epidermal-growth-factor and Enterocolitis--Necrotizing

Necrotizing enterocolitis (NEC) is a devastating disease of prematurity, with no current method for early diagnosis. Diagnosis is particularly challenging, frequently occurring after the disease has progressed to the point of significant and often irreversible intestinal damage. Biomarker research has tremendous potential to advance clinical management of NEC and our understanding of its pathogenesis. This review discusses the need for novel biomarkers in NEC management, evaluates studies investigating such biomarkers, and explains the difficulties associated with translating biomarker discovery into clinical use.

Topics: Alpha-Globulins; Biomarkers; Blood Cell Count; Breath Tests; C-Reactive Protein; Claudins; Complement C5a; Cytokines; Early Diagnosis; Enterocolitis, Necrotizing; Epidermal Growth Factor; Fatty Acid-Binding Proteins; Gastrointestinal Microbiome; Genomics; Humans; Hydrogen; Infant, Newborn; Infant, Premature; Leukocyte L1 Antigen Complex; Metabolomics; Platelet Activating Factor; Procalcitonin; Prognosis; Proteomics; Risk Assessment; S100A12 Protein; Sensitivity and Specificity; Serum Albumin, Human; Serum Amyloid A Protein; Spectroscopy, Near-Infrared; Trefoil Factor-3; Ultrasonography; Volatile Organic Compounds

Growth factors have important roles in gastrointestinal tract development, maintenance, and response to injury. Various experiments have been used to demonstrate growth factor influence in multiple disease processes. These studies demonstrated enhancement of mucosal proliferation, intestinal motility, immune modulation, and many other beneficial effects. Select growth factors, including epidermal growth factor and heparin-binding epidermal growth factor like growth factor, demonstrate some beneficial effects in experimental and clinical intestinal injury demonstrated in necrotizing enterocolitis. The roles of glucagon-like peptide 2, insulin-like growth factor 1, erythropoietin, growth hormone, and hepatocyte growth factor in necrotizing enterocolitis are summarized in this article.

Topics: Enterocolitis, Necrotizing; Epidermal Growth Factor; Glucagon-Like Peptide 2; Growth Hormone; Heparin-binding EGF-like Growth Factor; Hepatocyte Growth Factor; Humans; Infant, Newborn; Infant, Premature; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Intestines

This review will summarize the clinical and experimental studies evaluating the role of epidermal growth factor (EGF) in prophylaxis and treatment of necrotizing enterocolitis (NEC).. Clinical studies have suggested the importance of EGF in protection of the intestine against NEC, as well as its safety for infants suffering from NEC. The recent experimental studies identified the molecular mechanisms EGF uses for intestinal protection, which involves regulation of intestinal epithelial homeostasis and barrier function. Further studies are necessary to identify the optimal dose, timing, and route of administration of EGF to NEC patients. No clinical studies are currently underway.. NEC is a devastating problem for preterm neonates, but the exact disease pathogenesis remains unclear. Growing clinical evidence supports the use of EGF as a predictive marker of NEC and its use for prevention and treatment of NEC. In addition, experimental data indicate potential mechanisms of EGF prevention against NEC. These include reduction of inflammation, improvement of barrier function, and regulation of epithelial apoptosis and autophagy.

Topics: Animals; Apoptosis; Autophagy; Biomarkers; Enterocolitis, Necrotizing; Epidermal Growth Factor; Humans; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Intestinal Absorption; Intestinal Mucosa; Liver

Maternal milk is a complex fluid, with multifunctional roles within the developing gastrointestinal tract. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are members of the family of EGF-related peptides. Biological actions of these growth factors are mediated via interaction with the EGF-receptor (EGF-R). In the early postnatal period, breast milk is the major source of EGF for the developing intestinal mucosa. HB-EGF is also detected in breast milk, but in concentrations 2 to 3 times lower than EGF. With normal physiological conditions, the intestinal epithelium undergoes a continuing process of cell proliferation, differentiation, and maturation. EGF plays an important role in these processes. In pathophysiologic situations, EGF contributes to epithelial protection from injury and post-injury mucosal repair. Necrotizing enterocolitis (NEC) is a devastating disease affecting infants born prematurely. The pathogenesis of NEC is not known, and there is no effective treatment for this disease. In an experimental NEC model, oral administration of a physiological dose of EGF significantly reduces the incidence and severity of NEC. HB-EGF provides similar protection against NEC, but only when pharmacological doses are used. Further studies are necessary before EGF can be introduced as an efficient therapeutic approach of intestinal injury.

Topics: Animals; Enterocolitis, Necrotizing; Epidermal Growth Factor; Female; Gastrointestinal Tract; Heparin-binding EGF-like Growth Factor; Humans; Infant; Infant, Newborn; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Milk, Human

Necrotizing enterocolitis (NEC) is primarily a disease process of the gastrointestinal (GI) tract of premature neonates that results in inflammation and bacterial invasion of the bowel wall. Despite advances in the care of premature infants, NEC remains one of the leading causes of morbidity and mortality in this population. It occurs in 1-5% of all neonatal intensive care admissions and 5-10% of all very low birthweight (<1500 g) infants. Although research has presented an interesting array of potential contributing factors, the precise aetiology of this multifactorial disease process remains elusive. Historically, it was believed that NEC arose predominantly from ischaemic injury to the immature GI tract, yet alternate plausible hypotheses indicate that many factors are likely to be involved. These may include issues related to the introduction and advancement of enteric feeding, alterations in the normal bacterial colonization of the GI tract, bacterial translocation and activation of the cytokine cascade, decreased epidermal growth factor, increased platelet activating factor, and mucosal damage from free radical production. Clinical manifestations of NEC may be vague, including increased episodes of apnoea, desaturations, bradycardia, lethargy and temperature instability. There may also be GI-specific symptoms such as feeding intolerance, emesis, bloody stools, abdominal distention and tenderness, and abdominal wall discolouration. Laboratory values may be indicative of infection, coagulation abnormalities and fluid retention. Radiographic signs may include ileus, dilated or fixed intestinal loops, air in the intestinal wall or free air in the abdomen. Medical treatment typically consists of bowel rest and decompression, antibacterial therapy, and management of other haematological or electrolyte imbalances. Increased respiratory and cardiovascular support is sometimes needed. In neonates who do not respond adequately to medical management, or if pneumoperitoneum is present, surgical intervention may occur with either use of a peritoneal drain or laparotomy. Advances in antenatal and neonatal care have resulted in increased survival of extremely preterm neonates. As this at-risk population continues to increase, an effective preventative strategy for NEC is needed. One preventative strategy is the use of antenatal corticosteroids to enhance maturation of the fetus if preterm delivery is likely. Recommendation of use of breast milk, early initiation o

Topics: Adrenal Cortex Hormones; Amino Acids; Anti-Bacterial Agents; Antioxidants; Dietary Supplements; Enterocolitis, Necrotizing; Epidermal Growth Factor; Female; Humans; Immunoglobulins; Infant; Infant, Newborn; Infant, Premature; Pregnancy; Prenatal Care; Probiotics

Necrotizing enterocolitis (NEC) presents as the most common gastrointestinal emergency during the neonatal period and results in ulceration and necrosis of the distal small intestine and proximal colon. The etiology of NEC remains unknown. Based on the complexity of gut development, multiple growth factors and cytokines may be needed to synergistically support the developing gut. Epidermal growth factor (EGF) has been shown to play an important role in intestinal cell restitution, proliferation, and maturation. EGF is found in abundant quantities in many fluids, including the gastrointestinal tract, amniotic fluid, breast milk, and saliva. Preliminary clinical trials using EGF in neonates diagnosed with NEC have been shown to promote repair of intestinal epithelium. Additionally, other growth factors are also emerging as potential treatment modalities, including erythropoietin, granulocyte colony stimulating factor, and heparin-binding EGF. The role of EGF and other growth factors in the pathogenesis and prevention of NEC will be reviewed.

Topics: Enterocolitis, Necrotizing; Epidermal Growth Factor; Humans; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Intestinal Mucosa

The occurrence in the neonatal period and into early infancy of two inflammatory conditions, necrotising enterocolitis (NEC), and allergic colitis, that do not occur in later life highlight the peculiar vulnerability of the gastrointestinal tract in the newborn period to otherwise innocuous insults. The pathogenesis of the relatively benign allergic colitis as a mucosal inflammatory process driven by dietary antigens is relatively well characterised, and its treatment with dietary manipulation is well established. For NEC, hypoxic/ischaemic insult, mucosal immaturity, and its interaction with the intestinal microflora are understood to be the main factors in pathogenesis. Thus far, the most productive interventions have been in preventative approaches, in particular feeding strategies, to reduce the incidence of the condition whilst establishing adequate growth and progression onto enteral feeding. For established NEC, supportive medical therapy or surgical intervention remains the mainstay or treatment, although novel therapies, such as platelet-activating factor (PAF) inhibitors and epidermal growth factor (EGF), have shown some promise in animal models of the condition.

Topics: Colitis; Enterocolitis, Necrotizing; Epidermal Growth Factor; Gastrointestinal Tract; Humans; Infant, Newborn; Platelet Activating Factor

Neonatal necrotizing enterocolitis (NEC) is an increasingly frequent condition encountered in premature infants for which the etiology is not well understood. Epidermal growth factor (EGF) is abundant in many fluids bathing the fetal and neonatal gastrointestinal tract, including amniotic fluid, saliva, and breast milk. EGF is acknowledged to be important for normal intestinal development as well as repair following injury to the gastrointestinal mucosa. There appears to be mounting evidence to support a possible link between deficient EGF production and the development of NEC. The relevant evidence for the role of EGF in intestinal development and mucosal repair, as well as its potential involvement in the genesis of NEC will be reviewed.

Topics: Animals; Enterocolitis, Necrotizing; Epidermal Growth Factor; Humans; Infant, Newborn; Infant, Premature; Intestines; Models, Animal; Organogenesis; Regeneration

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease that occurs predominantly in premature infants. Despite various advances in management, the mortality of this disease remains high. During the last decade, studies from our laboratory have shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells (IEC) from various forms of injury in vitro. Furthermore, we have used both an intestinal I/R injury model in adult rats, and a neonatal rat pup model of NEC, to show that HB-EGF can protect the intestines from injury. On administration of HB-EGF in the neonatal rat model, the incidence of NEC is reduced from 65% to 27.3% (P < 0.05), and the histological injury score is decreased from 2 to 1.1 (P < 0.05). In addition, the survival rate is increased from 25% to 63.6% and the survival time extended from 59 hours to 73 hours (P < 0.05). In addition, using human specimens from newborns undergoing bowel resection for NEC, we found that the expression of endogenous HB-EGF mRNA in normal areas of the intestine at the resection margins was higher than that of the intestine afflicted with acute NEC. Endogenous HB-EGF may be involved in epithelial cell repair, proliferation, and regeneration during recovery from injury. Exogenous administration of HB-EGF potentiates recovery from intestinal injury in vitro and in vivo. Taken together, these results support a potential therapeutic role for HB-EGF in the treatment of NEC in the future.

Topics: Animals; Enterocolitis, Necrotizing; Enterocytes; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Humans; Infant, Newborn; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Models, Animal; Protective Agents; Rats

As the number of extremely low-birth-weight infants increases,necrotizing enterocolitis remains a critical eminent problem. Supplementation of enteral feeds with biologically active substances normally present in breast milk, such as epidermal growth factor, seems to be a logical and safe way to reduce the incidence of intestinal inflammation and necrotizing enterocolitis. Continuing basic research and clinical studies are essential before epidermal growth factor can be introduced as an efficient therapeutic approach in the treatment of neonatal necrotizing enterocolitis.

Topics: Animals; Cytoprotection; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; Humans; Intestines; Milk, Human; Regeneration

Neonatal necrotizing enterocolitis (NEC) is a common and serious acquired gastrointestinal tract condition. This clinical study assessed the potential clinical efficacy and microscopic effects of recombinant human epidermal growth factor 1-48 (EGF(1-48)) in neonates with NEC.. This prospective, double-blind, randomized controlled study included 8 neonates with NEC. The study compared the effects of a 6-day continuous intravenous infusion of EGF(1-48) at 100 ng kg(-1) h(-1) against placebo. Clinical outcomes and morphological evaluation of serial rectal mucosal biopsies were assessed at baseline and 4, 7, and 14 days after starting EGF infusions.. There was no difference between the clinical safety outcomes recorded for EGF(1-48) or placebo patients. Quantitative morphologic differences in the rectal mucosa biopsies were noted with EGF(1-48) treatment compared with baseline or placebo and included a statistically significant increase in the number of mitoses per mucosal crypt on study day 4, significantly increased thickness of rectal mucosa from baseline on study days 4 and 7, and increased crypt surface area of rectal mucosa in parallel with increased mucosa thickness on day 14.. This study of EGF(1-48) in neonates with severe NEC showed that growth factor treatment was well tolerated and produced positive and measurable remodeling trophic effects on the gastrointestinal mucosa.

Topics: Double-Blind Method; Enterocolitis, Necrotizing; Epidermal Growth Factor; Female; Gastrointestinal Agents; Humans; Infant; Infant, Newborn; Intestinal Mucosa; Male; Peptide Fragments; Prospective Studies

This study examined the effects of enterally administered epidermal growth factor (EGF) on nutrient absorption and tolerance of enteral feeds in pediatric patients with short bowel syndrome (SBS).. Patients identified with severe SBS (<25% bowel length predicted for age) were prospectively enrolled in treatment using human recombinant EGF (1-53); 100 microg/kg per day given mixed with enteral feeds and patients were treated for 6 weeks. End points followed were patient weight, tolerance of enteral feeds, nutrient absorption, and intestinal permeability as determined using carbohydrate probes and hematologic values for liver function parameters.. Five patients were treated with EGF; all showed a significant improvement in carbohydrate absorption (3-0 methylglucose): absorption 24.7% +/- 9.7% pretreatment vs 34.1% +/- 13.8% posttreatment and improved tolerance of enteral feeds (enteral energy as % of total energy, 25% +/- 28% pretreatment vs 36% +/- 24% posttreatment; mean +/- SD; P < .05 by Wilcoxon's signed rank test). Epidermal growth factor treatment was not associated with significant changes in intestinal permeability, the rate of weight gain, or liver function tests. During the treatment phase, no patients developed episodes of sepsis; however, within 2 weeks of discontinuation of EGF treatment, 3 patients developed septic episodes. No adverse effects of EGF administration were noted.. These results suggest that enteral treatment with EGF in pediatric SBS improves nutrient absorption, increases tolerance with enteral feeds, and may improve the infection rate. Further studies exploring treatment strategies including the timing and duration of EGF administration are indicated.

Topics: 3-O-Methylglucose; Child, Preschool; Dietary Carbohydrates; Enteral Nutrition; Enterocolitis, Necrotizing; Epidermal Growth Factor; Gastroschisis; Humans; Infant; Infant Food; Intestinal Absorption; Intestinal Volvulus; Lactulose; Liver Function Tests; Male; Mannitol; Pilot Projects; Postoperative Complications; Recombinant Proteins; Sepsis; Short Bowel Syndrome; Weight Gain

Epidermal Growth Factor (EGF) reduces necrotizing enterocolitis (NEC). However, its high cost virtually prohibits clinical use. To reduce cost, soybean expressing human EGF was developed. Here we report effectiveness of soybean-derived EGF in experimental NEC.. Newborn rats were subjected to the NEC-inducing regimen of formula feeding and hypoxia. Formula was supplemented with extract from EGF-expressing or empty soybeans. NEC pathology was determined microscopically. Localization of tight junction proteins JAM-A and ZO-1 was examined by immunofluorescence and levels of mucosal COX-2 and iNOS mRNAs by real time PCR.. Soybean extract amounts corresponding to 150μg/kg/day EGF caused considerable mortality, whereas those corresponding to 75μg/kg/day EGF were well tolerated. There was no significant difference in NEC scores between animals fed plain formula and formula supplemented with empty soybean extract. Soybean-EGF-supplemented formula at 75μg/kg/day EGF significantly decreased NEC, attenuated dissociation of JAM-A and ZO-1 proteins from tight junctions, and reduced intestinal expression of COX-2 and iNOS mRNAs.. Supplementation with soybean-expressed EGF significantly decreased NEC in the rat model. Soybean-expressed EGF may provide an economical solution for EGF administration and prophylaxis of clinical NEC.

Topics: Animals; Animals, Newborn; Cyclooxygenase 2; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; Glycine max; Humans; Infant Formula; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Intestinal Mucosa; Intestines; Junctional Adhesion Molecules; Nitric Oxide Synthase Type II; Plant Extracts; Protective Agents; Rats, Sprague-Dawley; Recombinant Proteins; RNA, Messenger; Zonula Occludens Proteins

Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother's breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N' terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.

Topics: Enterocolitis, Necrotizing; Epidermal Growth Factor; ErbB Receptors; Gastrointestinal Microbiome; Glycine max; HeLa Cells; Humans; Phosphorylation; Plants, Genetically Modified; Seeds

Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease that is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), whereas selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Overexpression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of glycogen synthase kinase-3β (GSK3β). Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis.

Topics: Animals; Apoptosis; Cell Line; Enterocolitis, Necrotizing; Enterocytes; Epidermal Growth Factor; ErbB Receptors; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Mice; Milk; Signal Transduction; Toll-Like Receptor 4

To evaluate epidermal growth factor (EGF) levels in cord blood as a possible marker for predicting the subsequent development of necrotizing enterocolitis (NEC) in very low birth weight (VLBW) infants.. A total of 241 VLBW infants (38 infants developed NEC and 203 had no NEC) were enrolled. EGF concentrations were measured by enzyme-linked immunosorbent assay.. VLBW infants who subsequently developed NEC had significantly lower cord serum concentrations of EGF than those who did not (p < 0.0001). Moreover, cord blood concentrations of EGF were significantly lower in infants with stage III than those with stage II or I NEC (p < 0.001). Logistic regression analysis demonstrated that low cord blood EGF was independently associated with the subsequent risk of NEC (OR = 0.978 [95% CI: 0.959-0.997]; p = 0.02).. Low cord blood EGF concentrations may predict the subsequent development of NEC in VLBW infants.

Topics: Enterocolitis, Necrotizing; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Fetal Blood; Humans; Infant, Premature; Infant, Very Low Birth Weight; Prospective Studies

CD40, a co-stimulatory molecule, plays a critical role in coordinating enteric inflammatory immune responses. In necrotizing enterocolitis (NEC), upregulation of IL-10, a CD40-modulated cytokine, has been described, but the role of the IL-10 receptor (IL-10Rβ), CD40, and its ligand CD40L in disease pathogenesis is unknown. The study herein investigates ileal expression of CD40, CD40L, and IL-10R in a rat model of NEC.. NEC was induced in newborn rats using established methods of formula feeding, asphyxia, and cold stress. Expression of CD40, CD40L, IL-10Rβ, and other proinflammatory molecules, including Toll-like receptor-4 (TLR-4) and IL-18, was assessed by immunoblotting. Tissue infiltration by macrophages, monocytes, and T cells was examined by confocal immunohistochemistry.. Ileum from rat pups with NEC showed increased expression of TLR-4, IL-18, and IL-10Rβ. Sections from both NEC and control pups demonstrated preservation of ileal cells expressing CD40/CD40L. The distal ileum of controls expressed both CD40 and CD40L; conversely, neither molecule was detected in ileal tissue from NEC pups. Additional studies showed that treatment with epidermal growth factor (EGF), previously shown to ameliorate the severity of NEC in an animal model, did not restore CD40 expression.. Ileal cytokine dysregulation, manifested by decreased CD40/CD40L and increased IL-10Rβ expression, may be involved in the pathogenesis of NEC. Dampened CD40 signaling may be related to enhanced IL-10 expression and a suppressed T-cell response to injury. We speculate that augmenting CD40-CD40L interactions may achieve a protective effect in this NEC model.

Topics: Animals; Blotting, Western; CD40 Antigens; CD40 Ligand; Enterocolitis, Necrotizing; Epidermal Growth Factor; Ileum; Inflammation; Interleukin-10 Receptor beta Subunit; Interleukin-18; Macrophages; Models, Animal; Monocytes; Rats; Rats, Sprague-Dawley; T-Lymphocytes; Toll-Like Receptor 4

Necrotizing enterocolitis (NEC) afflicts extremely low birth weight neonates, and probiotics reduces its incidence and severity. NO is involved in the pathogenesis of NEC, and caveolin-1 regulates NO signaling. We tested the hypothesis that intestinal caveolin-1 and NOS are deficient in formula-fed neonatal rats and that supplementation with "Florastar Kids" and/or galacto-oligosaccharides and fructo-oligosaccharides preserves caveolin-1 and NOS. At birth (P0), neonatal rat pups were maternally fed or hand-gavaged with or without supplemented formula. Samples from the terminal ileum were analyzed for total NO metabolites, growth factors, and gene expression of caveolin-1, NOS isoforms, and antioxidants. Our data showed that formula feeding with and without supplementation resulted in significant growth restriction. Despite suboptimal nutrition, growth factors involved in intestinal repair and regeneration were increased in the neonatal rat ileum. Caveolin-1, endothelial NOS, and neuronal NOS were simultaneously down-regulated with formula feeding while inducible NOS was up-regulated. Superoxide dismutase and glutathione peroxidase were up-regulated with supplementation. These data provide a probable mechanism for the benefits of supplemented formula for decreasing the severity of NEC by preserving the antioxidant systems.

Topics: Aging; Animals; Animals, Newborn; Antioxidants; Body Size; Caveolin 1; Enterocolitis, Necrotizing; Epidermal Growth Factor; Gene Expression Regulation; Glutathione Peroxidase; Humans; Ileum; Infant Formula; Infant, Newborn; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Prebiotics; Probiotics; Rats; Rats, Sprague-Dawley; RNA, Messenger; Superoxide Dismutase

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. Ultrastructural 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 EGF-mediated protection against NEC injury is associated with regulation of intestinal autophagy.

Topics: Administration, Oral; Animals; Animals, Newborn; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Cell Line; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; Gene Expression Regulation; Humans; Incidence; Intestinal Mucosa; Microtubule-Associated Proteins; Rats; Rats, Sprague-Dawley

We have previously demonstrated that enterally administered heparin-binding EGF-like growth factor (HB-EGF) produced in Escherichia coli decreases the incidence and severity of intestinal injury in a neonatal rat model of necrotizing enterocolitis (NEC). In preparation for upcoming human clinical trials, large-scale production of HB-EGF according to Good Manufacturing Practice (GMP) has been successfully accomplished using a Pichia pastoris yeast system. The current studies used a neonatal rat model of NEC to elucidate several important preclinical characteristics of HB-EGF therapy. We found that enteral administration of HB-EGF (800 microg/kg/dose) four times a day effectively reduced the incidence and severity of NEC, that Pichia-derived HB-EGF was not significantly different from E. coli-derived HB-EGF in preventing NEC, that EGF was not superior to HB-EGF in preventing NEC, and that prophylactic administration of HB-EGF added to formula starting with the first feed or 12 h later significantly reduced the incidence of NEC, with no change in the incidence of NEC noted if HB-EGF was added to the formula starting 24, 48, or 72 h after birth. Thus, large-scale production of GMP-grade HB-EGF in Pichia pastoris yeast produces a biologically active molecule suitable for human clinical trials.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Enteral Nutrition; Enterocolitis, Necrotizing; Epidermal Growth Factor; Escherichia coli; Gastrointestinal Agents; Heparin-binding EGF-like Growth Factor; Humans; Hypoxia; Intercellular Signaling Peptides and Proteins; Intestines; Lipopolysaccharides; Pichia; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Severity of Illness Index; Time Factors

Holder pasteurization renders donor human milk safe for consumption. Because human milk reduces the risk of necrotizing enterocolitis in preterm infants, we tested whether Holder pasteurization affects certain factors in human milk that protect the intestines: epidermal growth factor (EGF), transforming growth factor (TGF)-beta1, erythropoietin (EPO), and interleukin (IL)-10. Donor human milk from a milk bank was examined.. The aqueous phase of 17 samples of donor term human milk (mean duration of lactation, 8 +/- 3.5 months) was examined before and after Holder pasteurization. In the case of IL-10, lesser degrees of pasteurization were also evaluated. The agents were quantified using enzyme immunoassays. The function of IL-10 was also tested.. Concentrations of EGF and IL-10 were markedly lower than previously reported values in human milk from earlier phases of lactation. Holder pasteurization significantly reduced the concentrations of EPO and IL-10, whereas lesser degrees of heating increased the detection of IL-10. The immunosuppression of T-cell proliferation by human milk, thought to be attributed to IL-10 alone, persisted after Holder pasteurization.. Holder pasteurization greatly decreased concentrations of EPO and IL-10 in human milk. These decreases may impact the ability of human milk to protect against necrotizing enterocolitis. Evidence of possible binding of IL-10 to other proteins in human milk was also found. Experiments to test whether Holder pasteurization affects the function of IL-10 in human milk produced evidence for an agent in human milk other than IL-10 that inhibits T-cell proliferation and resists Holder pasteurization.

Topics: Adult; Enterocolitis, Necrotizing; Epidermal Growth Factor; Erythropoietin; Female; Food Preservation; Hot Temperature; Humans; Infant, Newborn; Infant, Premature; Infant, Premature, Diseases; Interleukin-10; Lactation; Milk, Human; Transforming Growth Factor beta1

Necrotizing enterocolitis (NEC) is a devastating disease of premature babies. Previously, we have shown that EGF reduces NEC and that overproduction of hepatic TNF-alpha is associated with intestinal damage. Leakage of TNF-alpha may be a consequence of epithelial hepatic cellular junction dysfunction. The aim of this study was to investigate changes in the composition of hepatic tight junctions (TJs) and adherens junctions (AJs). Using an established rat model of NEC, animals were divided into the following groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with EGF (EGF). Serum EGF and histologic localization of major TJ and AJ proteins were evaluated. Distribution patterns of hepatic TJ and AJ proteins were significantly altered in the NEC group compared with those in DF or EGF groups. Cytoplasmic accumulation of occludin, claudin-2, and ZO-1 with reduction of claudin-3 signal was detected in the liver of NEC rats. Localization of beta-catenin was associated with the hepatocyte membrane in EGF and DF groups, but diffused in the NEC group. These data show that hepatic cellular junctions are significantly altered during NEC pathogenesis. EGF-mediated reduction of experimental NEC is associated with protection of hepatic integrity and structure.

Topics: alpha Catenin; Animal Feed; Animals; beta Catenin; Cadherins; Claudin-1; Claudin-3; Claudins; Diet; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; Hepatocytes; Humans; Intercellular Junctions; Membrane Proteins; Occludin; Phosphoproteins; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha; Zonula Occludens-1 Protein; Zonula Occludens-2 Protein

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurely born infants. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) have protective effects against intestinal injury. The aim of this study was to compare the effect of oral administration of HB-EGF, EGF, or both on the incidence of NEC in a neonatal rat model.. Premature rats were fed by hand and exposed to asphyxia and cold stress to develop NEC. Four diets were used: formula (NEC), formula supplemented with 500 ng/mL HB-EGF (HB), 500 ng/mL EGF (EGF), or a combination of both (E+HB). Ileal injury, endogenous HB-EGF production, expression of EGF receptors, goblet cell density, and expression of apoptotic proteins were evaluated.. Oral administration of either EGF or HB-EGF significantly reduced the incidence of NEC; however, EGF provided better protection in physiologically relevant doses. Simultaneous administration of both growth factors did not result in any synergistic protective effect against NEC. There were no significant differences between treatment groups in ileal gene expression of EGF receptors or HB-EGF. However, the balance of apoptotic proteins in the ileum was shifted in favor of cell survival in EGF-treated rats. This mechanism may be responsible for the higher efficiency of EGF protection against NEC.. These data suggest that a physiological dosage of EGF or a pharmacological dosage of HB-EGF could be used for prevention of NEC.

Topics: Administration, Oral; Animals; Animals, Newborn; Apoptosis; Dose-Response Relationship, Drug; Drug Synergism; Enterocolitis, Necrotizing; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Ileum; Intercellular Signaling Peptides and Proteins; Random Allocation; Rats; Rats, Sprague-Dawley; Treatment Outcome

We have shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF) decreases experimental necrotizing enterocolitis (NEC). Intestinal epithelial cell (IEC) migration (restitution) and proliferation are key elements in recovery from intestinal injury. Here, we investigated whether the beneficial effects of HB-EGF are mediated, in part, by its ability to affect these processes.. Necrotizing enterocolitis was induced in newborn rats by exposure to stress (hypoxia, hypothermia, hypertonic feedings, and lipopolysaccharide), with pups receiving different doses of HB-EGF (0, 25, 50, 100, 200, 400, 600, and 800 microg/kg). To investigate the effect of HB-EGF on enterocyte proliferation and migration, bromodeoxyuridine was administered intraperitoneally 18 hours before sacrifice, with intestine subjected to bromodeoxy-uridine immunohistochemistry.. The incidence and severity of experimental NEC decreased, and the survival rate increased, with increasing doses of HB-EGF. Results were confirmed using scanning electron microscopy. Migration of IEC in breast-fed pups was 7.07 microm/h, decreased significantly to 2.29 microm/h in stressed pups, and was significantly improved at 5.95 microm/h in pups subjected to stress but treated with HB-EGF (P < .05). Quantification of IEC proliferation revealed 208 (+) cells per high-power field (HPF) in breast-fed pups, which decreased significantly to 99 (+) cells per HPF in stressed pups and increased to 190 (+) cells per HPF in stressed pups treated with HB-EGF (P < .05).. These results demonstrate that HB-EGF protects newborn rats from experimental NEC in a dose-dependent fashion. The ability of HB-EGF to protect the intestines from NEC is due, in part, to the ability of HB-EGF to preserve enterocyte migration and proliferation.

Topics: Animals; Animals, Newborn; Cell Movement; Cell Proliferation; Disease Models, Animal; Enterocolitis, Necrotizing; Enterocytes; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Rats; Rats, Sprague-Dawley

Topics: Age Factors; Animals; Biomarkers; Dietary Supplements; Enterocolitis, Necrotizing; Epidermal Growth Factor; Gestational Age; Humans; Infant, Newborn; Predictive Value of Tests

To examine the ontogeny of salivary epidermal growth factor (sEGF) in premature infants and to determine the relation of sEGF to the development of necrotizing enterocolitis (NEC).. Salivary EGF was prospectively measured in 327 infants with gestational ages from 23 weeks to term. Infants of < or = 32 weeks' gestation (n = 261) were followed with weekly sEGF measurements through 3 weeks of life. Multivariable regression analyses were used to determine variables significantly related to sEGF levels and to identify predictors of NEC.. Over the first 3 weeks of life, sEGF increased across gestational age and postnatal age categories. In multivariable models, gestational age was a significant predictor of sEGF levels (P < .009). In a cohort of 27 infants who had NEC, gestational age, race, and changes in sEGF levels between weeks of life 1 and 2 were predictive of the development of NEC. These infants had lower sEGF at week 1 and greater increases from week 1 to week 2 compared with infants without NEC.. There is a positive relation between sEGF levels and gestational age. Patterns of sEGF levels over the first 2 weeks of life were significantly related to development of NEC in very low birth weight infants.

Topics: Age Factors; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Apgar Score; Cohort Studies; Enterocolitis, Necrotizing; Epidermal Growth Factor; Female; Gestational Age; Humans; Indomethacin; Infant Formula; Infant, Newborn; Infant, Premature; Male; Milk, Human; Multivariate Analysis; Odds Ratio; Ohio; Prospective Studies; Racial Groups; Regression Analysis; Risk Factors; Saliva

Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We recently demonstrated that the gut/liver axis plays an important role in the pathophysiology of NEC through the release of inflammatory mediators into the intestinal lumen. We have also shown that supplementation of formula with epidermal growth factor (EGF) dramatically decreases ileal pathology associated with experimental NEC. In this study, we examined the effects of EGF on the liver portion of the gut/liver axis in the neonatal rat model of NEC.. Newborn rats were divided into three experimental groups, NEC, hand-fed with growth-factor free formula; NEC + EGF, hand-fed with formula supplemented with 500 ng/ml rat EGF; or DF, dam fed. All animals were exposed to asphyxia and cold stress twice daily for 4 days to develop NEC.. EGF receptor expression was significantly (p

Topics: Animals; Animals, Newborn; Cytokines; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Liver; NF-kappa B; Protein Transport; Rats; Rats, Sprague-Dawley

We have previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent. The aim of this study was to determine the effect of enterally administered HB-EGF on the incidence of necrotizing enterocolitis (NEC) in neonatal rats.. Necrotizing enterocolitis was induced in neonatal rats delivered by C-section on day 21 of gestation by exposure to repeated cycles of hypoxia and hypothermia plus administration of hypertonic formula feeding (HHHTF) plus enteral administration of lipopolysaccharide (LPS) (2 mg/kg). Neonatal rats were randomly assigned to breast-feeding, hypertonic formula feeding, HHHTF + LPS, and HHHTF + LPS with HB-EGF (600 mug/kg) supplementation in the formula. Animals were monitored until 96 hours of life and assessed for death, histological NEC, and intestinal mucosal permeability.. The incidence of NEC in the HHHTF group was higher than that in the breast-feeding or hypertonic formula feeding groups. With administration of HB-EGF, the incidence and severity of NEC were significantly decreased. Administration of HB-EGF also increased rat pup survival rate and extended survival time. In addition, treatment with HB-EGF significantly decreased intestinal permeability to fluorescein isothiocyanate-dextran.. We conclude that HB-EGF reduces the incidence and severity of NEC in a neonatal rat model, with simultaneous preservation of gut barrier integrity. These results support our contention that HB-EGF administration may represent a useful therapeutic and prophylactic therapy for the treatment of NEC.

Topics: Administration, Oral; Animals; Animals, Newborn; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Permeability; Random Allocation; Rats; Rats, Sprague-Dawley; Severity of Illness Index

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. While the effect of bile acids (BAs) on intestinal mucosal injury is known, we investigated the contribution of BAs during the development of NEC in neonatal rats.. Premature rats were fed with cow's milk-based formula and subjected to asphyxia and cold stress to develop NEC. Jejunal and ileal luminal BAs, portal blood BAs, and messenger RNA and protein for the apical sodium-dependent bile acid transporter, the ileal bile acid binding protein, and the heteromeric organic solute transporter (Ostalpha/Ostbeta)were evaluated.. Ileal luminal BAs levels were increased significantly during disease development and the removal of ileal BAs significantly decreased the incidence and severity of disease. Furthermore, when NEC was reduced via treatment with epidermal growth factor (EGF), BA levels were reduced significantly. Jejunal luminal BA levels were similar between animals with NEC and controls, but portal/ileal luminal BA ratios were decreased significantly in animals with NEC. The apical sodium-dependent bile acid transporter was up-regulated at the site of injury in animals with NEC and decreased after EGF treatment; however, the ileal bile acid binding protein was up-regulated only in the NEC and EGF group. Ostalpha/Ostbeta expression was low in all groups, and only slightly increased in the NEC group.. These data strongly suggest that BAs play a role in the development of ileal damage in experimental NEC and that alterations in BA transport in the neonatal ileum may contribute to disease development.

Topics: Animals; Bile Acids and Salts; Disease Models, Animal; DNA Primers; Enterocolitis, Necrotizing; Epidermal Growth Factor; Humans; Ileum; Infant, Newborn; Infant, Premature; Intestinal Mucosa; Jejunum; Portal System; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

We have previously demonstrated that enterally administered heparin-binding epidermal growth factor-like growth factor (HB-EGF) decreases the incidence and severity of necrotizing enterocolitis (NEC) in a neonatal rat model. Because apoptosis contributes to gut barrier failure in this model, the aim of this study was to investigate the effect of HB-EGF on apoptosis during the development of NEC.. NEC was induced in neonatal rats by exposure to hypoxia, hypothermia, hypertonic formula feeding (HHHTF) plus enteral administration of lipopolysaccharide (LPS). Fifty-one neonatal rats were randomly divided into the following groups: (1) breast-fed (BF), (2) HHHTF + LPS, and (3) HHHTF + LPS with HB-EGF (600 microg/kg) added to the formula. NEC was evaluated using a standard histological scoring system. Apoptotic cells in intestinal tissues were detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and by active caspase 3 immunohistochemical staining.. The incidence of NEC in the HHHTF + LPS group was higher than that in the BF group (65% vs 0%, P < .05). With administration of HB-EGF, the incidence of NEC significantly decreased to 23.8% (P < .05). The median TUNEL and active caspase 3 scores in the HHHTF + LPS group were higher than those in the BF group (1.9 vs 0.9 and 1.75 vs 0.6, respectively, P < .05). The median TUNEL and active caspase 3 scores were significantly decreased in the HHHTF + LPS + HB-EGF group compared with the HHHTF + LPS group (1.24 vs 1.9 and 1.0 vs 1.75, respectively, P < .05).. HB-EGF reduces the incidence of NEC in a neonatal rat model in part by decreasing apoptosis. These results support the use of HB-EGF-based clinical regimens for the treatment of NEC.

Topics: Animals; Animals, Newborn; Apoptosis; Enterocolitis, Necrotizing; Epidermal Growth Factor; Heparin; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Rats; Rats, Sprague-Dawley

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.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Count; Claudin-3; Enterocolitis, Necrotizing; Epidermal Growth Factor; Fluorescent Antibody Technique; Goblet Cells; Immunohistochemistry; Intestinal Mucosa; Membrane Proteins; Microscopy, Electron, Scanning; Mucin-2; Mucins; Occludin; Permeability; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA; Survival Rate

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.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cell Division; Corticosterone; Enterocolitis, Necrotizing; Epidermal Growth Factor; Ileum; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; RNA, Messenger

To determine whether diminished levels of epidermal growth factor (EGF) were present in neo-natal rats with intestinal injury and related with the degree of intestinal injury, so we modeled a model in neo-natal rats of intestinal injury and to examine the dynamic levels of EGF on injury of intestine.. One-day-old Wistar rat pups received an intraperitoneally injection with 4 mg/kg lipopolysaccharide (LPS), followed by collection of ileum tissue at 1, 3, 6, 12, and 24 h following LPS administration. The ileum was for histological evaluation of NEC and for measurements of EGF using ABC-ELISA. The correlation between the degree of intestinal injury and levels of EGF was determined.. The LPS-injected pups also showed a significant increase in injury scores at 1, 3, 6, 12, and 24 h (respectively, (1.08+/-0.61), (1.63+/-0.84), (1.95+/-0.72), (2.42+/-0.43) and (2.21+/-0.53)) vs the control (0.12+/-0.17) (P<0.01). EGF levels at 1, 3, 6, 12 h (respectively, (245.6+/-49.0), (221.4+/-39.0), (223.4+/-48.1), (246.0+/-46.6)) pg/mg were significantly loss than the control (275.6+/-50.4) pg/mg (P<0.05). There was a significant negative correlation between the EGF levels and the grade of intestinal injury within 24 h (P<0.05).. Neo-natal rats with intestinal injury have significantly lower levels of ileum EGF. Reduced levels of this growth factor might be related to the pathogenesis of NEC.

Topics: Animals; Animals, Newborn; Enterocolitis, Necrotizing; Epidermal Growth Factor; Ileum; Intestinal Mucosa; Lipopolysaccharides; Rats; Rats, Wistar

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We have shown in previous studies that proinflammatory interleukin-18 and interleukin-12 are up-regulated in the ileum of rats with experimental NEC and that epidermal growth factor (EGF) reduces the development of disease. Here we investigated whether the protective effects of EGF are a result of changes in ileal interleukin-18, interleukin-12 and/or antiinflammatory interleukin-10.. Newborn rats were artificially fed with either growth-factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/mL EGF (RMS + EGF) and NEC was induced via exposure to asphyxia and cold stress. Cytokine expression and localization were assessed using reverse-transcription real-time polymerase chain reaction and immunohistology/confocal microscopy.. Enteral administration of EGF (RMS + EGF) decreased overproduction of interleukin-18 and increased interleukin-10 production in the ileum. Furthermore, increased interleukin-10 production was associated with up-regulation of the transcription factor Sp1 in RMS + EGF rats.. These data suggest that EGF may reduce NEC via increased interleukin-10 and decreased interleukin-18 and that EGF-mediated up-regulation of Sp1 may account for the increased interleukin-10.

Topics: Animals; Animals, Newborn; Cytokines; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; Ileum; Immunohistochemistry; Interleukins; Microscopy, Confocal; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction

Necrotizing enterocolitis (NEC) seems to result from the inflammatory response of an immature intestine. Human milk is protective against NEC via an unknown mechanism. We hypothesized that specific factors found in human milk would decrease stimulated IL-8 secretion in intestinal epithelial cells. HT29-cl19A and Caco2 cells were compared with the fetal human primary intestinal epithelial cell line H4 and temperature-sensitive conditionally immortalized fetal human intestinal (tsFHI) cells. Cells were pretreated with transforming growth factor-beta (TGF-beta), erythropoietin (Epo), IL-10, or epidermal growth factor (EGF) at physiologic concentrations before stimulation with tumor necrosis factor-alpha (TNF-alpha) or IL-1beta, and then IL-8 was measured by ELISA. The fetal cells produced significantly more IL-8 when stimulated by TNF-alpha or IL-1beta. There were also differences in the pattern of alteration of IL-8 secretion by human milk factors. In HT29-cl19A cells, IL-10 inhibited TNF-alpha-stimulated IL-8 secretion by 52%, and EGF increased secretion by 144%. In H4 cells, TGF-beta1 and Epo inhibited TNF-alpha-stimulated IL-8 secretion to control levels, and EGF increased secretion by 29%. IL-1beta-stimulated IL-8 secretion was inhibited 25% by TGF-beta1 in Caco2 cells and in H4 cells was inhibited by TGF-beta1, Epo, and TGF-beta2. TsFHI cells confirmed H4 cell results. Fetal human enterocytes have an exaggerated IL-8 secretion in response to TNF-alpha and IL-1beta. TGF-beta and Epo decrease this stimulated IL-8 secretion, which may partially explain the protective effect of human milk in NEC.

Topics: Age Factors; Caco-2 Cells; Enterocolitis, Necrotizing; Epidermal Growth Factor; Epithelial Cells; Erythropoietin; Fetus; HT29 Cells; Humans; In Vitro Techniques; Interleukin-1; Interleukin-10; Interleukin-8; Intestinal Mucosa; Milk, Human; Transforming Growth Factor beta

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.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Enterocolitis, Necrotizing; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Ileum; Incidence; Milk; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor alpha; Weight Gain

Topics: Animals; Animals, Newborn; Enterocolitis, Necrotizing; Epidermal Growth Factor; Milk; Rats; Saliva

Because epidermal growth factor (EGF) is trophic to the intestinal mucosa, and neonatal necrotizing enterocolitis (NEC) is associated with a disrupted intestinal mucosal barrier, the authors sought to determine whether diminished levels of EGF were present in infants with NEC.. Saliva, serum, and urine specimens were obtained from infants with NEC during a 3-year period (February 1995 to May 1998). Control patients without NEC were chosen based on similar postnatal age and birthweight. EGF levels were determined by enzyme-linked immunosorbent assay (ELISA). Differences between groups were compared using Mann-Whitney Rank sum test with P less than .05 considered significant. Results are presented as mean values +/-SEM.. Twenty-five infants with NEC were compared with 19 control patients. Birth weight (1,616+/-238 g control v. 1,271+/-124 g NEC) and postnatal age (23+/-6 days control v. 22+/-3 days NEC) were similar. Infants with NEC had significantly lower levels of EGF in both saliva (590+/-80 pg/mL control v. 239+/-41 pg/mL NEC; P<.001) and serum (35+/-8 pg/mL control v. 5.6+/-1.9 pg/mL NEC; P<.001). Urinary EGF was also lower in the NEC group, but was not statistically significant.. Premature infants with NEC have significantly diminished levels of salivary and serum EGF. Reduced levels of this growth factor may distinguish infants at risk for NEC and play a pivotal role in the pathogenesis of the perturbed intestinal mucosal barrier that is central to this condition.

Topics: Enterocolitis, Necrotizing; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Humans; Infant; Prospective Studies; Saliva