstrate increased IL-8 production by foetal myofibroblasts that is partially mediated by Toll-like receptor 4, mitogen activated protein kinase and NF-κB cell signalling pathways. Intestinal myofibroblasts cells may contribute to the dysregulated inflammatory response in the immature intestine and may form targets that lead to new therapies to prevent neonatal intestinal inflammatory bowel diseases.

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Transition from parenteral to enteral nutrition induces immediate diet-dependent gut histological and immunological responses in preterm neonates

Necrotizing enterocolitis (NEC) in preterm infants develops very rapidly from a mild intolerance to enteral feeding into intestinal mucosal hemorrhage, inflammation, and necrosis. We hypothesized that immediate feeding-induced gut responses precede later clinical NEC symptoms in preterm pigs. Fifty-six preterm pigs were fed total parenteral nutrition (TPN) for 48 h followed by enteral feeding for 0, 8, 17, or 34 h with either colostrum (Colos, n = 20) or formula (Form, n = 31). Macroscopic NEC lesions were detected in Form pigs throughout the enteral feeding period (20/31, 65%), whereas most Colos pigs remained protected (1/20, 5%). Just 8 h of formula feeding induced histopathological lesions, as evidenced by capillary stasis and necrosis, epithelial degeneration, edema, and mucosal hemorrhage. These immediate formula-induced changes were paralleled by decreased digestive enzyme activities (lactase and dipeptidylpeptidase IV), increased nutrient fermentation, and altered expression of innate immune defense genes such as interleukins (IL-1α, IL-6, IL-18), nitric oxide synthetase, tight junction proteins (claudins), Toll-like receptors (TLR-4), and TNF-α. In contrast, the first hours of colostrum feeding induced no histopathological lesions, increased maltase activity, and induced changes in gene expressions related to tissue development. Total bacterial density was high after 2 days of parenteral feeding and was not significantly affected by diet (colostrum, formula) or length of enteral feeding (8-34 h), except that a few bacterial groups (Clostridium, Enterococcus, Streptococcus species) increased with time. We conclude that a switch from parenteral to enteral nutrition rapidly induces diet-dependent histopathological, functional, and proinflammatory insults to the immature intestine. Great care is required when introducing enteral feeds to TPN-fed preterm infants, particularly when using formula, because early feeding-induced insults may predispose to NEC lesions that are difficult to revert by later dietary or medical interventions.

Gene‐expression Profile Analysis in the Mid‐gestation Human Intestine Discloses Greater Functional Immaturity of the Colon as Compared With the Ileum

The occurrence of many neonatal inflammatory intestinal diseases in preterm infants highlights the susceptibility of the immature intestine to responding inadequately to nutrients and microbes. A better understanding of functional intestinal development is essential for the design of optimal treatments ensuring survival and growth of premature infants. The purpose of this study was to evaluate the gene expression profiles of the human ileum and colon at mid-gestation because these 2 segments are considered to be similar at this stage and are the sites of the most frequent pathologies in preterm infants. We compared the gene-expression profiles of human fetal small and large intestines using a cDNA microarray and analyzed the data with Ingenuity Pathway Analysis software. We found that a significant proportion of the genes was differentially expressed in the 2 segments. Gene cluster analysis revealed an even higher level of transcriptional dissimilarity at the functional level. For instance, segment-specific/overexpressed gene clusters in the ileum included genes involved with amino acid, vitamin, and mineral metabolism, reflecting the higher level of maturity of the small intestine as compared with the colon in which genes involved with cell cycle, cell death, and cell signaling were the predominant clusters of genes expressed. Functional clustering analysis of the differentially expressed genes revealed important functional differences between the 2 segments and a relative immaturity of the colon, suggesting that already at mid-gestation, the 2 intestinal segments should be considered as 2 distinct organs.

Motherʼs Milk-Induced Hsp70 Expression Preserves Intestinal Epithelial Barrier Function in an Immature Rat Pup Model

Preterm infants face many challenges in transitioning from the in utero to extrauterine environment while still immature. Failure of the preterm gut to successfully mature to accommodate bacteria and food substrate leads to significant morbidity such as neonatal necrotizing enterocolitis. The intestinal epithelial barrier plays a critical role in gut protection. Heat shock protein 70 (Hsp70) is an inducible cytoprotective molecule shown to protect the intestinal epithelium in adult models. To investigate the hypothesis that Hsp70 may be important for early protection of the immature intestine, Hsp70 expression was evaluated in intestine of immature rat pups. Data demonstrate that Hsp70 is induced by exposure to mother's milk. Hsp70 is found in mother's milk, and increased Hsp70 transcription is induced by mother's milk. This Hsp70 colocalizes with the tight junction protein ZO-1. Mother's milk-induced Hsp70 may contribute to maintenance of barrier function in the face of oxidant stress. Further understanding of the means by which mother's milk increases Hsp70 in the ileum will allow potential means of strengthening the intestinal barrier in at-risk preterm infants.

The mechanism of excessive intestinal inflammation in necrotizing enterocolitis: an immature innate immune response.

Necrotizing enterocolitis (NEC) is a devastating neonatal intestinal inflammatory disease, occurring primarily in premature infants, causing significant morbidity and mortality. The pathogenesis of NEC is associated with an excessive inflammatory IL-8 response. In this study, we hypothesized that this excessive inflammatory response is related to an immature expression of innate immune response genes. To address this hypothesis, intestinal RNA expression analysis of innate immune response genes was performed after laser capture microdissection of resected ileal epithelium from fetuses, NEC patients and children and confirmed in ex vivo human intestinal xenografts. Changes in mRNA levels of toll-like receptors (TLR)-2 and -4, their signaling molecules and transcription factors (MyD88, TRAF-6 and NFκB1) and negative regulators (SIGIRR, IRAK-M, A-20 and TOLLIP) and the effector IL-8 were characterized by qRT-PCR. The expression of TLR2, TLR4, MyD88, TRAF-6, NFκB1 and IL-8 mRNA was increased while SIGIRR, IRAK-M, A-20 and TOLLIP mRNA were decreased in fetal vs. mature human enterocytes and further altered in NEC enterocytes. Similar changes in mRNA expression were observed in immature, but not mature, human intestinal xenografts. Confirmation of gene expression was also validated with selective protein measurements and with suggested evidence that immature TRL4 enterocyte surface expression was internalized in mature enterocytes. Cortisone, an intestinal maturation factor, treatment corrected the mRNA differences only in the immature intestinal xenograft. Using specific siRNA to attenuate expression of primary fetal enterocyte cultures, both TOLLIP and A-20 were confirmed to be important when knocked down by exhibiting the same excessive inflammatory response seen in the NEC intestine. We conclude that the excessive inflammatory response of the immature intestine, a hallmark of NEC, is due to a developmental immaturity in innate immune response genes.

Enteral Feeding In Utero Induces Marked Intestinal Structural and Functional Proteome Changes in Pig Fetuses

Intestinal adaptation from parenteral to enteral nutrition is crucial for survival and growth of newborns. Rapid feeding-induced gut maturation occurs immediately after birth in both preterm and term neonates, but it remains unclear whether the responses depend on factors related to birth transition (e.g. bacterial colonization, endocrine, and metabolic changes). We hypothesized that enteral feeding matures the immature intestine, even in fetuses before birth. Hence, control pig fetuses were compared with fetuses fed with milk formula for 24 h in utero. Gel-based proteomics showed that feeding-induced changes in 38 proteins, along with marked increases in intestinal mass and changes in activities of brush border enzymes. Physiological functions of the identified proteins were related to enterocyte apoptosis (e.g. caspase 1) and nutrient metabolism (e.g. citric acid cycle proteins). Many of the differentiated proteins were similar to those identified previously in preterm pigs fed with the same formula after birth, except that effects on proteins related to inflammatory lesions (e.g. heat shock proteins) were absent. Our results show that enteral feeding, independently of the birth transition, induces marked gut maturation and proteome change in the immature intestine. Hence, immediate postnatal feeding-induced gut changes are largely independent of factors related to the birth transition.

Bovine colostrum is superior to enriched formulas in stimulating intestinal function and necrotising enterocolitis resistance in preterm pigs

Milk contains immunomodulatory compounds that may be important to protect the immature intestine in preterm neonates from harmful inflammatory reactions involved in disorders like necrotising enterocolitis (NEC). We hypothesised that bovine colostrum and milk formulas enriched with sialic acids (SL), gangliosides (Gang) or osteopontin (OPN) would improve gastrointestinal function and NEC resistance in preterm neonates. Forty-seven caesarean-delivered preterm pigs were given total parenteral nutrition for 2d followed by 1·5d of enteral feeding. In Expt 1, a control formula was compared with an OPN-enriched formula (n 13), while Expt 2 compared a control formula with bovine colostrum or formulas enriched with Gang or SL (n 4-6). OPN enrichment decreased NEC severity relative to control formula (P<0·01), without any significant effects on NEC incidence, digestive enzyme activities and hexose absorption. Neither SL- nor Gang-enriched formulas improved NEC resistance or digestive functions, while all the intestinal functional parameters were significantly improved in pigs fed bovine colostrum, relative to formula. The effects in vivo were supported in vitro by bacteria- and dose-dependent modulation by colostrum whey of the cytokine response from bacteria-stimulated murine bone marrow-derived dendritic cells (DC). In conclusion, OPN had only moderate NEC-protective effects, while formulas enriched with Gang or SL were ineffective. The observed modulation of DC cytokine response by bovine colostrum whey in vitro may be due to a synergistic action of various milk bioactives, and it may explain its beneficial effects on NEC development and intestinal function in a piglet model of preterm infants.

Dietary-dependent changes in the enteric nervous system and endothelium of premature piglets

After birth, intestinal morphology and function have to adapt at a high pace. This remodelling is more challenging in low birth weight neonates or in preterms, where it may result in necrotizing enterocolitis (NEC). We hypothesized that in the preterm piglet, feeding induces maladaptations of the enteric nervous system (ENS) and vasculature. Using image analysis, the densities of neurons expressing VIP, glial cells containing GFAP and the endothelium containing eNOS on immunohistochemically stained small intestinal sections of preterm 1) unfed piglets, 2) piglets receiving total parenteral nutrition (TPN) for 2–3 days and 3) piglets fed 2 days sow's colostrum (SOW) or formulated milk (FOR) following TPN were estimated. After enteral feeding, the ENS and vascular endothelium grew in the same order as the intestine. However, feeding formula increased the density of VIP'ergic myenteric neurons, lowered eNOS in the endothelium and resulted in a reactive gliosis. In conclusion, formula induces destructive changes in the immature small intestine, whereas colostrum prevents their occurrence. These conditions may be among the factors that predispose to NEC.

Commensal Escherichia coli Reduces Epithelial Apoptosis through IFN-αA–Mediated Induction of Guanylate Binding Protein-1 in Human and Murine Models of Developing Intestine

Appropriate microbial colonization protects the developing intestine by promoting epithelial barrier function and fostering mucosal tolerance to luminal bacteria. Commensal flora mediate their protective effects through TLR9-dependent activation of cytokines, such as type I IFNs (alpha, beta) and IL-10. Although IFN-beta promotes apoptosis, IFN-alpha activates specific antiapoptotic target genes whose actions preserve epithelial barrier integrity. We have recently identified guanylate binding protein-1 (GBP-1) as an antiapoptotic protein, regulated by both type I and type II IFNs, that promotes intestinal epithelial barrier integrity in mature intestine. However, the mechanisms by which commensal bacteria regulate epithelial apoptosis during colonization of immature intestine and the contributions of GBP-1 are unknown. The healthy newborn intestine is initially colonized with bacterial species present in the maternal gastrointestinal tract, including nonpathogenic Escherichia coli. Therefore, we examined the influence of commensal E. coli on cytokine expression and candidate mediators of apoptosis in preweaned mice. Specifically, enteral exposure of 2 wk-old mice to commensal E. coli for 24 h selectively increased both IFN-alphaA and GBP-1 mRNA expression and prevented staurosporine-induced epithelial apoptosis. Exogenous IFN-alphaA treatment also induced GBP-1 expression and protected against staurosporine-induced apoptosis in a GBP-1 dependent manner, both in vitro and ex vivo. These findings identify a role for IFN-alphaA-mediated GBP-1 expression in the prevention of intestinal epithelial apoptosis by commensal bacteria. Thus IFN-alphaA mediates the beneficial effects of commensal bacteria and may be a promising therapeutic target to promote barrier integrity and prevent the inappropriate inflammatory responses seen in developing intestine as in necrotizing enterocolitis.

Tight Junction Expression Reflects Changes in Intestinal Permeability in the Developing Mouse Intestine

Necrotizing Enterocolitis (NEC) is a devastating disease of preterm infants. Immature intestinal barrier function has been implicated in its pathogenesis. Tight junctions (TJ), comprised of transmembrane Claudins which seal the paracellular space, are a vital component of the intestinal barrier and its regulation of intestinal permeability. However, little is known about how TJ development in the immature intestine influences changes in intestinal permeability. We screened mRNA expression of 29 TJ proteins by qRT‐PCR in the ileum of developing mice over the first 8 weeks of life. Selective TJ proteins were further analyzed by immunoblot analysis and localized by immunofluorescence labeling/confocal microscopy (IF). Temporal changes in expression were correlated with FITC‐dextran intestinal permeability assays. Most TJ proteins demonstrated decreased expression over time. However, Claudin‐3 and Claudin‐7 expression peaked at 3 weeks. IF demonstrated appropriate localization of Claudins in TJ after 3 weeks. Over the same period, intestinal permeability decreased. Changes in Claudin expression likely drive changes in intestinal permeability by tightening the paracellular space. Further studies are needed to elucidate what factors influence temporal changes in TJ expression and intestinal permeability and how they may affect predisposition to NEC.

Localization of the lipopolysaccharide recognition complex in the human healthy and inflamed premature and adult gut

Microbiota in the intestinal lumen provide an abundant source of potentially detrimental antigens, including lipopolysaccharide (LPS), a potent immunostimulatory product of Gram-negative bacteria recognized by the host via TLR-4 and MD-2. An aberrant immune response to LPS or other bacterial antigens has been linked to inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). We investigated which cells express MD-2 in the normal and inflamed ileum from neonates and adults by immunohistochemistry. Moreover, MD-2 and TLR4 mRNA expression in normal adult ileum was studied by reverse-transcription polymerase chain reaction (RT-PCR) on cells isolated by laser capture microdissection. Premature infants did not show MD-2 expression either in epithelial cells or in the lamina propria. Similarly, MD-2 was absent in epithelial cells and lamina propria inflammatory cells in preterm infants with NEC. MD-2 protein in the healthy term neonatal and adult ileum was predominantly expressed by Paneth cells and some resident inflammatory cells in the lamina propria. MD-2 and TLR-4 mRNA expression was restricted to crypt cells. Also in IBD, Paneth cells were still the sole MD-2-expressing epithelial cells, whereas inflammatory cells (mainly plasma cells) were responsible for the vast majority of the MD-2 expression. The absence of MD-2 in the immature neonatal gut suggests impaired LPS sensing, which could predispose neonates to NEC upon microbial colonization of the immature intestine. The apparent expression of MD-2 by Paneth cells supports the critical concept that these cells respond to luminal bacterial products in order to maintain homeostasis with the intestinal microbiota in vivo.

The Use of Probiotics in the Prevention of Necrotizing Enterocolitis in Preterm Infants

Necrotizing enterocolitis (NEC) is defined as inflammation and necrosis of the bowel. It is the final manifestation of vascular, mucosal, and metabolic insults to the immature intestine (1, 2). NEC is a common gastrointestinal emergency in newborns and one of the leading causes of morbidity and mortality in neonatal intensive care units (NICU), with a mortality rate of 15% to 30% (3, 4). No single unifying theory exists for the pathogenesis of NEC that explains all the clinical observations associated with the disorder. Proposed mechanisms for the development of NEC include immature intestinal motility, digestion, circulatory regulation, barrier function, innate immunity, and abnormal bacterial colonization (2, 3). The most frequent risk factors associated with NEC include prematurity and low birth weight. Over 90% of neonates who develop NEC are born preterm. Extremely low birth weight (<1000 g) infants <28 weeks' gestational age (GA) are the most susceptible. Other risk factors for the development of NEC include asphyxia, acute cardiopulmonary disease, polycythemia, hyperviscosity syndromes, exchange transfusions, aggressive enteral feeding, and abnormal colonization of the intestine with enteric pathogens (1–5). Many nonspecific signs may trigger the suspicion of NEC, such as abdominal tenderness or distention, feeding intolerance, bloody stools, and apnea. The definite signs of NEC include pneumatosis intestinalis and portal venous gas, which are revealed by x-rays. Abdominal x-rays are performed to diagnose and determine progression of NEC. Laboratory values, such as complete blood count with differential, arterial blood gas measurements, and electrolytes, are measured as well as obtaining blood and stool cultures to look for other signs of NEC and rule out other conditions (2). To better classify and diagnose NEC using nonspecific and definite signs and symptoms of NEC, Bell's staging criteria were developed: suspected NEC is classified as stage 1, proven NEC as stage 2, and severe cases as stage 3 (1, 5) (Table ​(Table11). Table 1 Bell's staging criteria for necrotizing enterocolitis∗

Lactobacillus rhamnosus prevents inflammatory signaling in immature murine intestines via generation of reactive oxygen species

Immature intestinal defense plays a key role in the pathogenesis of necrotizing enterocolitis (NEC), a devastating disease of premature infants. Here, we show that inflammatory responses in neonatal mouse intestines increase during postnatal maturation with peak responses occurring at 2 weeks of life. Furthermore, we show that these inflammatory responses can be prevented by prior oral administration of the probiotic Lactobacillus rhamnosus (LGG). Probiotic bacteria can block inflammatory responses in adult intestinal epithelia by inducing generation of reactive oxygen species (ROS) which inhibit NF‐¿B activation through oxidative inactivation of the Cul‐1 neddylation enzyme, Ubc12. To determine whether LGG can reduce exaggerated inflammatory responses in immature intestines via a similar mechanism, we measured the effect of LGG on GSH oxidation and loss of neddylated Cul‐1 in immature murine small intestines. Intestines from immature mice gavage fed LGG exhibited increased GSH oxidation and Cul‐1 deneddylation reflecting local ROS generation and its resultant Ubc12 inactivation, respectively. These studies indicate that LGG can reduce inflammatory signaling in immature intestines by inducing local ROS generation and may be a mechanism by which probiotic bacteria can prevent NEC in premature infants.

Prevention and Treatment of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality among premature infants. Although the pathogenesis of NEC remains unclear, recent researches revealed several associated factors of the immature intestine, with an emphasis of delayed maturation of motor and digestive function, impairment of regulation of vascular flow and intestinal barrier function, and defective immune defense. Many clinical trials have investigated the preventive role of possible disease-modification factors, but only breast feeding and antenatal steroid were proven to decrease the incidence of NEC in metaanalyses. Recent multicenter studies demonstrated a promising outcome of probiotics supplementation in the prevention of NEC, which emphasized the role of abnormal bacterial colonization in the pathogenesis of NEC. Studies on optimal choice for surgically indicated infants with NEC (laparatomy versus primary peritoneal drainage) still remain inconclusive. As NEC is a disease with a multifactorial etiology, combinations of current evidence in practice are required to reduce the incidence of NEC.

A roadmap for understanding and preventing necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is the most common severe gastrointestinal emergency that affects newborns. Its etiology is still undetermined. Most of our knowledge of this disease stems from epidemiologic studies, clinical experience, and studies using animal models that do not directly represent the disease in premature infants. An immature intestine that is colonized by bacteria is a prerequisite for the pathogenesis of NEC. Although causative microbes have not been found, newly developed analytic techniques now allow the detection of noncultivatable microbes and the determination of their overall diversity and metabolic capabilities. The National Institutes of Health Human Microbiome Roadmap Project is likely to generate new information highly germane to understanding NEC. Newly developed proteomic and metabolomic approaches also offer exciting new potential for early diagnosis and prevention of this disease in infants at highest risk.

Glucagon-like peptide 2 has limited efficacy to increase nutrient absorption in fetal and preterm pigs

Exogenous glucagon-like peptide 2 (GLP-2) prevents intestinal atrophy and increases nutrient absorption in term newborn pigs receiving total parenteral nutrition (TPN). We tested the hypothesis that the immature intestine of fetuses and preterm neonates has a diminished nutrient absorption response to exogenous GLP-2. This was accomplished using catheterized fetal pigs infused for 6 days (87-91% of gestation) with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 7) or saline (n = 7), and cesarean-delivered preterm pigs (92% of gestation) that received TPN with GLP-2 (25 nmol.kg(-1).day(-1) iv; n = 8) or saline (n = 7) for 6 days after birth. Responses to GLP-2 were assessed by measuring intestinal dimensions, absorption of nutrients (glucose, leucine, lysine, proline) by intact tissues and brush border membrane vesicles, and abundance of sodium-glucose cotransporter mRNA. Infusion of GLP-2 increased circulating GLP-2 levels in fetuses, but did not increase intestinal mass or absorption of nutrients by intact tissues and brush border membrane vesicles, except for lysine. Administration of exogenous GLP-2 to preterm TPN-fed pigs similarly did not increase rates of nutrient absorption, yet nutrient absorption capacities of the entire small intestine tended to increase (+10-20%, P < 0.10) compared with TPN alone due to increased intestinal mass (+30%, P < 0.05). GLP-2 infusion did not increase sodium-glucose cotransporter-1 mRNA abundance in fetuses or postnatal preterm pigs. Hence, the efficacy of exogenous GLP-2 to improve nutrient absorption by the intestine of fetal and preterm pigs is limited compared with term pigs and more mature animals and humans.