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Infection and Immunity, August 1999, p. 4237-4242, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Helicobacter pylori Induces Gastric
Epithelial Cell Apoptosis in Association with Increased Fas
Receptor Expression
Nicola L.
Jones,1,2,3
Andrew S.
Day,2,3
Hilary A.
Jennings,3 and
Philip
M.
Sherman1,2,3,*
Departments of Molecular Microbiology and
Medical Genetics1 and
Pediatrics,2 University of Toronto, and
Research Institute, The Hospital for Sick
Children,3 Toronto, Canada
Received 19 April 1999/Accepted 12 May 1999
 |
ABSTRACT |
The mechanisms involved in mediating the enhanced gastric
epithelial cell apoptosis observed during infection with
Helicobacter pylori in vivo are unknown. To determine
whether H. pylori directly induces apoptosis of gastric
epithelial cells in vitro and to define the role of the Fas-Fas ligand
signal transduction cascade, human gastric epithelial cells were
infected with H. pylori for up to 72 h under
microaerophilic conditions. As assessed by both transmission electron
microscopy and fluorescence microscopy, incubation with a
cagA-positive, cagE-positive, VacA-positive clinical H. pylori isolate stimulated an increase in
apoptosis compared to the apoptosis of untreated AGS cells (16.0% ± 2.8% versus 5.9% ± 1.4%, P < 0.05) after 72 h. In contrast, apoptosis was not detected following infection with
cagA-negative, cagE-negative, VacA-negative
clinical isolates or a Campylobacter jejuni strain. In
addition to stimulating apoptosis, infection with H. pylori enhanced Fas receptor expression in AGS cells to a degree comparable to
that of treatment with a positive control, gamma interferon (12.5 ng/ml) (148% ± 24% and 167% ± 24% of control, respectively). The
enhanced Fas receptor expression was associated with increased sensitivity to Fas-mediated cell death. Ligation of the Fas receptor with an agonistic monoclonal antibody resulted in an increase in
apoptosis compared to the apoptosis of cells infected with the
bacterium alone (38.5% ± 7.1% versus 16.0% ± 2.8%,
P < 0.05). Incubation with neutralizing anti-Fas
antibody did not prevent apoptosis of H. pylori-infected
cells. Taken together, these findings demonstrate that the gastric
pathogen H. pylori stimulates apoptosis of gastric
epithelial cells in vitro in association with the enhanced expression
of the Fas receptor. These data indicate a role for Fas-mediated
signaling in the programmed cell death that occurs in response to
H. pylori infection.
| |
INTRODUCTION |
Infection with the gastric pathogen
Helicobacter pylori causes chronic active gastritis and
peptic ulcer disease (35). In addition, H. pylori
infection has been associated epidemiologically with the development of
gastric cancers, including adenocarcinoma (14) and lymphoma
(31). The mechanisms by which H. pylori mediates these host responses, however, remain unknown.
Apoptosis is a genetically programmed form of cell death characterized
by distinct morphologic and molecular features (32). Programmed cell death plays an important role in the regulation of
epithelial cell numbers in the gastrointestinal tract (15). In addition, deregulation of the apoptotic pathway is implicated in a
number of disease processes in the intestine, including carcinogenesis (26). Microbes have developed mechanisms to stimulate the
apoptotic signal transduction cascade which likely play a role in
pathogenesis (37). Microbial pathogens, or their products,
can directly activate the cell death signaling cascade. For example,
invasive enteric pathogens such as Salmonella can directly
induce apoptosis of intestinal epithelial cells (21).
Alternatively, immune responses, including infiltrating inflammatory
cells and production of inflammatory mediators directed against the
microbe, can activate the pathway to cell death. For example, induction
of the proinflammatory cytokine tumor necrosis factor alpha mediates in
part the cell death of epithelial cells which occurs during
Salmonella infection in vitro. Cytotoxic lymphocytes can
induce apoptosis of hepatitis C virus-infected cells (1).
One mechanism by which immune cells trigger apoptosis of target cells
occurs through binding of the Fas receptor to the Fas ligand
(32).
Fas, or CD95, is a member of the tumor necrosis factor receptor family
which, when bound by its natural ligand, stimulates an apoptotic signal
through activation of the caspase cascade (28). Under
physiologic conditions, the Fas system is involved in regulating the
immune response by eliminating activated lymphocytes (23).
Virus-infected cells are also eliminated through Fas-Fas ligand
interactions (1). In addition, present evidence links excessive activity of the Fas system with the pathogenic effects associated with infection by certain microbes. For example, the lymphocyte depletion observed in patients infected with the human immunodeficiency virus appears to be Fas mediated (3, 19).
Alterations in the gastric epithelial cell cycle, including both
enhanced proliferation and increased apoptosis of gastric cells, are
identified during infection with H. pylori (4,
27). These changes in cell turnover are present in both H. pylori-infected children (16) and adults
(25). Investigations of the molecular determinants mediating
apoptosis have identified both enhanced expression of the tumor
suppressor p53 (16) and increased expression of the
proapoptotic protein Bak in response to H. pylori infection (5). Among H. pylori-infected children, gastric
epithelial cell apoptosis returns to baseline levels only following
both eradication of the bacterium and resolution of the
accompanying gastritis (16). These findings suggest a role
for immune-mediated apoptosis of gastric epithelial cells during
H. pylori infection. Therefore, the aims of this study were
to determine if H. pylori can directly stimulate programmed
cell death of gastric epithelial cells and to characterize the role of
Fas-Fas ligand signaling in this cell death cascade.
| |
MATERIALS AND METHODS |
Bacteria and growth conditions.
H. pylori LC 11, a
cagA-positive, cagE-positive, vacuolating
cytotoxin (VacA)-producing H. pylori strain originally
isolated from a child with duodenal ulcer disease, and two
cagA-negative, cagE-negative, VacA-negative
strains, LC 3 and LC 20, were employed for these studies
(13). The presence of cagA and cagE
was determined by isolation of genomic DNA and PCR (24) with
the primer pair GCCTACTGGTGGGGATTG and
GCCTGTAGTTGGTCTTC for cagA and both primer pair
AGACATGCAAAAAGGTAT and CAATCTAGTGGGGTGGTA and
primer pair TGCTGATACGATTAGAGA and TAGTCCCTTAGTGATGAT
(kindly provided by Robin Beech, McGill University, Montreal,
Quebec, Canada) for cagE. The presence of the vacuolating
cytotoxin was determined by the method of Cover et al. (7).
Concentrated broth supernatants were incubated with HEp-2 cells for
24 h at 37°C, and vacuolation was assessed by bright-field
microscopy (24).
H. pylori strains were grown under microaerophilic
conditions on Columbia blood agar plates for 72 h at 37°C,
harvested, and resuspended in brucella broth (Difco) supplemented with
10% heat-inactivated fetal calf serum, vancomycin, and trimethoprim.
Bacterial cells were grown overnight at 37°C in an Erlenmeyer flask
with shaking at 120 rpm, as described previously (17). Cells
were then pelleted and resuspended in phosphate-buffered saline at a
concentration of 109 CFU/ml. Campylobacter
jejuni TGH 9011 was kindly provided by V. L. Chan (University
of Toronto).
Cell culture.
The human gastric adenocarcinoma cell line AGS
was grown as a monolayer in tissue culture flasks at 37°C in 5%
CO2. The tissue culture medium was Ham's F-12 medium (Life
Technologies, GIBCO BRL, Grand Island, N.Y.) supplemented with 10%
(vol/vol) heat-inactivated fetal calf serum (Cansera International,
Rexdale, Ontario, Canada) and 0.1% sodium bicarbonate. The human
gastric adenocarcinoma cell line KATO III was grown under similar
conditions in RPMI 1640 media supplemented with 10% fetal calf serum.
Cells were incubated for up to 72 h with H. pylori
(5 × 108 CFU/ml) under microaerophilic conditions.
Control cells were incubated under the same conditions in the absence
of bacteria.
Assessment of apoptosis. (i) Transmission electron
microscopy.
For transmission electron microscopy, cells were grown
to confluence in tissue culture flasks and incubated with H. pylori as described above. Cells incubated in the absence of
H. pylori served as controls. Both cells in suspension and
trypsinized cells were pelleted, fixed with 2% glutaraldehyde
(vol/vol) in 0.1 M phosphate buffer, postfixed in 2% osmium tetroxide,
and dehydrated through a series of graded acetone washes
(9). Samples were embedded in epoxy resin, and ultrathin
sections were placed onto 300-mesh copper grids. The grids were then
stained with uranyl acetate and lead salts, as described previously
(9). Grids were examined for the presence of apoptotic cells
with a transmission electron microscope at an accelerating voltage of
60 kV (20).
(ii) Fluorescent dye staining.
Cells in suspension and
trypsinized cells were pelleted and resuspended in 1 ml of
phosphate-buffered saline. Acridine orange-ethidium bromide in
phosphate-buffered saline (100 µg/ml) was added to the suspension
(11). A drop of the suspension was applied to a microscope
slide, and apoptotic cells were assessed by fluorescence microscopy, as
previously described (11). Apoptotic cells were enumerated
by counting 500 cells at multiple randomly selected fields. The
apoptotic index was expressed as the percent of apoptotic cells per 500 cells enumerated.
Determination of Fas receptor expression. (i) Fluorescence
microscopy.
AGS cells were grown on Lab-Tek chamber slides (Nunc,
Naperville, Ill.), as described above, until they were semiconfluent. The cells were then incubated in the presence or absence of gamma interferon (IFN-
) (12.5 ng/ml) for 24 h at 37°C
(12). Cells were washed with phosphate-buffered saline and
then fixed with 100% acetone for 5 min at room temperature. Cells were
next incubated with an anti-Fas receptor monoclonal antibody (clone
DX2; Oncogene Research Products, Cambridge, Mass.) at a concentration
of 2.5 µg/ml for 1 h at 37°C. Following the washings, cells
were incubated with fluorescein isothiocyanate-conjugated anti-mouse
immunoglobulin G (1:100) for 1 h at 37°C. After incubation and
further washings, slides of the cells were mounted with coverslips and
viewed under fluorescence microscopy.
(ii) Immunoassay.
A commercially available enzyme-linked
immunosorbent assay (Oncogene Research Products) was employed to
measure Fas receptor expression in tissue culture cells that had been
incubated in the presence or absence of H. pylori LC 11 for
24 h. Cells treated with IFN- (12.5 ng/ml) were used as a
positive control (12). Fas receptor expression was
calculated according to the manufacturer's instructions per
106 cells enumerated. Briefly, cell extracts were obtained
by incubating harvested cells in the supplied buffer with an antigen
extraction agent and then centrifuged to obtain a clear lysate. The
lysates were diluted and added to the supplied microtiter plates.
Following incubation with the detector antibody and streptavidin
conjugate, absorbances of wells in the plates were read
spectophotometrically at dual wavelengths of 450 to 490 nm.
Functional assessment of Fas receptor.
Untreated and
H. pylori-infected AGS cells were exposed for 24 h to
an activating anti-Fas monoclonal antibody (Upstate Biotechnology, Lake
Placid, N.Y.) at a concentration which had previously been shown to
induce apoptosis in Fas-sensitive cells (100 ng/ml) (12). Apoptotic cells were enumerated by fluorescence microscopy following staining with acridine orange and ethidium bromide, as described above.
H. pylori-infected cells were incubated with a neutralizing
anti-Fas antibody (Upstate Biotechnology) at 1,000 ng/ml. Dose-response studies (concentrations ranging from 100 to 2,000 ng/ml) determined that the maximal inhibition of apoptosis (50%) in AGS cells stimulated with the activating Fas antibody (100 ng/ml) was achieved at a dosage
of 1,000 ng/ml. Apoptotic cells were enumerated as described above.
Results are expressed as the means of the data obtained from two
independent experiments.
Statistical analysis.
Results are expressed as means ± standard errors (SE). To test the statistical significance between
multiple groups, a one-way analysis of variance (ANOVA) was used,
followed by post hoc comparisons with the Newman-Keuls test.
| |
RESULTS |
Evaluation of apoptosis in H. pylori-infected
gastric epithelial cells.
To determine if infection with H. pylori alone could stimulate apoptosis of gastric epithelial cells
in vitro, AGS and KATO III cells were incubated with the bacteria for
up to 72 h. AGS cells infected with a cagA-positive,
cagE-positive, VacA-positive isolate, strain LC 11, underwent apoptosis as assessed by fluorescence microscopy. As shown in
Fig. 1, apoptotic cells displayed the characteristic features of reduced size, cytoplasmic vacuolation, and
enhanced fluorescence of condensed and marginated nuclear chromatin.
These results were confirmed by transmission electron microscopy.
Unlike untreated cells, AGS cells infected with strain LC 11 demonstrated the ultrastructural features which characterize the
process of programmed cell death, including cytoplasmic vacuolation, condensed nuclear chromatin, and formation of apoptotic bodies (Fig.
2).
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FIG. 1.
Identification of apoptotic cells in untreated AGS cells
(A) and H. pylori-infected cells (B) after 72 h by
acridine orange-ethidium bromide staining and fluorescence microscopy.
(A) AGS cells demonstrate normal morphology. (B) H. pylori-infected AGS cells show morphologic features of apoptosis
(arrow), including condensed and marginated chromatin with apoptotic
body formation. Approximate magnifications, ×1,000.
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FIG. 2.
Transmission electron photomicrographs of uninfected (A)
and H. pylori-infected (B) AGS cells. (A) Control cells show
normal cellular morphology. (B) H. pylori-infected AGS cells
demonstrate the characteristic features of programmed cell death,
including cytoplasmic vacuolation (arrowhead) and apoptotic body
formation (arrow). Approximate magnifications, ×7,800.
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|
Quantitation of apoptotic AGS cells by fluorescence microscopy
demonstrated that
H. pylori LC 11-mediated cell death was
time
dependent. An increase in the death of gastric epithelial cells
was observed following 72 h of infection with the bacterium
(16.0%
± 2.8% versus 5.9% ± 1.4%,
P < 0.05)
(Fig.
3). When AGS cells
were infected
for 72 h with two clinical isolates which lack the
putative
virulence genes
cagE and
cagA and vacuolating
cytotoxin
activity, apoptosis of gastric epithelial cells was not
detected
(6.5% ± 1.3% versus 7% ± 2.1%). Similarly, infection
with the
related enteric pathogen
C. jejuni did not induce
apoptosis of
gastric cells (5.9% ± 2.7% versus 6.4% ± 2.3%). In
contrast to
AGS cells, KATO III cells more readily underwent necrosis
in response
to infection with
H. pylori strain LC 11 for
72 h; therefore,
the remaining studies were performed with AGS
cells.
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FIG. 3.
Quantitation of apoptotic AGS cells infected with
H. pylori for various lengths of time. Incubation with the
bacterium resulted in an increase in apoptosis compared to the
apoptosis of untreated cells at 72 h (P < 0.05
[ANOVA]). Results are expressed as the mean percentages of apoptotic
cells per 500 cells enumerated. Variations are represented as the SE.
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Expression of the Fas receptor during H. pylori
infection.
To determine if AGS cells had a basal expression of
Fas, which could be enhanced by IFN-, which is known to upregulate
expression of the receptor in other cell lines (12),
fluorescence microscopy with a monoclonal antibody to the Fas receptor
was employed. As shown in Fig. 4, AGS
cells had a low level of Fas expression which was enhanced following
stimulation by IFN- (12.5 ng/ml).
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FIG. 4.
Fluorescence micrograph demonstrating Fas expression.
(A) A low level of Fas expression is detected in untreated AGS cells.
(B) Fas expression is enhanced following incubation of AGS with IFN-
(12.5 ng/ml).
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Compared to uninfected (control) cells, infection with
H. pylori LC 11 also enhanced expression of Fas as determined by
enzyme-linked
immunosorbent assay (Fig.
5).
H. pylori-stimulated Fas
receptor
expression was comparable to that mediated by IFN-
(148% ± 24%
and 167% ± 24% of the control, respectively;
n = 3). The
H. pylori-mediated
Fas expression was not a
result of cross-reactivity with a bacterial
product, since assessment
of bacterial extracts alone showed no
detectable Fas expression.
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FIG. 5.
Effect of H. pylori on Fas receptor
expression in AGS cells. Incubation with H. pylori enhanced
Fas receptor expression in AGS cells to a degree comparable to that
obtained by treatment with IFN- (12.5 ng/ml). Results are expressed
as the percent increase in Fas receptor expression compared to the
expression in untreated (control) cells (+ SE).
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Sensitivity of H. pylori-infected cells to
Fas-stimulated cell death.
An agonistic monoclonal antibody to the
Fas receptor, which stimulates Fas-sensitive cells to undergo apoptosis
(12), was then employed to determine if the enhanced Fas
expression was functional. As shown in Fig.
6, incubation of AGS cells with the anti-Fas antibody mediated an increase in apoptosis compared to the
apoptosis of untreated cells (21.1% ± 1.8% versus 5.9% ± 1.4%). Furthermore, incubation of LC 11-infected gastric cells with the anti-Fas antibody resulted in a marked increase in programmed cell
death compared to the death of cells infected with the bacterium alone
(38.5% ± 7.1% versus 16.0% ± 5.5%, P < 0.05
[ANOVA]). Incubation of LC 11-infected AGS cells with a neutralizing
anti-Fas receptor antibody did not prevent cell death (7.7% versus
9.9%), indicating that the induction of apoptosis observed following
infection with the bacterium alone was not mediated by the enhanced Fas
receptor expression.
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FIG. 6.
Effect of H. pylori infection on
Fas-stimulated apoptosis. Untreated (control) and H. pylori-infected (strain LC 11) AGS cells were incubated in the
presence or absence of an agonistic monoclonal antibody (mab) to the
Fas receptor, and apoptosis was assessed. Incubation with the Fas
agonist enhanced programmed cell death in control cells (21.1% ± 1.8% versus 5.9% ± 1.4%, P < 0.05 [ANOVA]). The
apoptotic index of H. pylori-infected cells incubated with
an anti-Fas antibody increased compared to that of AGS cells incubated
with the bacterium alone (38.5% ± 7.1% versus 16.0% ± 2.8%,
P < 0.05 [ANOVA]). Results are expressed as the
percentages of apoptotic cells per 500 cells enumerated (+ SE).
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| |
DISCUSSION |
In vivo studies demonstrate that infection with H. pylori triggers apoptosis of gastric epithelial cells
(34). However, in this setting it is unclear whether immune
factors or bacterial factors contribute to cell death. This study
supports and extends recent evidence indicating that several mechanisms
are involved in stimulating apoptosis of gastric epithelial cells
during H. pylori infection (2, 33, 36).
These data demonstrate that H. pylori is capable of directly
inducing the death of gastric epithelial cells in vitro in the absence
of immune cells. The mechanism of cell death differed between the two
gastric cell lines. KATO III cells underwent necrosis in response to
prolonged infection with the bacterium, while AGS cells underwent
apoptosis. The response of AGS cells to infection with the bacterium
mimics the in vivo setting, indicating that the AGS cell line serves as
a better model system for investigating these apoptotic pathways than
the KATO III cell line.
The exact bacterial factors which directly mediate the death signal are
not known. Fan et al. (10) recently provided evidence that
binding of H. pylori to the class II major
histocompatibility complex expressed on gastric epithelial cells can
transduce the cell death signal in vitro. In this study, the presence
of factors considered to be associated with virulence, including
cagE and cagA, two genes found on the
pathogenicity island, as well as vacuolating cytotoxin activity, was
associated with apoptosis. In contrast, apoptosis was not detected
following infection with clinical isolates lacking these virulence
factors. These results are in agreement with the recent findings of
Rudi et al. (33), who detected apoptosis of gastric
epithelial cells following incubation with culture supernatants from a
cagA-positive H. pylori isolate with cytotoxic
activity but not with supernatants from a cagA-negative, noncytotoxic strain. In contrast, another study detected apoptosis during infection with both cagA-positive, VacA-producing
strains and cagA-negative, VacA-negative H. pylori strains (36). However, the cagE
status of the strains utilized in both of these studies was not
determined. Taken together, these findings suggest that the induction
of programmed cell death could play a role in mediating disease
outcome. Of interest, a preliminary study demonstrated that infection
with cagE-positive strains is associated with peptic ulcer
disease in children (8).
In addition to cell death triggered directly by infection with H. pylori, upregulation of the Fas receptor is observed in association with increased sensitivity to apoptosis upon ligation of
the receptor. Wagner and colleagues (36) also identified enhanced apoptosis in the gastric epithelial cell line HM02 following infection with H. pylori and Fas ligation. However, the
mechanism for the enhanced sensitivity to Fas signaling was not
determined. This study suggests that H. pylori infection
enhances expression of the Fas receptor in gastric epithelial cells,
thereby resulting in an increased sensitivity to Fas-triggered cell
death. H. pylori-mediated enhanced Fas expression does not
directly stimulate apoptosis since a neutralizing antibody did not
prevent cell death. These findings indicate that immune system-mediated
cell death through the Fas-Fas ligand pathway likely also contributes
to the apoptosis that is observed during infection in vivo.
The factors mediating enhanced Fas receptor expression and Fas-mediated
cell death during H. pylori infection are not known. A
recent study demonstrated that IFN-, which is increased in the
gastric mucosa during H. pylori infection (18),
augments apoptosis. IFN- also upregulates expression of the Fas
death receptor (30). Cytokines produced by inflammatory
cells in the lamina propria in response to H. pylori
infection could also modulate cell death. Further studies are required
to determine the factors which increase the susceptibility of gastric
cells to the Fas death cascade.
Our findings indicate that Fas-stimulated cell death could play a role
in H. pylori-mediated pathogenesis in vivo. During infection
with the bacterium, gastric epithelial cells exhibiting enhanced Fas
receptor expression could be eliminated by infiltrating lymphocytes
that express the Fas ligand. In support of this contention, a recent
study identified an increase in Fas ligand mRNA expression in
lymphocytes within the lamina propria and enhanced Fas receptor expression in both gastric epithelial cells and cells within the superficial lamina propria of H. pylori-infected gastric
biopsy tissue (33). Furthermore, the region of the gastric
mucosa with enhanced Fas ligand mRNA corresponded to areas of enhanced apoptosis.
Also, animal models suggest that Fas signaling plays a role in gastric
injury. In a murine model of autoimmune gastritis generated by either
thymectomy or adoptive transfer of a Th1 cell clone recognizing an
epitope of the H+, K+-ATPase, enhanced Fas
expression was detected on gastric parietal cells (29). In
contrast, gastric tissue from normal controls lacked detectable Fas.
The topographic expression of this death receptor in parietal cells
correlated with the induction of apoptosis as assessed by the terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
method (29). This indicates that one mechanism by which
autoimmune-mediated target cell destruction may be effected is Fas-Fas
ligand interactions. This is of particular interest since
autoantibodies directed against gastric parietal H+,
K+-ATPase are detected in sera from H. pylori-infected subjects and correlate with the presence of
gastric atrophy (6).
In summary, the present study shows that H. pylori infection
is capable of activating the apoptotic cell death cascade in gastric
epithelial cells by more than one mechanism. The bacterium can directly
stimulate programmed cell death and also enhances both expression of
the cell death receptor Fas and sensitivity to Fas-mediated apoptosis.
In vivo studies, including those with animal models of human disease
(22), should now be undertaken to further delineate the role
of Fas signaling in the pathogenesis of H. pylori-mediated disease.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Medical Research
Council of Canada. N. L. Jones is the recipient of a Research Initiative Award from the Canadian Association of
Gastroenterology/Astra Pharmaceuticals/Medical Research Council of
Canada. A. S. Day is a recipient of a Research Fellowship Award
from the Canadian Association of Gastroenterology/Solvay Pharma/Medical
Research Council of Canada. P. M. Sherman is a recipient of an AC
Finkelstein Award from the Medical Research Council of Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Gastroenterology/Nutrition, Room 8411, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. Phone: (416)
813-6185. Fax: (416) 813-6531. E-mail:
sherman{at}sickkids.on.ca.
Editor:
D. L. Burns
| |
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Abstract
Introduction
