1 Department of Pediatrics and Steele Memorial Children's Research Center, 3 Department of Microbiology and Immunology, and 2 Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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- mRNA expression was assessed by RT
competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of
NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression
was markedly increased in the RMS group compared with RMS+EGF. Enhanced
EGF-R expression in the RMS group was localized predominantly in the
epithelial cells of injured ileum. These data suggest a new potential
therapeutic approach for the prevention and treatment of NEC.
intestinal injury; inflammation; neonatal intestine; artificial feeding
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NEONATAL NECROTIZING ENTEROCOLITIS (NEC) is the most common gastrointestinal (GI) disease of premature infants affecting 3,000-5,000 neonates in the US each year (31). Prematurity, enteral feeding, bacterial colonization, and intestinal hypoxia-ischemia are considered the major risk factors for the development of this disease (6). In spite of extensive epidemiological, clinical, and basic research, the pathogenesis of NEC is unknown and there is no effective preventative treatment for this disease (40). During the past three decades, several animal models have been developed to study the etiology of NEC (9), with the neonatal rat model considered one of the best means to study this disease (5, 9, 45). In this model, NEC is induced in newborn rats by enteral feeding of artificial formula coupled with asphyxia stress. (2, 5, 7). This treatment simulates the most common conditions for NEC in humans; formula feeding, immature GI tract, and hypoxia. Since the majority of infants with NEC are fed enterally with formula before disease onset, it has been suggested that the composition of the milk is the key factor in preventing the development of NEC (24). Indeed, the protective role of maternal milk in NEC pathogenesis has been reported both in human (27, 41) and animal studies (2, 10). These findings stimulated the search for various components of milk that might be responsible for protection against NEC.
Mammalian milk contains a large number of biologically active substances that directly affect gut maturation and mucosal protection (14). One of these substances, epidermal growth factor (EGF), is a potent peptide that produces a variety of biological responses, such as enhanced proliferation and differentiation of epithelial cells. In addition, significant effects of EGF on the healing of damaged GI mucosa or on intestinal adaptation after injury have been reported in a number of studies (see, for review, Ref. 23). EGF is detected in many body fluids, including colostrum (60) and milk (18), and during the early postnatal period, colostrum and maternal milk are the major sources of EGF for the developing neonate (42). The biological actions of EGF are mediated through binding to its specific receptor, EGF-R, which is distributed throughout the fetal and neonatal GI tract (8). Diminished levels of salivary and serum EGF in human babies with NEC (19, 48) and the development of hemorrhagic enteritis pathologically similar to NEC in EGF-R knockout mice (28) suggest an important role of EGF insufficiency in the pathogenesis of NEC. In addition, a critically ill child with NEC-like symptoms was successfully treated by continuous intravenous infusion of EGF (51).
The aim of the present study was to test the effects of enteral
administration of EGF on the development of NEC in neonatal rats. Our
hypotheses were that enteral administration of EGF would reduce the
development and incidence of neonatal NEC and that the healing effect
of EGF would be mediated through interaction with the EGF-R at the site
of intestinal injury. We induced experimental NEC in neonatal rats
using artificial formula feeding coupled with exposure to asphyxia/cold
stress (2, 5). We evaluated the effects of enteral
administration of EGF on the development and incidence of NEC, gene
expression and cellular localization of EGF-R at the site of injury,
and changes in ileal endogenous synthesis of two major EGF-R ligands:
EGF and transforming growth factor (TGF)- peptides.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animal model and diets. The study protocol was approved by the Animal Care and Use Committee of the University of Arizona (A-324801-95081). Sixty Sprague-Dawley neonatal rats (Charles River Labs, Pontage, MI), originating from nine different litters, were used in three separate experiments. Newborn rats were collected from their mothers immediately after birth to prevent suckling of maternal milk. Animals were weighed and then placed in an infant incubator to control body temperature and assigned to three experimental groups: artificially fed with growth factor-free rat milk substitute (RMS), artificially fed with RMS supplemented with 500 ng/ml rat EGF (Harlan Bioproducts, Indianapolis, IN) (RMS+EGF), or littermates fed by their mothers (dam fed). In addition, nonstressed dam fed littermates served as physiological controls in these studies. To develop clinical and pathological signs of NEC, rat pups from all three experimental groups (RMS, RMS+EGF, and dam fed) were stressed twice daily with asphyxia by breathing 100% nitrogen gas for 60 s followed by cold stress at 4°C for 10 min, as originally described by Barlow et al. (1, 2) and recently modified by Caplan et al. (5).
For the first 48 h of life, rat pups were hand-fed every 3-4 h using a silicone rubber tube (0.2 mm) with 0.1 ml of RMS prepared as described previously (13, 50). This method is time and labor demanding yet essential because gastrostomy of newborn rats is associated with a very high surgery-related death rate. After 48 h, the hand-feeding method was replaced with mechanized artificial feeding for an additional 48 h as described previously (13). Body weights were recorded daily. Two times daily, urination and defecation were induced by gentle stimulation of the anogenital region and stool was collected for future measurements. Animals that developed abdominal distention, respiratory distress, and lethargy during the first 96 h of the experiment were killed. After 96 h, all surviving animals were killed via decapitation.NEC evaluation. After decapitation, the GI tract was removed. The small intestine was visually evaluated for typical signs of NEC such as intestinal discoloration, intestinal hemorrhage, ileal distention, and ileal stenosis. Results of macroscopic visual evaluation were recorded, and pictures of small intestine and colon was taken for objective comparison of samples from different studies using a digital still camera (MVC-FD91, Sony). The small intestine was then divided into two halves: jejunum and ileum. A 3-cm section of distal ileum 4 cm proximal to the ileocecal valve was cut, fixed in 70% ethanol, embedded in paraffin, sectioned (4-6 µm/section), and counterstained with hematoxylin and eosin for histological evaluation of NEC. The rest of the ileum was snap frozen in liquid nitrogen for DNA, protein, and mRNA measurements. Histological changes in the ileum were scored by a blinded evaluator and graded as follows: normal (0), no damage; mild (1+), slight submucosal and/or lamina propria separation; moderate (2+), moderate separation of submucosa and/or lamina propria and/or edema in submucosal and muscular layers; severe (3+), severe separation of submucosa and/or lamina propria and/or severe edema in submucosal and muscular layers and regional villus sloughing; necrosis (4+), loss of villi and necrosis (30).
Stool occult blood. The detection of occult blood in the stool was performed using the guaiac test. A small quantity of stool was smeared on filter paper and mixed with 100 µl of glacial acetic acid. Guaiac solution (100 µl of 1 g of gum guaiac in 5 ml of ethanol) and 100 µl of 3% H2O2 were added and mixed. The appearance and intensity of any blue or blue-green color was evaluated within 1 to 5 min and scored on a scale of 0-4 by two independent, blinded evaluators. Deep blue color appearing within 1 min was graded 4. Lesser degrees of color development occurring within 1-5 min were graded 1-3 (3, strong signal; 2, moderate signal; 1, small traces of blood in stools). No blood in stools (no color development) was scored as 0. To minimize the variability in analysis of grading, a no-blood sample (0) and a systemic blood sample (4) were assayed as a standard range.
DNA and protein measurements. Tissue DNA and protein content were measured as described previously (13). Briefly, total DNA content in the ileum was assayed by the diphenylamine method of Burton (4) and determined by spectrophotometry. Assays for total protein content in the ileum (26) were determined by spectrophotometry (SPECTRAmax PLUS, Molecular Devices, Sunnyvale, CA).
Immunohistology of the EGF-R. Samples of distal ileum (2-3 cm) were fixed overnight in 70% ethanol, processed, paraffin embedded, and microtome sectioned at 4-6 µm. Sections were deparaffinized in xylene and rehydrated in serial dilutions of 50-100% ethanol and water. Antigen unmasking was achieved using 0.05% saponin (Sigma, St. Louis, MO) for 30 min, followed by blocking of endogenous peroxidase using 0.1% hydrogen peroxide for an additional 30 min. Sections were blocked with 1.5% goat serum (Vector Laboratories, Burlingame, CA) in PBS for 30 min, then incubated with 1 µg/ml rabbit anti-EGF-R polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min, washed with PBS three times, and incubated with a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) for 30 min. After three PBS washes, the Vectastain Elite ABC reagent (Vector Laboratories) was applied for 30 min, slides were washed three times with PBS, then diaminobenzene (Sigma) was utilized as the substrate. Sections were then counterstained with hematoxylin, dehydrated, and coverslipped (58). Control sections were treated with the same procedure except that they were incubated with 5 µg/ml rabbit Ig (Sigma) instead of the anti-EGF-R antibody. No staining was detected in control slides. Sections from all three experimental groups were stained for EGF-R at the same time, and stained sections were evaluated by a blinded observer.
RNA extraction and RT competitive-PCR assay.
Total RNA was isolated from tissue using the RNeasy mini kit (Qiagen,
Santa Clarita, CA) as described in the manufacturer's protocol. All
samples were incubated with RNase-free DNase (20 U/reaction) for 10 min
at 37°C to eliminate DNA contamination. RNA concentration was
quantified by ultraviolet spectrophotometry at 260 nm
(A260) and the purity was determined by the
A260/A280 ratio (SPECTRAmax PLUS, Molecular
Devices). The integrity of RNA samples was verified by electrophoresis
on 1.2% agarose gel containing formaldehyde (2.2 M) and ethidium
bromide in 1× MOPS buffer [40 mM MOPS (pH 7.0), 10 mM sodium acetate,
and 1 mM EDTA (pH 8.0)]. The RT competitive-PCR assay was used to
quantify intestinal EGF-R, EGF, and TGF- mRNA levels as previously
described in detail (12, 15).
Statistics. Statistical analysis of the results was performed by one-way ANOVA followed by Fisher's protected least significant difference using the statistical program StatView for Macintosh computers (Abacus Concepts, Berkeley, CA). A value of P < 0.05 was considered significant at the 95% confidence level. All data in the figures are means ± SE.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of EGF on body weight gain and ileal parameters.
Body weights in the RMS group gradually decreased during the study
period, whereas rats fed RMS+EGF were able to maintain the same body
weight during the same time period (Fig.
1). Differences in body weight between
RMS and RMS+EGF groups were statistically significant at 72 h
(P < 0.05), with the difference increasing by 96 h (P < 0.01). Dam fed pups showed steady increases in
body weight throughout the experiments. There were no differences in body weight gain of pups in the asphyxia/cold stressed dam fed group
compared with their nonstressed dam fed littermates (results not
shown). Ileal protein content was similar in all three experimental groups (Table 1). DNA content was
significantly higher in dam fed rats compared with both RMS and RMS+EGF
groups, but there was no statistical difference between the RMS and the
RMS+EGF groups (Table 1).
|
|
Effects of EGF on the incidence of NEC. Animals developed visual signs characteristic of intestinal injury between 72 and 96 h after the beginning of the experiments. These signs included abdominal distention, discoloration of the abdominal wall, and occult blood in the stool. Because progression of intestinal damage typically resulted in death, we performed guaiac measurements at 72 rather than 96 h. Results from these measurements are summarized in Table 1. The RMS group exhibited a significantly stronger signal (P < 0.0001) for the presence of blood in the stool compared with the RMS+EGF group. Dam fed animals had no or minimal blood in the stool.
Macroscopic and microscopic examination of the GI tract showed clear evidence of intestinal damage similar to neonatal NEC. Macroscopically, the jejunum and ileum exhibited severe inflammation, hemorrhage, and discoloration (Fig. 2). The degree of damage varied from pink/red to dark purple/black discoloration in NEC rats (Fig. 2B) compared with the yellow/green appearance of normal gut from healthy rat pups (Fig. 2A). The ileum was generally more severely affected than the jejunum; typical intestinal distention and stenosis were located predominantly in ileum (Fig. 2B, detail). In addition, luminal contents of the ileum were dark and bloody. In animals with moderate progression of NEC, pathological changes in the small bowel were patchier, with scattered areas of hemorrhage, distention, and stenosis in the ileum. Histological analyses of ileal segments were performed using a scoring system from 0 to 4+ (Fig. 3) to determine the severity of NEC. Results from these measurements are shown in Fig. 4. In the RMS group, histological findings in most animals showed significant pathological changes in ileal structure (mean NEC score = 2.6). The degree of ileal damage was significantly reduced (P < 0.001) in rats fed with RMS+EGF (mean NEC score = 1.3) compared with the RMS group. Dam fed pups showed no or minimal pathological changes in ileal morphology (mean NEC score = 0.43). Comparison of the overall incidence of NEC revealed that 81% of RMS pups developed significant intestinal abnormalities described as moderate, severe, or full necrosis. Supplementation of EGF into formula resulted in a dramatic, 50% reduction in the incidence of NEC. Dam fed animals showed no changes or abnormalities in the small intestinal structure resembling the development of NEC (Table 2).
|
|
|
|
Ileal EGF-R, EGF, and TGF- mRNA levels.
To evaluate changes in gene expression of the EGF-R, ileal steady-state
mRNA levels were measured using RT competitive-PCR (Fig.
5A). Ileal EGF-R mRNA levels
were markedly increased (~7- to 8-fold) in the RMS group compared
with the dam fed group. Supplementation of the RMS diet with EGF was
associated with a statistically significant decrease in ileal EGF-R
content compared with RMS alone (P < 0.01), with the
mRNA level in this group intermediate between the mRNA levels of dam
fed and RMS fed rats. There was no difference in the EGF-R mRNA level
between dam fed and nonstressed dam fed littermates (results not
shown). Changes in endogenous synthesis of two major EGF-R ligands, EGF
and TGF-
, revealed no statistically significant changes in either
EGF and TGF-
mRNA levels in the ileum of RMS animals compared with
either RMS+EGF or dam fed pups (Fig. 5, B and
C).
|
Histological localization of the EGF-R.
Histological examination of ileal samples from dam fed pups revealed
low levels of EGF-R expression in the apical and basolateral membranes
of the villi (Fig. 6A and
D). The pattern of EGF-R localization in the RMS group was
similar to that seen in dam fed animals, except that there was very
strong staining of the apical membrane and moderate staining of the
basolateral membrane of the villi enterocytes (Fig. 6B and
E). In contrast, animals given RMS+EGF had markedly
decreased expression of the EGF-R in the apical and basolateral
membranes of the villi compared with the RMS group (Fig. 6C
and F).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our present study provides, for the first time, evidence that
enteral administration of EGF reduces the development and the incidence
of NEC in a neonatal rat model. Moreover, the increased expression of
EGF-R in damaged ileum indicates that the healing effect of exogenous
EGF is mediated directly at the site of intestinal injury.
Interestingly, the development of NEC has no effect on the endogenous
production of EGF and TGF- ligands in damaged ileum.
NEC is a disease of premature babies. The reasons for a predilection for prematurity are unclear, but an immature mucosal barrier and immune response likely contribute to the premature neonates' susceptibility. Amniotic fluid concentrations of EGF gradually increase during pregnancy, with the highest level achieved at the end of the normal gestation period (22). EGF is also present in high concentrations in colostrum and breast milk of many mammalian species (25, 34), and during the suckling period, milk-borne EGF is the major source of EGF for the developing neonate (42). In contrast, EGF is absent in all commercial formulas. In suckling animals, supplementation of EGF into formula enhances the growth of stomach and the small intestine (3), induces precocious maturation of intestinal brush-border disaccharidase activities (32), and modulates intestinal nutrient transport (33). Because the incidence of NEC is, in the majority of cases, related to formula feeding of newborn babies, we proposed that the absence of EGF in developing gut increases the potential to develop this disease. Results from the present study support this hypothesis and suggest a critical role of milk-borne EGF in the maintenance of gut integrity and healthy development.
The first report relating EGF and intestinal necrosis was reported in England, where an 8-mo-old child with intestinal necrosis similar to NEC was successfully treated with continuous infusion of EGF (51). Scott et al. (46) have shown significant elevation of urinary EGF levels in neonates diagnosed with NEC that they suggest might result from increased absorption of EGF through the damaged intestinal mucosa. Furthermore, markedly diminished serum and salivary EGF levels have been reported in premature infants with NEC (19, 48). These data suggest that administration of exogenous EGF may provide an effective means to prevent or treat this disease. In this study, we demonstrate that the supplementation of milk formula with EGF markedly reduces the development and severity of NEC. The importance of this finding is particularly significant because EGF was given orally. The use of growth factors for the treatment of GI disease is always associated with concerns over their potential risks. Systemically administered growth factors could induce proliferation in other regions of the body that harbor premalignant cells (34). Thus enteral administration of EGF enables use of a higher dose of EGF and helps deliver growth factor directly into the site of intestinal injury. Rat milk contains 30-50 ng/ml EGF (43, 55). Our preliminary study has indicated that supplementation of formula with a physiological dose of EGF (100 ng/ml) has markedly lower efficiency in the prevention and severity of NEC (data not shown). In addition, maternal milk contains a variety of proteins, such as casein, that can act as competitive substrates for proteolytic enzymes and specific proteolytic inhibitors that can protect luminal peptide growth factors from degradation (35, 38). Moreover, intestinal proteolytic activity is significantly lower in the early postnatal period compared with adulthood (20). Therefore, supplementation of a higher dose of EGF into milk formula can protect EGF against this inherent proteolytic degradation in the stomach and small intestine and improve the efficiency of injury treatment during the perinatal period of life.
The mechanisms underlying the protective effects of EGF in injured
mucosa are not clearly understood. However, the involvement of the
EGF-R in the biological action of EGF has been extensively studied
(8). The presence of EGF-R in human fetal intestine was
first demonstrated in crypt cells, epithelial cells at the base of the
villus, and the inner circular layer of the intestinal muscle
(37). In rats, EGF-R expression was detected throughout the entire small intestine (39) and localized originally
to the basolateral membrane of the crypt and villus epithelium only (44, 53, 54). However, recent studies have shown the
presence of EGF-R also on the apical membrane of villus epithelium in
rats (29) and rabbits (56). Indeed, results
from our study indicate that in neonatal rats, low expression of EGF-R
is localized in both apical and basolateral membrane of ileal
enterocytes. In addition, during mucosal injury EGF-R localization and
expression in intestinal mucosa is changed. In mice and rats that have
undergone small bowel resection, an increase in EGF-R production and
increased receptor activation was noted (47, 57).
Increased EGF-R production was reported in epithelial cells of the
ulcer margins (59) with EGF-R expression localized on the
basolateral and apical surfaces (52). These results
indicate that during mucosal injury, the EGF-R can effectively be
reached by its ligands (EGF or TGF-) from the luminal side. In
addition, during the early postnatal period high intestinal
permeability enables rapid transport of EGF across the jejunal
epithelium (39) and therefore EGF-R on the basolateral
side of the epithelium can be reached as well. Our results complement
the findings of these previous studies. Gene expression of EGF-R at the
site of injury (distal ileum) is increased, and treatment with orally
administered EGF downregulates EGF-R mRNA levels toward normal levels.
The presence of EGF-R in NEC tissues is localized predominantly in the
apical and basolateral membranes of the villi enterocytes, whereas in
healthy tissue EGF-R is minimally detected. Changes in EGF-R expression
are observed as soon as 24 h after asphyxia/cold stress exposure,
before any histopathological changes in intestinal architecture can be
detected (data not shown). Together, these data support the idea that
the EGF-R belongs to an essential defense mechanism of intestinal epithelial integrity wherein peptide growth factors such as EGF or
TGF-
play critical protective and healing roles (34,
36).
In suckling rats, intestinal contents of EGF and TGF- peptides are
similar but the origins are different. The major source of intestinal
EGF is maternal milk (42). TGF-
, however, is not
detectable in rat milk, and its intestinal content is likely the result
of endogenous synthesis by the small intestine and pancreatic secretion
(11). Recently, we have shown that endogenous intestinal
EGF and TGF-
production can be regulated nutritionally in suckling
rats. Feeding growth factor-free RMS diet for 4 days to neonatal rats
resulted in significant increases of TGF-
and EGF mRNA levels in the
developing duodenum and ileum (15). Studies have shown
increased mucosal TGF-
expression in adult rabbits (49)
and rats (21) with experimental colitis. Mice lacking TGF-
exhibited increased susceptibility to dextran sulfate-induced colitis (17), whereas mice overexpressing TGF-
had
markedly decreased susceptibility to experimentally induced colitis
(16). Our current observations that ileal EGF and TGF-
mRNA levels were unaffected by either NEC injury or EGF treatment may
indicate that highly immature intestinal tissue does not have the
potential to initiate the reparative process by increased endogenous
production of EGF-R ligands.
In the present study, we have shown that oral administration of EGF has beneficial effects in the prevention of NEC in an experimental rat model. Supplementation of commercial milk formula with recombinant EGF to stimulate intestinal repair processes may significantly reduce the incidence of neonatal NEC in prematurely born neonates and suggests new therapeutic approaches in the treatment of GI diseases of pediatric patients.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Anthony F. Philipps for editing the manuscript.
![]() |
FOOTNOTES |
---|
First published October 10, 2001;10.1152/ajpgi.00196.2001
Current address for R. Stepankova: The Academy of Sciences of the Czech Republic, Institute of Microbiology, Novy Hradek, Czech Republic.
This work was supported by National Institute of Child Health and Human Development Grants HD-26013 and HD-39657 (to B. Dvorak), the University of Arizona Vice President for Research and Graduate Studies Award (to B. Dvorak), and the Grant Agency of the Czech Republic 303/00/1370 (to R. Stepankova).
Address for reprint requests and other correspondence: B. Dvorak, Dept. of Pediatrics, Univ. of Arizona, 1501 N. Campbell Ave, PO Box 245073, Tucson, AZ 85724-5073 (E-mail: dvorakb{at}peds.arizona.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 May 2001; accepted in final form 5 October 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barlow, B,
and
Santulli TV.
Importance of multiple episodes of hypoxia or cold stress on the development of enterocolitis in an animal model.
Surgery
77:
687-690,
1975[ISI][Medline].
2.
Barlow, B,
Santulli TV,
Heird WC,
Pitt J,
Blanc WA,
and
Schullinger JN.
An experimental study of acute neonatal enterocolitisthe importance of breast milk.
J Pediatr Surg
9:
587-595,
1974[ISI][Medline].
3.
Berseth, CL.
Enhancement of intestinal growth in neonatal rats by epidermal growth factor in milk.
Am J Physiol Gastrointest Liver Physiol
253:
G662-G665,
1987
4.
Burton, K.
A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid.
Biochem J
62:
315-323,
1956[ISI].
5.
Caplan, MS,
Hedlund E,
Adler L,
and
Hsueh W.
Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis.
Pediatr Pathol
14:
1017-1028,
1994[ISI][Medline].
6.
Caplan, MS,
and
MacKendrick W.
Necrotizing enterocolitis: a review of pathogenetic mechanisms and implications for prevention.
Pediatr Pathol
13:
357-369,
1993[ISI][Medline].
7.
Caplan, MS,
Miller-Catchpole R,
Kaup S,
Russell T,
Lickerman M,
Amer M,
Xiao Y,
and
Thomson R, Jr.
Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model.
Gastroenterology
117:
577-583,
1999[ISI][Medline].
8.
Chailler, P,
and
Menard D.
Ontogeny of EGF receptors in the human gut.
Front Biosci
4:
D87-D101,
1999[Medline].
9.
Crissinger, KD.
Animal models of necrotizing enterocolitis.
J Pediatr Gastroenterol Nutr
20:
17-22,
1995[ISI][Medline].
10.
Crissinger, KD,
Burney DL,
Velasquez OR,
and
Gonzalez E.
An animal model of necrotizing enterocolitis induced by infant formula and ischemia in developing piglets.
Gastroenterology
106:
1215-1222,
1994[ISI][Medline].
11.
Dvorak, B,
and
Koldovsky O.
The presence of transforming growth factor-alpha in the suckling rat small intestine and pancreas and the absence in rat milk.
Pediatr Res
35:
348-353,
1994[Abstract].
12.
Dvorak, B,
Kolinska J,
McWilliam DL,
Williams CS,
Higdon T,
Zakostelecka M,
and
Koldovsky O.
The expression of epidermal growth factor and transforming growth factor- mRNA in the small intestine of suckling rats: organ culture study.
FEBS Lett
435:
119-124,
1998[ISI][Medline].
13.
Dvorak, B,
McWilliam DL,
Williams CS,
Dominguez JA,
Machen NW,
McCuskey RS,
and
Philipps AF.
Artificial formula induces precocious maturation of the small intestine of artificially reared suckling rats.
J Pediatr Gastroenterol Nutr
31:
162-169,
2000[ISI][Medline].
14.
Dvorak, B,
Philipps AF,
and
Koldovsky O.
Breast milk hormones: importance for the premature.
In: Seminars in Neonatal Nutrition and Metabolism, edited by Hay WW.. Columbus, OH: Ross Product Division, 1999, p. 1-4.
15.
Dvorak, B,
Williams CS,
McWilliam DL,
Shinohara H,
Dominguez JA,
McCuskey RS,
Philipps AF,
and
Koldovsky O.
Milk-borne epidermal growth factor modulates intestinal transforming growth factor-alpha levels in neonatal rats.
Pediatr Res
47:
194-200,
2000
16.
Egger, B,
Carey HV,
Procaccino F,
Chai NN,
Sandgren EP,
Lakshmanan J,
Buslon VS,
French SW,
Buchler MW,
and
Eysselein VE.
Reduced susceptibility of mice overexpressing transforming growth factor to dextran sodium sulphate induced colitis.
Gut
43:
64-70,
1998
17.
Egger, B,
Procaccino F,
Lakshmanan J,
Reinshagen M,
Hoffmann P,
Patel A,
Reuben W,
Gnanakkan S,
Liu L,
Barajas L,
and
Eysselein VE.
Mice lacking transforming growth factor have an increased susceptibility to dextran sulfate-induced colitis.
Gastroenterology
113:
825-832,
1997[ISI][Medline].
18.
Goodlad, RA,
and
Wright NA.
Epidermal growth factor (EGF).
Baillieres Clin Gastroenterol
10:
33-47,
1996[ISI][Medline].
19.
Helmrath, MA,
Shin CE,
Fox JW,
Erwin CR,
and
Warner BW.
Epidermal growth factor in saliva and serum of infants with necrotising enterocolitis.
Lancet
351:
266-267,
1998[ISI][Medline].
20.
Henning, SJ.
Postnatal development: coordination of feeding, digestion, and metabolism.
Am J Physiol Gastrointest Liver Physiol
241:
G199-G214,
1981
21.
Hoffmann, P,
Zeeh JM,
Lakshmanan J,
Wu VS,
Procaccino F,
Reinshagen M,
McRoberts JA,
and
Eysselein VE.
Increased expression of transforming growth factor precursors in acute experimental colitis in rats.
Gut
41:
195-202,
1997
22.
Hofmann, GE,
and
Abramowicz JS.
Epidermal growth factor (EGF) concentrations in amniotic fluid and maternal urine during pregnancy.
Acta Obstet Gynecol Scand
69:
217-221,
1990[Medline].
23.
Jones, MK,
Tomikawa M,
Mohajer B,
and
Tarnawski AS.
Gastrointestinal mucosal regeneration: role of growth factors.
Front Biosci
4:
D303-D309,
1999[Medline].
24.
Kliegman, RM.
Neonatal necrotizing enterocolitis.
In: Pediatric Gastrointestinal Disease, edited by Wyllie R,
and Hyams JS.. Philadelphia, PA: Saunders, 1999, p. 445-465.
25.
Koldovsky, O.
Hormones in milk.
Vitam Horm
50:
77-149,
1995[Medline].
26.
Lowry, OH,
Rosebgrough NJ,
Farr AL,
and
Randal RJ.
Protein measurement with the folin phenol reagent.
J Biol Chem
193:
265-275,
1951
27.
Lucas, A,
and
Cole TJ.
Breast milk and neonatal necrotising enterocolitis.
Lancet
336:
1519-1523,
1990[ISI][Medline].
28.
Miettinen, PJ,
Berger JE,
Meneses J,
Phung Y,
Pedersen RA,
Werb Z,
and
Derynck R.
Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.
Nature
376:
337-341,
1995[ISI][Medline].
29.
Montaner, B,
and
Perez-Tomas R.
Epidermal growth factor receptor (EGF-R) localization in the apical membrane of the enterocytes of rat duodenum.
Cell Biol Int
23:
475-479,
1999[ISI][Medline].
30.
Nadler, EP,
Dickinson E,
Knisely A,
Zhang XR,
Boyle P,
Beer-Stolz D,
Watkins SC,
and
Ford HR.
Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.
J Surg Res
92:
71-77,
2000[ISI][Medline].
31.
Neu, J.
Necrotizing enterocolitis: the search for a unifying pathogenic theory leading to prevention.
Pediatr Clin North Am
43:
409-432,
1996[ISI][Medline].
32.
O'Loughlin, EV,
Chung M,
Hollenberg M,
Hayden J,
Zahavi I,
and
Gall DG.
Effect of epidermal growth factor on ontogeny of the gastrointestinal tract.
Am J Physiol Gastrointest Liver Physiol
249:
G674-G678,
1985[ISI][Medline].
33.
Opleta-Madsen, K,
Meddings JB,
and
Gall DG.
Epidermal growth factor and postnatal development of intestinal transport and membrane structure.
Pediatr Res
30:
342-350,
1991[Abstract].
34.
Playford, RJ,
Macdonald CE,
and
Johnson WS.
Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders.
Am J Clin Nutr
72:
5-14,
2000
35.
Playford, RJ,
Woodman AC,
Clark P,
Watanapa P,
Vesey D,
Deprez PH,
Williamson RC,
and
Calam J.
Effect of luminal growth factor preservation on intestinal growth.
Lancet
341:
843-848,
1993[ISI][Medline].
36.
Playford, RJ,
and
Wright NA.
Why is epidermal growth factor present in the gut lumen?
Gut
38:
303-305,
1996[Abstract].
37.
Pothier, P,
and
Menard D.
Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon.
FEBS Lett
228:
113-117,
1988[ISI][Medline].
38.
Rao, RK,
Davis TP,
Williams C,
and
Koldovsky O.
Effect of milk on somatostatin degradation in suckling rat jejunum in vivo.
J Pediatr Gastroenterol Nutr
28:
84-94,
1999[ISI][Medline].
39.
Rao, RK,
Koldovsky O,
Korc M,
Pollack PF,
Wright S,
and
Davis TP.
Processing and transfer of epidermal growth factor in developing rat jejunum and ileum.
Peptides
11:
1093-1102,
1990[ISI][Medline].
40.
Schanler, RJ.
Overview: the clinical perspective.
J Nutr
130:
417S-419S,
2000[ISI][Medline].
41.
Schanler, RJ,
Shulman RJ,
and
Lau C.
Feeding strategies for premature infants: beneficial outcomes of feeding fortified human milk versus preterm formula.
Pediatrics
103:
1150-1157,
1999
42.
Schaudies, RP,
Grimes J,
Davis D,
Rao RK,
and
Koldovsky O.
EGF content in the gastrointestinal tract of rats: effect of age and fasting/feeding.
Am J Physiol Gastrointest Liver Physiol
256:
G856-G861,
1989
43.
Schaudies, RP,
Grimes J,
Wray HL,
and
Koldovsky O.
Identification and partial characterization of multiple forms of biologically active EGF in rat milk.
Am J Physiol Gastrointest Liver Physiol
259:
G1056-G1061,
1990
44.
Scheving, LA,
Shiurba RA,
Nguyen TD,
and
Gray GM.
Epidermal growth factor receptor of the intestinal enterocyte. Localization to laterobasal but not brush border membrane.
J Biol Chem
264:
1735-1741,
1989
45.
Schiffrin, EJ,
Carter EA,
Walker WA,
Frieberg E,
Benjamin J,
and
Israel EJ.
Influence of prenatal corticosteroids on bacterial colonization in the newborn rat.
J Pediatr Gastroenterol Nutr
17:
271-275,
1993[ISI][Medline].
46.
Scott, SM,
Watterberg K,
Rogers C,
Hartenberger C,
Merker L,
and
Gifford KL.
Positive relationship of cortisol concentrations and oral nutrition to epidermal growth factor concentrations in preterm infants.
Biol Neonate
74:
259-265,
1998[ISI][Medline].
47.
Shin, CE,
Falcone RA, Jr,
Duane KR,
Erwin CR,
and
Warner BW.
The distribution of endogenous epidermal growth factor after small bowel resection suggests increased intestinal utilization during adaptation.
J Pediatr Surg
34:
22-26,
1999[ISI][Medline].
48.
Shin, CE,
Falcone RA, Jr,
Stuart L,
Erwin CR,
and
Warner BW.
Diminished epidermal growth factor levels in infants with necrotizing enterocolitis.
J Pediatr Surg
35:
173-177,
2000[ISI][Medline].
49.
Sottili, M,
Sternini C,
Reinshagen M,
Brecha NC,
Nast CC,
Walsh JH,
and
Eysselein VE.
Up-regulation of transforming growth factor alpha binding sites in experimental rabbit colitis.
Gastroenterology
109:
24-31,
1995[ISI][Medline].
50.
Stepankova, R,
Dvorak B,
Sterzl J,
and
Trebichavsky I.
Effects of essential fatty acids deficiency in milk diets on the development of germ-free and conventional rats.
Physiol Bohemoslov
39:
135-146,
1990[ISI][Medline].
51.
Sullivan, PB,
Brueton MJ,
Tabara ZB,
Goodlad RA,
Lee CY,
and
Wright NA.
Epidermal growth factor in necrotising enteritis.
Lancet
338:
53-54,
1991[ISI][Medline].
52.
Tarnawski, A,
Stachura J,
Durbin T,
Sarfeh IJ,
and
Gergely H.
Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats.
Gastroenterology
102:
695-698,
1992[ISI][Medline].
53.
Thompson, JF,
Lamprey RM,
and
Stokkers PC.
Orogastric EGF enhances c-neu and EGF receptor phosphorylation in suckling rat jejunum in vivo.
Am J Physiol Gastrointest Liver Physiol
265:
G63-G72,
1993
54.
Thompson, JF,
van den Berg M,
and
Stokkers PC.
Developmental regulation of epidermal growth factor receptor kinase in rat intestine.
Gastroenterology
107:
1278-1287,
1994[ISI][Medline].
55.
Thornburg, W,
Matrisian L,
Magun B,
and
Koldovsky O.
Gastrointestinal absorption of epidermal growth factor in suckling rats.
Am J Physiol Gastrointest Liver Physiol
246:
G80-G85,
1984
56.
Wallace, LE,
Hardin JA,
and
Gall DG.
Expression of EGF and ERBB receptor proteins in small intestinal epithelium (Abstract).
Gastroenterology
120:
A511,
2001[ISI].
57.
Warner, BW,
Vander Kolk WE,
Can G,
Helmrath MA,
Shin CE,
and
Erwin CR.
Epidermal growth factor receptor expression following small bowel resection.
J Surg Res
70:
171-177,
1997[ISI][Medline].
58.
Whiteland, JL,
Shimeld C,
Nicholls SM,
Easty DL,
Williams NA,
and
Hill TJ.
Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues.
J Immunol Methods
210:
103-108,
1997[ISI][Medline].
59.
Wright, NA,
Pike C,
and
Elia G.
Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells.
Nature
343:
82-85,
1990[ISI][Medline].
60.
Xu, RJ.
Development of the newborn GI tract and its relation to colostrum/milk intake: a review.
Reprod Fertil Dev
8:
35-48,
1996[ISI][Medline].