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.

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at "canonical" position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1' substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.

Cytokines and growth factors play diverse roles in the uninflamed fetal/neonatal intestinal mucosa and in the development of inflammatory bowel injury during necrotizing enterocolitis (NEC). During gestational development and the early neonatal period, the fetal/premature intestine is exposed to high levels of many "inflammatory" cytokines and growth factors, first via swallowed amniotic fluid in utero and then, after birth, in colostrum and mother's milk. This article reviews the dual, seemingly counter-intuitive roles of cytokines, where these agents play a "trophic" role and promote maturation of the uninflamed mucosa, but can also cause inflammation and promote intestinal injury during NEC.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease resulting in major neonatal morbidity and mortality. The pathology is poorly understood, and the means of preventing and treating NEC are limited. Several endogenous growth factors have been identified as having important roles in intestinal growth as well as aiding intestinal repair from injury or inflammation. In this review, we will discuss several growth factors as mediators of intestinal regeneration and repair as well as potential therapeutic agents for NEC.

In addition to supplying essential nutrients, some food proteins can confer additional health benefits beyond nutrition. The presence of bioactive proteins and peptides in different foods is a factor not currently taken into consideration when assessing the dietary quality of food proteins. The range of described physiological benefits attributed to bioactive proteins and peptides is diverse. Multiple factors can potentially impact on the ability of a bioactive peptide or protein to elicit an effect. Although some food proteins act directly in their intact form to elicit their effects, generally it is peptides derived from digestion, hydrolysis or fermentation that are of most interest. The levels of bioactive peptides generated must be sufficient to elicit a response, but should not be so high as to be unsafe, thus causing negative effects. In addition, some peptides cause systemic effects and therefore must be absorbed, again in sufficient amounts to elicit their action. Many studies to date have been carried out in vitro; therefore it is important that further trials are conducted in vivo to assess efficacy, dose response and safety of the peptides, particularly if health related claims are to be made. Therefore, methods must be developed and standardised that enable the measurement of health benefits and also the level of bioactive peptides which are absorbed into the bloodstream. Once standardised, such methods may provide a new perspective and an additional mechanism for analysing protein quality which is currently not encompassed by the use of the protein digestibility-corrected amino acid score (PDCAAS).

Premature birth rates and premature infant morbidity remain discouragingly high. Improving nourishment for these infants is the key for accelerating their development and decreasing disease risk. Dietary protein is essential for growth and development of infants. Studies on protein nourishment for premature infants have focused on protein requirements for catch-up growth, nitrogen balance, and digestive protease concentrations and activities. However, little is known about the processes and products of protein digestion in the premature infant. This review briefly summarizes the protein requirements of term and preterm infants, and the protein content of milk from women delivering preterm and at term. An in-depth review is presented of the current knowledge of term and preterm infant dietary protein digestion, including human milk protease and anti-protease concentrations; neonatal intestinal pH, and enzyme activities and concentrations; and protein fermentation by intestinal bacteria. The advantages and disadvantages of incomplete protein digestion as well as factors that increase resistance to proteolysis of particular proteins are discussed. In order to better understand protein digestion in preterm and term infants, future studies should examine protein and peptide fragment products of digestion in saliva, gastric, intestinal and fecal samples, as well as the effects of the gut micro biome on protein degradation. The confluence of new mass spectrometry technology and new bioinformatics programs will now allow thorough identification of the array of peptides produced in the infant as they are digested.

Maternal milk plays an important role in breast milk jaundice (BMJ) development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to investigate whether there is a relationship between EGF levels in the infant serum and in the milk of nursing mothers and BMJ. Two groups were defined: study group (n = 30), newborns who were followed up for BMJ without any identifiable pathologic cause; control group, healthy newborns whose serum total bilirubin levels were <10 mg/dL. Milk and infant plasma samples were collected between the third and the fourth postpartum week. EGF concentrations in all of the samples were determined by using ELISA. The infants with BMJ had higher concentrations of EGF in the serum and in the breast milk compared with that of the infants without BMJ. The milk concentrations of EGF were significantly correlated with neonatal bilirubin and blood EGF concentrations. The degree of BMJ was associated with the increased levels of milk borne EGF. Although the exact mechanisms of the hyperbilirubinemic action of EGF are not completely known, the inhibition of gastric motility, increased absorption, and activation of bilirubin transport have been suggested as possible mechanisms.

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.

The human fetus and neonate swallow biologically significant quantities of IL-8/CXC ligand 8 (CXCL8) in amniotic fluid and breast milk, and this remains measurable through simulated neonatal gastric and proximal intestinal digestions. We sought to confirm the structural and functional integrity of IL-8/CXCL8 in digestates and determine the mechanisms underlying this protease resistance. We observed that in comparison with BSA, IL-8/CXCL8 is highly resistant to pepsin and can be detected intact in assays for structural, immunologic, and functional integrity. In a computational molecular docking simulation, IL-8/CXCL8 was observed to fit poorly in the pepsin active site. On the basis of simulated mutation analyses, we hypothesized that this protease resistance is due to disulfide bond-related tertiary folding in IL-8/CXCL8. This was confirmed on chemical reduction of these groups.

Villous development of the intestine is beginning to be understood in terms of the underlying molecular mechanisms. There is increasing information on the role of cytokines as extrinsic regulators in this process. This article summarizes information available on various cytokines that have been studied in this context.

Maternal milk is the major source of nutrients and growth-promoting substances in the first weeks of life for the majority of neonates. Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are trophic peptides present in human milk with significant healing effects on injured gastrointestinal mucosa. Decreasing gestational age of neonates is associated with higher risk of developing gastrointestinal disorders, and human milk provides better protection against these diseases compared with formula. The aim of this study was to evaluate the concentrations of EGF and TGF-alpha in human milk collected from mothers with infants born: extremely preterm, preterm, and full term. Milk samples were collected at the end of first, second, and fourth week postpartum from each mother of infants born in one of the three gestational age groups: extremely preterm (23-27 wk, n = 16), preterm (32-36 wk, n = 16), and full term (38-42 wk, n = 15). Milk concentrations of EGF and TGF-alpha were quantified with a homologous RIA in the milk aqueous fraction. Concentrations of EGF in human milk from the extremely preterm group (23-27 wk) were significantly higher compared with values from the preterm and full-term groups throughout the first month of lactation. A similar pattern was observed with human milk TGF-alpha; however, milk TGF-alpha levels were lower than EGF. In conclusion, we have found higher concentrations of EGF and TGF-alpha in human milk of mothers with extremely preterm babies. These data may indicate the potential importance of milk-borne EGF and TGF-alpha for the development of extremely premature infants.

By 20 wk of gestation, the human fetal gastrointestinal (GI) tract morphologically resembles that of the term infant, but functional development is limited before 26 wk. By 30 wk of gestation, the fetus has the capacity for limited digestion and enteral absorption. GI growth and development continue postnatally. Trophic factors, including nutrients, peptides, hormones and growth factors, are recognized as having important influences on the morphology and histology of the developing GI tract. Other trophic factors are important in adaptation and repair following injury. Many such factors are provided in utero via amniotic fluid swallowing and later by human colostrum and milk.

This review discusses cytokines with known GI trophic effects, either in vitro or in vivo, and focuses on those cytokines that have been used in the neonatal intensive care unit.

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB(0) mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (V(max)) without altering the transporter apparent affinity (K(m)). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units.

Epidermal growth factor (EGF) in intestinal lumen regulates many gut epithelial cell functions. Influenced by growth factors at various differentiation stages, enterocytes execute the major task of absorbing nutrient amino acids. The purpose of this study was to investigate the effects of EGF on Na(+)-independent L-alanine transport in intestinal epithelial cells. Na(+)-independent [3H]-L-alanine transport was measured in the differentiating Caco-2 cells. In both the undifferentiated and differentiated states, L-alanine uptake occurred via a single saturable Na(+)-independent system L plus simple passive diffusion. System L activity decreased as the cells progressed from the undifferentiated to the differentiated state. Prolonged incubation with EGF (>30 hours) resulted in a 70% increase in system L activity in both undifferentiated and differentiated cells. EGF stimulated the system L V(max) without affecting K(m). System L activity stimulation was inhibited by chelerythrine chloride, cycloheximide, or actinomycin D. These data suggest that intestinal epithelial cell differentiation is associated with a decrease in system L transport capacity. EGF activates system L transport activity through a signaling mechanism involving protein kinase C, independent of cell differentiation state. Both cell differentiation and EGF regulation of system L activity occur via alteration of functional copies of the system L transporter.

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.

The maintenance of gastrointestinal epithelium integrity requires a fine balance between proliferation and differentiation as well as protection against gastric acid secretion. Transforming growth factor-alpha (TGF-alpha) regulates these functions by binding to epidermal growth factor receptor (EGF-R). This study was designed to identify the localization of TGF-alpha and EGF-R in the rat gastroduodenal region. In the stomach, the surface and gastric pit cells showed staining for TGF-alpha antibodies in the cytoplasm and basolateral and apical membranes. TGF-alpha and EGF-R were observed in the supranuclear region of the cells lining the gland. In the duodenum, the enterocytes coexpressed both TGF-alpha and EGF-R in the supranuclear area. The EGF-R was also observed in the apical membrane. Brunner's glands were positive for both TGF-alpha and EGF-R antibodies. Our results demonstrate the coexpression of TGF-alpha and EGF-R in the rat gastroduodenal area, which suggests a functional role for them in the establishment and maintenance of the epithelial renewal.

The presence of motilin in human milk and the influence of human milk on the degradation of [125I][Nle13] porcine motilin by gastric and duodenal fluids were investigated. Milk and plasma samples were collected from 14 mothers, and motilin was measured by radioimmunoassay. Plasma levels were 416 +/- 37 pg/mL. In 8 defatted samples the motilin level was 105 +/- 14 pg/mL, in the six others levels were above 1000 pg/mL but dilution curves were non-linear. After solid-phase extraction milk levels were 108 +/- 21 pg/mL in 13 samples, in 1 sample the dilution curve was still non-linear. The stability of motilin after ingestion was studied in vitro by incubating [121I][Nle13] porcine motilin with gastric and intestinal juices obtained from newborns (10 times diluted). Incubations were performed at 37 degrees C at pH 1.8, 3.2 and 5.8 for the gastric fluid and at pH 7.4 for the duodenal fluid. After different times of intervals (5, 10, 20 and 30 minutes) intact motilin was precipitated with trichloroacetic acid and the radioactivity of the supernatant was determined. Motilin was rapidly degraded by gastric juice. The breakdown was greatest at pH 3.2 (74% after 30 minutes) and lowest at pH 5.8 (29%), the pH after milk feeding in neonates. Degradation by intestinal juice at pH 7.4 was also very rapid (77% after 30 minutes). Human milk and BSA inhibited partially the gastric digestion at pH 3.2 (17 and 29%, respectively). Digestion by intestinal juice was not affected by human milk and BSA. These results suggest that digestion of motilin in the stomach may be sufficiently retarded by human milk in the newborn to exert a biological role.

Stability and distribution of orally administered epidermal growth factor (EGF) were examined in newborn and 5-day-old pigs. Forty-five minutes after oral administration of iodine-125 labeled EGF, 60 and 50% of the radioactivity administered were recovered from the internal organs in newborn and 5-day-old pigs, respectively. In both age groups, over 95% of the recovered radioactivity was found in the gastrointestinal tract, of which 78-86% was found in the luminal contents with the remaining found in the gastrointestinal wall. Within the gastrointestinal tract, 65-71% of radioactivity was found in the stomach, 27-30% in the proximal and mid small intestine and 3-4% was found in the distal part of the small intestine. There were no significant differences in the overall distribution of orally administered radioactivity between two age groups. Based on liquid chromatography and trichloroacetic acid precipitation, a substantial amount of EGF recovered from the luminal contents (63-86%) and the gastrointestinal wall (42-81%) remained "intact". The receptor binding ability of the EGF recovered from the gastric contents was 96-102% comparable to the native EGF tracer. The receptor binding ability remained high (40-58%) in the proximal small intestinal lumen and it decreased to 15% in the distal small intestinal lumen in newborn pigs. In 5-day-old pigs, EGF recovered from the small intestinal contents had 5 to 24% receptor binding ability when compared with native EGF tracer. The receptor binding ability of the EGF recovered from all other organs was below 5% with an exception of the gastric wall, from which recovered EGF retained 9 to 26% receptor binding ability. These results indicate that most of orally ingested EGF remained in the gastrointestinal tract in neonatal pigs 45 min after oral ingestion, and significant amount of the ingested EGF remained biologically active. It suggests that milk-borne EGF can survive in the gastrointestinal tract and may play a role in regulating gut development in neonatal animals.

In addition to its content of traditional nutrients, milk is a rich source of hormones and peptides, which survive digestion in the neonatal gastrointestinal tract secondary to lower proteolytic activity and increased protein permeability. Previous studies have shown accelerated erythropoiesis or elevated serum erythropoietin (Epo) levels in neonatal (suckling) animals after maternal phlebotomy or maternal hypoxia exposure. We sought to determine whether significant quantities of Epo are present in human milk and whether Epo remains intact under physiologic digestion conditions. Immunoreactive Epo concentrations were determined in 409 human milk samples obtained from mothers of term and premature infants. Samples collected between birth and postpartum d 134 were divided into 11 postpartum day groups. Mean milk-borne Epo concentrations were within the normal range for plasma Epo concentrations and rose with postpartum day (F10,398 = 5.82, p < 0.0001). No differences were observed between milk collected from mothers of premature versus term infants. Estimated weekly human milk-borne Epo intakes approximated the lower range of published parenteral therapeutic doses. In simulated digestion at physiologic pH levels of 3.2, 5.8, and 7.4, milk-borne Epo resisted degradation at 1 and 2 h, compared with baseline. Therefore, we conclude that human milk contains considerable amounts of Epo which resist degradation after exposure to gastric juices at physiologic pH levels. These results support continued investigation into the fate and developmental roles of Epo in human milk.

Degradation of epidermal growth factor (EGF) in human gastric and duodenal lumen was analyzed by incubating 125I-labeled or unlabeled human recombinant EGF with human gastric or duodenal luminal fluids in vitro. Degradation of EGF was assessed by measuring the generation of acid soluble radioactivity or by reversed-phase high-performance liquid chromatography (HPLC). Incubation with gastric luminal fluids resulted in a time- and dose-dependent degradation of labeled and unlabeled EGF at pH 2.5 but not at pH 7.5. Duodenal luminal fluids, on the other hand, degraded EGF at pH 7.5 but not at pH 2.5. The rate of degradation of unlabeled EGF in gastric luminal fluids was nearly 12-fold higher than the rate of degradation of labeled EGF, whereas only a slight difference in rates of degradation of labeled and unlabeled EGF was observed in duodenal luminal fluids. High-performance liquid chromatography analysis detected three major degradation products that eluted with retention time of 17.5 min, 20.0 min, and 22.5 min that was associated with a reduction of intact EGF (retention time 23.5 min). Defatted and decaseinated supernatant of bovine milk effectively inhibited the degradation of EGF in both gastric and duodenal luminal fluids. Dietary derived protease inhibitors, such as soya bean trypsin inhibitor, lima bean trypsin inhibitor, egg white protease inhibitor, and Bowman-Birk protease inhibitor prevented EGF degradation in duodenal luminal fluids but failed to inhibit EGF degradation in gastric luminal fluids. These results suggest that bovine milk may contain specific inhibitors that protect EGF from proteolytic degradation in human gastric lumen.

This study was performed to define the biologically active growth modulators in human gastric juice. Mitogenic activity was evaluated by the incorporation of [3H]thymidine into 3T3 fibroblasts. A negative correlation was observed between pH and mitogenic activity in gastric juice (r = -0.45, P < 0.01). The concentrations of epidermal growth factor (EGF), transforming growth factor-alpha and -beta 1 (TGF-alpha and -beta 1), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) in gastric juice did not explain these changes in mitogenic activity. Gel filtration identified growth-stimulating activity due to small molecule mitogens (less than 13 kDa), and growth inhibitory activity only in neutral samples due to a macromolecular substance (larger than 240 kDa) susceptible to trypsin digestion and heat and acid treatments. We conclude that acidity-dependent changes in mitogenic activity observed in this study are due to appearance of acid-unstable, high-molecular-weight, growth-inhibitory substance.

To determine whether epidermal growth factor (EGF) or the related transforming growth factor alpha (TGF alpha) may have a role in the developing human stomach; to substantiate the presence of EGF in human liquor in the non-stressed infant and whether EGF in amniotic fluid is maternally or fetally derived.

The temporal expression and localisation of EGF, TGF alpha, and their receptors during fetal and neonatal life were examined in 20 fetal and five infant stomachs. Simultaneously, samples of amniotic fluid and fetal urine from 10 newborn infants were collected and assayed for EGF by radioimmunoassay.

EGF immunoreactivity was not noted in any of the specimens examined. In contrast, TGF alpha immunoreactivity was shown in mucous cells from 18 weeks of gestation onwards. EGF receptor immunoreactivity was seen on superficial mucous cells in gastric mucosa from 18 weeks of gestation onwards. The median concentration of EGF was 30 and 8.5 pg/ml in amniotic fluid and fetal urine, respectively, suggesting that EGF is not produced by the fetus.

This study adds weight to the hypothesis that swallowed EGF, probably produced by the amniotic membranes, and locally produced TGF alpha, may have a role in the growth and maturation of the human stomach.

To determine if milk-borne insulinlike growth factor-I (IGF-I) is absorbed in neonatal piglets, recombinant human IGF-I was iodinated and then administered via an oral-gastric tube to newborn and 3-day-old piglets together with fluorescent isothiocyanate dextran (FITC-D70s, a macromolecular marker for assessment of gut closure). Results showed that total radioactivity and trichloroacetic acid (TCA)-precipitable radioactivity rose significantly in the plasma of both newborn and 3-day-old piglets 1 hour after oral-gastric administration of 125I-IGF-I, although the magnitude of increase was greater in newborns than in 3-day-old piglets. In contrast, significant absorption of FITC-D70s was observed only in newborn piglets but not in 3-day-old piglets. Chromatographic analysis revealed that 125I-IGF-I represented up to 20% of total plasma radioactivity in the newborns and up to 10% in the 3-day-old piglets. The 125I-IGF-I found in the plasma was mostly bound to IGF-binding proteins. These results indicate that milk-borne IGF-I can be absorbed in neonatal piglets and the absorption is independent of gut closure.

This article reviews the presence and potential physiological significance of hormones and hormonally active substances (including growth factors) in human milk. Human milk has been found to contain several nonpeptide hormones and many peptide hormones and growth factors. In contrast to human breast milk, infant formulae lack some hormonally active peptides. There is little data concerning the effects of these agents on human neonates. Studies in immature experimental animals showing effects of orogastically administered hormones are summarized. The problems of supplementation of infant formula are discussed. Since hormones are present in the milk as a "cocktail" of potentially agonistic and antagonistic substances, one question is whether supplementation with a single agent would disturb this balance.

The stability of epidermal growth factor (EGF) in the gastrointestinal humen of 3-day-old suckling and 45-day-old weaned pigs was examined by incubating iodine labeled recombinant human EGF (hEGF) in the gastrointestinal fluids at 37 degrees C and monitoring the generation of acid soluble radioactivity. Chromatographic analysis and receptor binding study were also undertaken. The results showed that hEGF was quite stable in the gastric fluids in both suckling and weaned pigs with less than 20% degradation after 20 min incubation. The degradation rate in the intestinal fluids varied with the region of the small intestine and the age of the animals. At the proximal and mid regions of the small intestine in suckling pigs the degradation rate of hEGF after 20 min incubation was 5 and 20% respectively, while the degradation rate at the distal region was up to 50%. In the small intestinal lumen in weaned pigs the degradation rate of hEGF was much greater than that in suckling pigs, and the degradation rates at the proximal, mid and distal regions were 33, 51 and 56% respectively. Addition of acid soluble or casein fractions of porcine colostrum markedly reduced the degradation of hEGF in the intestinal fluids. These results indicate that milk-borne EGF is stable in the gastric and proximal intestinal humen in suckling pigs, and may play a role in regulating postnatal development in the suckling young.

Salivary epidermal growth factor (EGF) retains its biologic function in gastric juice and may play a physiologic role. Little is know, however, about the existence of mitogens other than EGF and the constitutional alterations of these factors in gastric juice by anti-secretagogues.

The mitogenic activity was evaluated by measuring [3H]-thymidine incorporation, and the EGF contribution was determined by using a specific anti-rat EGF antibody. An H2-receptor antagonist (IT-066) and a proton pump inhibitor (omeprazole) were used to determine whether these drugs alter the relative composition of active mitogens in gastric juice.

Normal gastric juice significantly increased DNA synthesis. This activity was suppressed by antibody (87-88%). Both drugs increased EGF concentrations and activity dose-dependently IT-066 specifically increased total amount and activity of EGF. Approximately 50% of this activity was reduced by boiling or antibody.

The major mitogenic activity of normal rat gastric juice depends on EGF, and antisecretory drugs enhance the mitogenic activity by preserving and including intraluminal mitogens than EGF.

The regulation of plasma membrane L-arginine transport activity was investigated in differentiated and undifferentiated states of the human intestinal cell line, Caco-2. The sodium-independent, leucine-insensitive uptake of L-arginine measured in this study has been assigned by us previously to system y+ in Caco-2 cells. Treatment of cells with serum-free media containing epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated system y+ arginine transport activity in Caco-2 cells. Transport upregulation by these growth factors or by TPA was blocked by cycloheximide or the PKC inhibitor chelerythrine. Arginine uptake was diminished during the course of differentiation, attributable to a reduction in the transport system y+ capacity (Vmax) with no change in apparent affinity (Km). TPA stimulated arginine uptake required at least 3 h of continual exposure, and increased the membrane's transport capacity (Vmax) in both undifferentiated and differentiated cells. TPA elevated the diminished transport Vmax of differentiated cells TPA to the elevated Vmax value associated with undifferentiated cells. We conclude that upregulation of arginine transport is part of a pleiotropic response to EGF/TGF alpha, and that this involves PKC and de novo synthesis of polypeptides associated with system y+ transport activity.

Reflux of gastric acid and pepsins into the lower oesophagus causes symptoms such as heartburn and nausea, and tissue injury leading to erosive oesophagitis and stricture formation. This article reviews the mechanisms involved in protecting the oesophagus against acid-mediated injury, including the role of the lower oesophageal sphincter, secondary oesophageal peristalsis and swallowed saliva. The oesophageal mucosa has inherent abilities to resist acid damage, and recent data from three laboratories suggest a secretory function with local production of bicarbonate and mucus responsive to local acidification. The evidence for these putative oesophageal defence mechanisms is discussed.

Epidermal growth factor (EGF) is present in gastric juice and has potent mitogenic properties. The stability of EGF in gastric juice under various physiological and pathophysiological conditions was examined.

Recombinant human EGF1-53 was incubated with HCl containing pepsin. We also determined the forms of EGF present in the gastric juice of patients under basal conditions, patients taking the acid suppressant omeprazole, patients with achlorhydria, and volunteers undergoing intragastric neutralization with NaHCO3 (n = 6 per group). Samples were analyzed using mass spectroscopy and/or high-pressure liquid chromatography followed by radioimmunoassay. The effect of acid and pepsin digestion on EGF bioactivity was determined using an in vitro hepatocyte bioassay and an in vivo cytoprotection assay in the rat stomach.

EGF1-53 was digested to the EGF1-49 and EGF1-46 forms in all samples containing pepsin when the pH was < 4. In gastric juice samples with pH > 4, the proportion of intact EGF increased to about 60%. For both methods of bioassay, intact EGF1-53 was about 3-4 times as potent as acid and pepsin-treated EGF.

EGF is produced in the 1-53 form but is rapidly cleaved to smaller, less active forms in acidic gastric juice. In contrast, only a small proportion of the EGF is cleaved if the pH is maintained above 4. This mechanism may be relevant to the healing process of acid suppressants.

Human milk contains many hormone and hormone-like peptides. The gastrointestinal tract of newborn infants exhibits lower proteolytic activity than in adults and higher "permeability" for macromolecules. Studies in experimental animals demonstrate that several peptides (epidermal growth factors, insulin-like growth factor I and bombesin) after orogastric administration exhibit effects on the small intestine and other organs (liver or pancreas). Few studies performed in human neonates suggest a "survival" of epidermal growth factor in their gastric content. Further studies are needed to evaluate the role of milk-borne hormonally active peptides. This need is stressed by the fact that several of those known to be present in human milk were found to be low or not detectable in infant formulae (epidermal growth factor, insulin-like growth factor I, insulin, parathyroid hormone-related peptide.

Urinary epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) concentrations were determined by radioimmunoassay in a longitudinal study analyzing 348 24-h urine specimens of 32 infants (16 breast-fed, 16 formula-fed) during the first 16 weeks of life. EGF excretion showed a statistically significant increase from 6.0 +/- 2.5 to 14.1 +/- 4.9 micrograms/g creatinine (mean +/- 1 SD) during the investigation period. TGF-alpha levels were fairly constant throughout this period. Comparing breast-fed infants, with more than 100-fold higher ingestion of EGF and TGF-alpha, with formula-fed infants, no significant differences in urinary EGF and TGF-alpha excretion were observed. These results do not rule out a systemic effect of EGF and TGF-alpha after intestinal absorption in breast-fed infants. The results suggest, however, that urinary EGF and TGF-alpha originate mainly from sources other than intestinal absorption.

The present investigation was undertaken to study the localization and accessibility of epidermal growth factor binding sites in the human fetal gut (15-19 weeks of gestation) using light-microscopic and quantitative autoradiography. Exposure of colonic explants to 5 nmol/L 125I-labeled epidermal growth factor for 60 minutes at 22 degrees C revealed extensive accumulation at binding sites in undifferentiated cells of the crypts and at the base of the villus, as well as in the inner circular layer of the muscularis externa bordering the submucosa. Some labeling was also present in the mesenchymal and vascular elements of the lamina propria. Labeling was virtually absent on the brush border at all levels of the epithelium. Quantitative analysis revealed a distinct gradient in grain density along the various compartments of the crypt-villus axis. Epithelial cells in the deep portions of the crypts showed the highest grain density (9.2 grains/microns 2) with values gradually decreasing to 6.5 in upper crypt and 3.9 in lower villus cells. The upper third of the villus showed very little labeling (0.4 grains/microns 2). Cellular distribution of silver grains in lower villous cells revealed a polarization of labeling in the basolateral infranuclear region. Experiments performed at 4 degrees C and at various incubation times showed similar results. Using isolated loops of intact colon and jejunum, segments in which labeled epidermal growth factor was only accessible on the serosal side showed extensive labeling and distribution similar to that found in explanted tissue. On the other hand, labeled epidermal growth factor could not access these same receptor sites when infused into the lumen, either at 22 degrees C or 4 degrees C. These results show that in the human fetal gut (a) the greatest concentration of epidermal growth factor binding sites is found in regions of high proliferative activity and (b) binding sites are absent from the brush border, suggesting that, under normal circumstances, systemic but not luminal epidermal growth factor has free access to its specific receptor.

This study was undertaken to evaluate the effect of epidermal growth factor (EGF) on the morphological changes and polyamine metabolism in the atrophic small intestinal mucosa of rats caused by feeding elemental diet (ED; Elental, Ajinomoto, Tokyo) for several weeks. Four-week-old Wistar male rats were given ad libitum ED (1 kcal/ml) for 4 weeks. The body weight increased to the same extent as the control group fed a pellet diet. However, the small intestine became atrophic: the mucosal wet weight of the jejunum decreased to 70%, while that of the ileum decreased to 60%. EGF (10 micrograms/kg) was subcutaneously injected into these rats every 8 hours. Ornithine decarboxylase (ODC) activities of the jejunal and ileal mucosa rose within 12 hours of the initial EGF administration. Mucosal DNA specific activities tended to increase. Next, EGF (30 micrograms/kg/day) was intraperitoneally administered with a Mini-osmotic pump for one week. The wet weight, protein and DNA contents of the ileal mucosa increased significantly compared with those of the saline administered controls, while the crypt cell production rate (CCPR) also increased. Histologically, increases in both villus height and crypt depth were confirmed. These findings indicate that EGF causes mucosal proliferation through polyamine metabolism even in the atrophic small intestine of mature rats after ED administration for 4 weeks.