Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario L8N 3Z5, Canada
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ABSTRACT |
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The association between oral contraceptives
or pregnancy and inflammatory bowel disease is unclear. We investigated
whether 17-estradiol modulates intestinal inflammation in two models of colitis. Female mice were treated with 17
-estradiol alone or with
tamoxifen, tamoxifen alone, 17
-estradiol, or placebo. Dinitrobenzene
sulfonic acid (DNB)- or dextran sodium sulfate (DSS)-induced colitis
were assessed macroscopically, histologically, and by myeloperoxidase
(MPO) activity. Malondialdehyde and mRNA levels of intercellular
adhesion molecule-1 (ICAM-1), interferon-
(IFN-
), and
interleukin-13 (IL-13) were determined. In DNB colitis, 17
-estradiol
alone, but not 17
-estradiol plus tamoxifen, or 17
-estradiol
reduced macroscopic and histological scores, MPO activity and
malondialdehyde levels. 17
-Estradiol also decreased the expression
of ICAM-1, IFN-
, and IL-13 mRNA levels compared with placebo. In
contrast, 17
-Estradiol increased the macroscopic and histological
scores compared with placebo in mice with DSS colitis. These results
demonstrate anti-inflammatory and proinflammatory effects of
17
-estradiol in two different models of experimental colitis. The
net modulatory effect most likely reflects a combination of estrogen
receptor-mediated effects and antioxidant activity and may explain, in
part, conflicting results from clinical trials.
inflammation; antioxidant activity; sex steroids
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INTRODUCTION |
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CLINICAL DATA ON THE ASSOCIATION between inflammatory bowel disease (IBD) and female sex hormones are conflicting. On one hand, IBD is believed to flare during a first pregnancy and postpartum and, on the other hand, to be better controlled during subsequent pregnancies (14, 43, 44). Others have proposed that the course of IBD during pregnancy depends on disease activity at conception (42, 64). Oral contraceptives have been suggested as a risk factor for relapse in Crohn's disease (60), but this is not supported by other studies (13). A recent report cited female gender, but not oral contraceptive use, as a risk factor for relapse in ulcerative colitis (5). Although study design, inclusion criteria, and different formulations of oral contraceptives may explain some of the discrepancies between studies, a causal relationship between sex hormones and IBD has not been clearly established. It also remains unknown whether sex hormones and, in particular, estrogen, possess immunomodulating effects in the gastrointestinal system.
Females have stronger humoral and cell-mediated immune responses than males (3, 18, 22, 59) and, in general, have a higher incidence of autoimmune diseases (31, 33, 40, 48, 57). Although estrogen has been reported to enhance immunity, estrogen therapy has been shown to attenuate inflammation in carrageenan-induced pleurisy in rats and myocardial reperfusion injury and to improve outcome after cerebral ischemia (15, 47, 56). This discrepancy may reflect recently described divergent effects of estrogen based on dose, tissue specificity, and cellular environment (63).
Most of the effects of estrogen are mediated by binding of the hormone to specific estrogen receptors (ERs) that act as nuclear transcriptor activators (25). T cells, B cells, and macrophages are known targets of estrogen (24, 62). Classical ERs have been described in vascular endothelium, fibroblasts, smooth muscle cells, and gastrointestinal mucosa, including epithelial cells (8, 25, 27, 54). Thus it is reasonable to expect that estrogen modulates inflammation in the gastrointestinal system.
In this study, we examined the effect of supraphysiological doses of
17-estradiol (those achieved during pregnancy) on colitis induced in
female mice. In light of the conflicting clinical data regarding sex
hormones and Crohn's disease and ulcerative colitis, we investigated
whether the effect of 17
-estradiol differed according to the manner
in which colitis is induced. For this purpose, we used two different
murine models of colitis: 1) a lymphocyte-dependent model by
intracolonic administration of dinitrobenzene sulfonic acid (DNB) and
2) a lymphocyte-independent model by oral administration of
dextran sodium sulfate (DSS). We found that supraphysiological doses of
17
-estradiol have anti-inflammatory effects in DNB colitis and
proinflammatory effects in DSS colitis, demonstrating complex immunomodulatory effects of estrogen during intestinal inflammation.
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MATERIALS AND METHODS |
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Animals
Female C57Bl/6 mice were obtained from Taconic and kept under specific pathogen-free conditions. Female, macrophage colony-stimulating factor (MCSF-1) deficient mice (op/op) and heterozygotes (op/+) were obtained from a colony maintained at McMaster Animal Care Facility as described previously (21). All experimental protocols were approved by the McMaster Animal Care Committee and the Canadian Council on the Use of Laboratory Animals.DNB Colitis
Study design and treatment groups.
Female C57Bl/6 mice were divided into five groups (n = 5-13 per group) that received 17-estradiol alone (0.5 mg/pellet) or with tamoxifen (5 mg/pellet), tamoxifen alone (5 mg/pellet), 17
-estradiol (0.5 mg/pellet), or placebo via 21-day
release pellets implanted subcutaneously (IRA, Sarasota, Florida). The
dose of 17
-estradiol used in this study markedly elevates the
plasma hormone level in mice (~1,000 pg/ml) by 7 days after
implantation, and these levels remain constant for 4 wk (49,
50).
Induction of colitis. Mice were anaesthetized with enflurane. A 10-cm long PE-90 tubing (Clay Adams, Parsippany, NJ), attached to a tuberculin syringe, was inserted 3.5 cm into the distal colon. Colitis was induced by the administration of 100 µl of a 4-mg DNB solution (ICN Biomedicals, Aurora, Ohio) in 50% ethanol. Control mice (without colitis) received saline administered as above. Mice with colitis were supplied with 8% sucrose and 0.1% saline in drinking water to prevent dehydration during the first week post-DNB.
Macroscopic and histological scores. The colon was removed and opened longitudinally, and macroscopic damage was immediately assessed. Tissue adjacent to the ulcer was taken, fixed in 10% formalin and stained with hematoxylin and eosin for subsequent histological examination. In the event that no macroscopic ulceration was observed, tissue was taken from hyperemic areas. Histological examinations were performed by the same investigator in a blinded fashion. Macroscopic and microscopic scores were performed using previously described scoring systems for hapten-induced colitis (1).
Myeloperoxidase activity.
Myeloperoxidase (MPO) activity was used as an index of
polymorphonuclear (PMN) infiltration as described previously
(6). Colonic tissue from mice 3 and 14 days post-DNB
colitis was frozen in liquid nitrogen and stored at 70°C until
assayed. All experiments were performed within 1 wk of collection of
tissue. MPO was expressed in units per milligram of tissue, where 1 unit corresponds to the activity required to degrade 1 mmol of hydrogen
peroxide in 1 min at 24°C.
Lipid peroxidation.
17-Estradiol is a potent antioxidant that could directly interfere
with MPO measurements and may not reflect the in vivo situation.
Therefore, malondialdehyde (MDA) levels were determined as an indicator
of lipid peroxidation in vivo as described previously (46). Colonic tissue from mice treated with
17
-estradiol alone and placebo 3 days post-DNB colitis was frozen in
liquid nitrogen and stored in
70°C until assayed. The
remaining groups were not tested due to insufficient tissue samples.
All experiments were performed within 1 wk of tissue collection. The
level of lipid peroxides is expressed as nanomoles of MDA per gram of
tissue calculated from the absorbance at 532 nm using an external standard.
RT-PCR.
mRNA expression of intercellular adhesion molecule-1 (ICAM-1) was
measured in colonic tissue obtained 3 and 14 days post-DNB colitis.
Because the cytokines that characterize DNB colitis are usually
detected during later phases, interferon- (IFN-
) and interleukin-13 (IL-13), were only measured in tissue obtained 14 days
post-DNB colitis.
DSS Colitis
In contrast to DNB colitis, acute DSS colitis does not require the presence of T cells to induce damage (17). DSS damage seems to be more dependent on the perpetuation of inflammation by translocation of intestinal flora (2). Macrophages have also been involved in DSS-induced damage (17). To investigate some of the possible mechanisms whereby estradiol can affect severity of DSS colitis, we used op/op mice that totally lack the population of macrophages dependent on MCSF-1, in addition to C57Bl/6 mice.Treatment groups and induction of colitis.
Female C57Bl/6 op/op and
op/+ mice (n = 5 to n = 10) received 17-estradiol alone (0.5 mg/pellet) or placebo via 21-day release pellets implanted
subcutaneously (IRA).
Disease severity and histological scores. The colon was removed and opened longitudinally, and macroscopic damage was immediately assessed. Tissue was obtained from the sigmoid colon and processed as above for histological assessment. Macroscopic and histological scores were performed using a previously described scoring system for DSS colitis (12) slightly modified to score separately for rectal and gross colonic bleeding.
TNF- measurement.
Serum samples were obtained from C57Bl/6 mice with and without DSS
colitis and stored at
20° until TNF-
protein measurement by
ELISA using a commercial kit (Quantikine M murine; R&D Systems, Minneapolis, MN). MCSF-1 mutant mice lack the population of macrophages that are the main source for proinflammatory cytokines, such as TNF-
. Because a number of other cells may also produce this
cytokine, we used RT-PCR to measure mRNA as described above and ELISA
to measure TNF-
protein in colonic tissue.
Statistical Analysis
Nonparametric data are presented as medians and 95% confidence intervals and parametric data as means ± SD as stated in the table legends. Box plots depict 5th, 25th, 50th (median), 75th, and 95th percentiles. On the basis of data distribution, statistical testing was performed using the Mann-Whitney U-test for unpaired nonparametric data or Student's t-test for unpaired parametric data. Multiple comparisons were performed using the Friedman test followed by Wilcoxon-Wilcox. ![]() |
RESULTS |
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Effects of Estrogen on Mice Without Colitis
In mice without colitis, estrogen pretreatment resulted in more similar macroscopic and histological scores and MPO activities than in placebo-treated mice (data not shown).Effects of Estrogen on Body Weight
Eighteen days after pellet implantation and before the induction of colitis with DNB or DSS, treatment with 17
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Effects of Estrogen at Day 3 Post-DNB-Induced Colitis
In mice with DNB colitis, treatment with 17
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17-Estradiol treatment decreased PMN infiltration in mice with DNB
colitis as reflected by a 15-fold reduction in MPO activity compared
with placebo-treated mice with colitis (Fig. 1,C). This marked reduction in MPO activity, suggested a direct antioxidant effect
of 17
-estradiol in addition to the histological finding of a reduced
PMN infiltration. The antioxidant effect was confirmed by a reduction
in lipid peroxidation from 580 (420-640) nmol MDA/g in placebo-treated mice with DNB colitis, to 282 (75-388) nmol MDA/g in 17
-estradiol-treated mice
with DNB colitis (P < 0.04).
The ICAM-1-to-GAPDH ratios 3 days post-DNB colitis are shown in Table
2. ICAM-1 mRNA expression was decreased
in 17-estradiol-treated mice compared with placebo-treated mice with
DNB colitis. ICAM-1 mRNA expression was similar in
17
-estradiol-treated mice with DNB colitis and in mice with no
colitis.
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Effects of Estrogen at Day 14 Post-DNB-Induced Colitis
Figure 2, C and D, depicts minimal inflammation in a mouse treated with 17
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The decrease in macroscopic and histological scores 14 days postcolitis
in mice treated with 17-estradiol alone was also accompanied by a
reduction in cytokine mRNA expression compared with placebo-treated
mice with colitis. ICAM-1 mRNA expression 14 days post-DNB colitis was
detected in all groups including mice without colitis, suggesting
constitutive expression (Fig. 4)
(46). Mice with DNB colitis treated with 17
-estradiol
alone had an ICAM-1-to-GAPDH ratio 1.5-fold higher than in mice without DNB colitis. There was, however, a 33% decrease in the ICAM-1-to-GAPDH ratio, in 17
-estradiol-treated mice with DNB colitis compared with
mice treated with tamoxifen plus
-estradiol or with placebo. There
was a 98% reduction in the IFN-
-to-GAPDH ratio in
17
-estradiol-treated mice with DNB colitis compared with
placebo-treated mice with DNB colitis. The IL-13-to-GAPDH ratio was
also decreased in 17
-estradiol-treated mice with DNB colitis
compared with placebo-treated mice with DNB colitis (Fig. 4).
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Although the present study was not designed to investigate the role of physiological levels of estrogen on intestinal inflammation, we did examine vaginal swabs to determine the phase of the estrous cycle at the time of DNB colitis induction and of death, in mice without pellet implantation. No differences were found in the severity of DNB colitis between the high estrogen (proestrous-estrous) or low estrogen (metestrous-diestrous) phases of the cycle (data not shown).
Effects of Estrogen on DSS-Induced Colitis
C57Bl/6 mice.
Table 3 shows the disease severity
and histological scores at day 5 in mice with DSS colitis.
17-Estradiol treatment had opposite effects on the severity of DSS
colitis when compared with DNB colitis. In mice with DSS colitis
treated with 17
-estradiol, the disease severity score and
histological scores increased by 100 and 67%, respectively, with
respect to placebo-treated mice with DSS colitis (P = 0.01).
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MCSF-1 deficient mice (op/op). Although there was a trend toward lower disease severity scores and histological scores in op/op mice with DSS colitis as a whole, these differences did not achieve statistical significance.
17
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TNF- in C57Bl/6 Mice and MCSF-1 Deficient Mice (op/op) Mice
Low levels of mRNA for TNF- and protein TNF-
measured by ELISA
were detected in op/op mice with DSS colitis. Overall,
op/op mice treated with 17
-estradiol tended to have
higher cytokine levels in colonic tissue than mice treated with placebo
(Fig. 6).
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DISCUSSION |
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In light of the conflicting clinical data regarding sex hormones
and IBD, we examined the effect of supraphysiological doses of
17-estradiol on DNB- and DSS-induced colitis, two experimental models with different underlying pathophysiological mechanisms. Three
days after induction of DNB colitis, 17
-estradiol reduced the
severity of colitis, the infiltration of colonic tissue with PMN cells
(assessed both by histology and MPO activity), the degree of lipid
peroxidation, and the level of mRNA expression of ICAM-1. 17
-Estradiol also reduced the severity of tissue damage, the infiltration of tissue with mononuclear cells, and the mRNA
expression of IFN-
, IL-13, and ICAM-1, 14 days postcolitis. In
contrast, 17
-estradiol increased the disease severity and
histological scores in DSS colitis in C57Bl/6 and in MCSF-1-deficient
mice (op/op).
Our results indicate that the anti-inflammatory effect of
17-estradiol in DNB colitis was mediated by ERs. Tamoxifen is a selective ER modulator with both agonist and antagonist activities depending, in part, on the target tissue and the estrogenic milieu (29). Because the effect of tamoxifen as an ER antagonist
in the gastrointestinal tract is unknown and because we wished to evaluate the role of ER, we also used 17
-estradiol, an inactive estrogen that binds to ERs without activating transcription. Tamoxifen alone or with 17
-estradiol inhibited the protective effect of 17
-estradiol on macroscopic and histological scores 3 days
postcolitis. At later stages, tamoxifen with 17
-estradiol
significantly inhibited the protective effect of 17
-estradiol on
histological scores, but there was only a trend toward inhibiting the
protective effect in macroscopic scores. This may be because, in
contrast to histological damage, macroscopic damage 14 days post-DNB
colitis is minimal, and differences between groups are difficult to
detect. The absence of a protective effect in mice with DNB colitis
treated with tamoxifen or 17
-estradiol supports the fact that the
anti-inflammatory effect of 17
-estradiol involves ERs.
Sex steroids have antioxidant properties that are not blocked by ER
antagonists (4, 15) and that could contribute to the low
MPO activity and lipid peroxidation seen during 17-estradiol treatment. Our results suggest that a pure antioxidant effect is not,
however, the sole anti-inflammatory mechanism of 17
-estradiol. 17
-Estradiol did not decrease the severity of DNB colitis, although it possesses antioxidant activity. We believe that the major
anti-inflammatory effect of 17
-estradiol in DNB colitis is the
inhibition of neutrophil recruitment during the early stages of
colitis. This is supported by the following findings. Three days
postcolitis, ICAM-1 mRNA expression was lower in mice treated with
17
-estradiol, and histological examination revealed a marked
decrease in PMN infiltration. Also, MPO activity was close to zero and
lipid peroxidation levels were markedly reduced in
17
-estradiol-treated mice compared with placebo-treated mice. Our
results are in accordance with previous reports in systems other than
the gastrointestinal tract, where an inhibitory effect of estrogen on
PMN infiltration has also been suggested. 17
-Estradiol was shown to
reduce PMN chemotaxis and PMN infiltration at several sites of
inflammation (7, 15, 56, 66) and to decrease the level of
adhesion molecules, such as sICAM-1 and sVCAM-1 in cardiovascular
disease (9, 61).
Immune response in DNB colitis results from a delayed-type
hypersensitivity (DTH) response reaction against haptenized colonic proteins and is characterized by a T helper (Th1) cytokine response (19). In contrast, the mechanism by which DSS induces
acute colitis is unclear. It seems to result from an alteration in the colonic epithelium with resulting bacterial translocation that perpetuates inflammation rather than from an alteration of B and T cell
responses because acute DSS colitis develops in SCID mice. Innate immunity and macrophages have, therefore, been suggested to play
a role (17). Estrogen has been reported to suppress T
cell-dependent DTH and activation of inflammatory cells producing TNF- and IFN-
(51-53, 58). Our data suggest a
downregulation of the Th1 response associated to DNB colitis as
reflected by decreased IFN-
mRNA expression 14 days postcolitis in
17
-estradiol-treated mice. The finding that IL-13 mRNA expression
was also decreased in mice treated with 17
-estradiol alone was
somewhat unexpected. On the basis of a recent report that estrogen has
biphasic effects on immune responses (63), 17
-estradiol
at high doses would suppress Th1 responses and promote Th2 responses,
and this could also occur in the gut during an episode of inflammation.
It is possible, however, that Th3 or Th2 cytokines, other than IL-13, not measured in this study, are increased by high doses of estrogen. We
believe that in this study, the effect of 17
-estradiol on mRNA
expression of Th1 and Th2 cytokines is a consequence of the lower
initial acute inflammation, rather than a direct effect of estradiol on
Th cells. This could also explain the fact that ICAM-1 expression was
lower in estradiol-treated mice than in placebo-treated mice with DNB
colitis, both 3 and 14 days postcolitis. Although no differences were
found in ICAM-1 expression between estradiol-treated mice and mice
without colitis 3 days post-DNB colitis, ICAM-1 expression was higher
14 days postcolitis in estradiol-treated mice than in mice without
colitis. This may reflect the different inflammation pattern found at
day 3 (predominantly neutrophilic) and at day 14 post-DNB colitis (predominantly lymphocytic). The effect of estradiol
on early integrin expression on endothelial cells may differ from the
effect of estradiol on integrin expression on lymphoid cells. This
matter should be further investigated.
A number of studies have suggested that estrogen modifies macrophage
function and the production of inflammatory mediators (16, 26,
39, 55). However, we found an increase in disease severity score
and histological scores in both
op/+ and op/op mice
with DSS colitis treated with 17-estradiol compared with mice with
DSS colitis treated with placebo. The enhanced colitis severity seen in
17
-estradiol-treated op/op mice, excludes a role for
MCSF-1 macrophages and implicates other mechanisms. Although there was
a clear increase in disease severity score and histological scores, in
17
-estradiol-treated mice with DSS colitis, MPO activity was low
(data not shown). In fact, in placebo-treated mice, MPO activity was
overall 10-fold lower in mice with DSS colitis than in mice with DNB
colitis. This suggests that the antioxidant effect and/or the
inhibition of neutrophil infiltration by 17
-estradiol would be less
prominent in DSS colitis than in DNB colitis. Op/op mice have resident MCSF-1-independent macrophages with preserved antigen presentation and phagocytosis (65). Estradiol has
been shown to sensitize immune cells to lipopolysaccharide
(LPS), and females treated with pharmacological doses of
estradiol, are more sensitive to liver damage by LPS (20, 30,
37). Because translocation of luminal bacteria may play an
important role in perpetuating DSS colitis, it is possible that the
increased severity may relate to the sensitizing effects of estradiol
to bacterial endotoxins. Interestingly, TNF-
plasma levels were
increased in estradiol-treated C57Bl/6 mice at day 5 post-DSS colitis compared with placebo-treated mice with colitis and
mice without colitis. Consistent with previous reports on plasma
TNF-
levels in op/op mice (65), we detected low levels of TNF-
in colonic tissue from op/op mice.
Despite the low levels, TNF-
tended to be higher in
estradiol-treated op/op mice with DSS colitis than in
placebo-treated op/op mice with colitis. Natural killer
cells, lymphocytes, mast cells, basophils, and eosinophils may be an
alternative source of TNF-
in these mice.
Estradiol treatment alone, or with tamoxifen, led to a 15% increase in
body weight before the induction of colitis compared with mice treated
with placebo. The effect on body weight was more pronounced if estrogen
pretreatment was more prolonged (18 instead of 7 days). However, it is
very unlikely that this increase in body weight could have affected the
severity of colitis significantly. First, when estradiol was
administered with tamoxifen, an increase in body weight was also noted.
Still, colitis severity was increased compared with 17-estradiol
treatment alone, and significant weight loss was observed after the
induction of colitis. Second, despite an increase in body weight before
the administration of DSS in 17
-estradiol-treated mice, severity of
DSS colitis and weight loss were increased when compared with
placebo-treated mice with DSS colitis.
In conclusion, this is, to our knowledge, the first demonstration that
estrogen has complex effects on intestinal inflammation. In DNB
colitis, supraphysiological doses of 17-estradiol decreased recruitment of PMN cells at early stages of colitis, probably through
an ER-mediated mechanism. An antioxidant effect of 17
-estradiol also
contributed to the reduced tissue damage. At later stages of DNB
colitis, a downregulation of the DNB-associated Th1 response was
observed. Our data do not support the hypothesis that
17
-estradiol-induced increased severity of DSS colitis involves
activation of MCSF-1 macrophages. Other mechanisms whereby estrogen may
affect intestinal inflammation remain to be investigated. These include
a possible effect on neuropeptide release and transmission, effects on
smooth muscle contractility, and effects on the healing and repair
process. Recent reports on the genetic changes that may induce
susceptibility for IBD suggest a link between a defective innate immune
response to bacterial components and the development of chronic
inflammation (28, 44). The NOD2 gene encodes
for a protein that makes nuclear factor-
B responsive to bacterial
LPS. It could be hypothesized that the effect of sex hormones could
also depend on the genetic background or specific mutations that
underlie the development of IBD in a given individual. Some mutations
may involve mechanisms modulated by sex hormones and some may not.
Taken together, our results indicate that the effect of estrogen on
colitis depends on the manner in which inflammation is induced. This
may explain the apparent conflicting clinical literature on gender,
pregnancy, and sex hormones in IBD.
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ACKNOWLEDGEMENTS |
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This study was supported, in part, by a grant from the Canadian Institutes for Health Research (to S. M. Collins).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M. Collins, Rm. 4W8, McMaster Univ. Medical Center, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada (E-mail: scollins{at}mcmaster.ca).
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.
First published February 20, 2002;10.1152/ajpgi.00460.2001
Received 31 October 2001; accepted in final form 4 February 2002.
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