Role of T cells in development of chronic pancreatitis in male Wistar Bonn/Kobori rats: effects of tacrolimus

Tamaki Yamada1, Takashi Hashimoto1, Mitsue Sogawa2, Sawako Kobayashi2, Kenji Kaneda2, Soichi Nakamura1, Atsushi Kuno1, Hitoshi Sano1, Tomoaki Ando1, Shinya Kobayashi1, Shigeru Aoki1, Takahiro Nakazawa1, Hirotaka Ohara1, Tomoyuki Nomura1, Takashi Joh1, and Makoto Itoh1

1 First Department of Internal Medicine, Nagoya City University Medical School, Nagoya 467-8601; and 2 Second Department of Anatomy, Osaka City University Medical School, Osaka 545-8585, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We assessed T cell association with acinar cell apoptosis and a preventive effect of tacrolimus, a T cell suppressant, on the development of chronic pancreatitis in male Wistar Bonn/Kobori rats. At 15 wk, cellular infiltrates composed of F4/80-positive cells (monocytes/macrophages), CD4-positive cells, and CD8-positive cells were extensive in the interlobular connective tissue and parenchyma. In particular, CD8-positive cells invaded pancreatic lobules and formed close associations with acinar cells, some of which demonstrated features of apoptosis. At 20 wk, CD8-positive cells were still abundant in the fibrotic tissue formed with loss of acinar cells. Repeated subcutaneous injection of 0.1 mg · kg-1 · day-1 but not 0.025 mg · kg-1 · day-1 of tacrolimus for 10 wk completely prevented the occurrence of acinar cell apoptosis, infiltration of CD4- and CD8-positive cells, and development of pancreatitis at the age of 20 wk, but these maneuvers did not recover the decreased plasma corticosterone levels, which may be responsible for the development of disease. We demonstrated that T cells, possibly CD8-positive cells, are involved in inducing apoptosis of acinar cells, raising the possibility that tacrolimus might find clinical application in the treatment of autoimmune chronic pancreatitis.

autoimmune pancreatitis; acinar cells; apoptosis; corticosterone


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HUMAN CHRONIC PANCREATITIS is an irreversible and progressive disease characterized by acinar cell destruction and fibrosis. Causes are various, and cell-mediated autoimmunity might also be involved, as indicated by infiltration of CD4- and CD8-positive cells and high expression of major histocompatibility complex class I and class II antigens by exocrine epithelial cells, which is suggestive of cell-mediated autoimmune reactions (2, 3, 7, 14). Recently, autoimmune pancreatitis has been proposed as an entity in humans as defined by lymphocyte infiltration, autoantibody production, and effectiveness of steroid therapy (13, 29, 31, 37).

Male Wistar Bonn/Kobori (WBN/Kob) rats spontaneously develop chronic pancreatitis and diabetes. The disease starts with edema of the interlobular and intralobular connective tissue with focal infiltration of inflammatory cells, progresses with increasing extent of inflammation and acinar cell injury, and finally exhibits pancreas replacement by granulation composed of fibroblasts, neutrophils, and lymphocytes, resulting in insufficiencies of exocrine and endocrine functions (24, 26, 28). We previously found (11) that in these rats, apoptosis of acinar cells significantly increases at the ages of 15 and 20 wk in parallel with inflammatory cell infiltration, and we proposed that this might profoundly contribute to the development of chronic pancreatitis. It was also noted that acinar cell apoptosis was related to the decreased levels of endogenous corticosterone, and administration of prednisolone considerably alleviated the disease (11), suggesting the involvement of autoimmune mechanisms in this rat model. Autoimmune mechanisms have also been postulated for the animal models of spontaneous diabetes mellitus, such as BioBreeding/Worcester rats, nonobese diabetes (NOD) mice, and MRL/Mp strain mice (8, 17, 20, 21). Furthermore, Vallace et al. (35) demonstrated that the aged major histocompatibility complex class II-deficient mice develop an immune-based chronic pancreatitis with selective loss of exocrine pancreatic function.

It was reported (16, 30) that cytotoxic T cells bearing Fas ligand are capable of inducing apoptotic cell death. In this study, to assess the involvement of cytotoxic T cells in induction of acinar cell apoptosis in male WBN/Kob rats, we immunohistochemically investigated the kinetic and spatial relationships of T cell accumulation with apoptotic acinar cells. We further investigated the in vivo effects of tacrolimus, a potent T cell suppressant, on the induction of acinar cell apoptosis, infiltration of CD4- and CD8-positive T cells, development of chronic pancreatitis, and decreased levels of endogenous corticosterone.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male WBN/Kob rats were purchased from SLC (Hamamatsu, Japan) and kept in a temperature-controlled room under a constant light cycle. They were allowed free access to water and standard laboratory diet. The study protocol was approved by the Animal Care Committee of Nagoya City University.

Administration of tacrolimus. Tacrolimus and its placebo were generously donated by Fujisawa (Tokyo, Japan). WBN/Kob rats at the age of 10 wk were randomly divided into three groups: 1) a placebo group (n = 7), 2) a low-dose (0.025 mg · kg-1 · day-1) group (n = 6), and 3) a high-dose (0.1 mg · kg-1 · day-1) group (n = 6). The placebo and tacrolimus dissolved in saline were subcutaneously injected every other day for 10 wk. Body weights were recorded once a week.

In a separate series of experiments, to quantitatively assess the effect of tacrolimus on T cells, 10-wk-old WBN/Kob rats were divided into two groups, 1) a placebo group (n = 4) and 2) a high-dose (0.1 mg · kg-1 · day-1) group (n = 8), and were treated for 10 wk in the same manner. Then, pancreatic specimens were immunohistochemically stained to identify CD4- and CD8-positive cells and thymocytes, and peripheral lymphocytes were analyzed by flow cytometry as described in Flow cytometric analysis.

Histology. Animals aged 10, 15, and 20 wk (6 animals/age) were killed with an overdose of pentobarbital sodium (Abbott Laboratories, North Chicago, IL). Placebo- (n = 7), low dose of tacrolimus- (n = 6), and high dose of tacrolimus- (n = 6) treated rats were also killed after 10 wk treatment in the same manner at 4 PM after blood was drawn from the abdominal aorta to minimize the influence of circadian rhythm on the endogenous glucocorticoid levels. Blood was heparinized, and plasma was kept at -40°C to measure plasma corticosterone levels as described in Measurement of plasma corticosterone levels.

For immunohistochemistry, the pancreata of three animals at each age were taken out and fixed with 4% paraformaldehyde in PBS overnight at 4°C. After successive transfer in 8-20% sucrose in 0.1 M phosphate buffer (pH 7.4), the pancreata were frozen in liquid nitrogen. Sections were cut with a CM3000 cryostat (Leika Instruments, Nussloch, Germany) and immediately air dried. To block endogenous peroxidase activity, they were incubated with 0.3% hydrogen peroxide in methanol for 20 min at room temperature and further incubated with diluted mouse anti-rat CD4 monoclonal antibody (MAb) (1:1,000; Serotec, Oxford, UK), mouse anti-rat CD8 MAb (1:1,000; Serotec), and mouse anti-rat F4/80 MAb (1:300, A3-1; Cosmo Bio, Tokyo, Japan) overnight at 4°C. After being washed with PBS, they were treated with diluted biotinylated secondary rabbit anti-mouse IgG antibody (1:500; DAKO, Copenhagen, Denmark) for 60 min at room temperature, followed by incubation with avidin-biotin-peroxidase complex (Vectastain kit; Vector Laboratories, Peterborough, UK). Reaction products were visualized after incubation with 0.025% diaminobenzidine and 0.003% hydrogen peroxide. Counterstaining was achieved with methyl green. Other sections were stained with hematoxylin and eosin (H-E).

For detection of apoptosis, the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method was employed. Three untreated animals at each age and placebo- (n = 7) and tacrolimus- (low dose; n = 6 and high dose; n = 6) treated animals after a 10-wk administration were examined. The pancreas was perfusion fixed with 1.5% glutaraldehyde in 0.067 M cacodylate buffer (pH 7.4) plus 1% sucrose via the left ventricle for 5 min and then immersed in 10% formalin. Paraffin sections were treated with 20 µg/ml proteinase K for 15 min. To block endogenous peroxidase, pancreata were incubated with 3% hydrogen peroxide for 5 min. TUNEL staining was performed using a complete ApopTag kit (Oncor, Gaithersburg, MD). Reaction products were visualized with the diaminobenzidine reaction combined with cobalt and nickel.

Scoring of pancreatitis. Histological alterations, including inflammatory cell infiltration, interstitial edema and fibrosis, acinar cell injury, and hemorrhage were graded on a scale of negligible to maximal (0-3), as reported previously (11).

Counting of apoptotic acinar cells and CD4- or CD8-positive T cells. The numbers of TUNEL-positive acinar cells in the placebo (n = 7) and tacrolimus (low dose; n = 6 and high dose; n = 6) groups were counted in 20 blindly selected areas for each animal at 200-fold magnification as reported previously (11). The numbers of CD4- or CD8-positive T cells were counted in the immunohistochemically stained sections derived from the placebo (n = 4) and high dose of tacrolimus (n = 8) groups at 400-fold magnification in the same manner.

Measurement of pancreatic myeloperoxidase activity. Portions of fresh tissue obtained from the placebo (n = 7) and tacrolimus (low dose; n = 6 and high dose; n = 6) groups were stored at -40°C. Pancreatic myeloperoxidase (MPO) activity, an indirect quantitative index of granulocyte infiltration, was determined by using a minor modification of the method of Grisham et al. (9). Briefly, pancreatic tissue was homogenized in 20 mM phosphate buffer (pH 7.4) and centrifuged at 6,000 g for 20 min at 4°C. The pellet was then homogenized and sonicated with an equivalent volume of 50 mM acetic acid (pH 6.0) containing 0.5% (wt/vol) hexadecyltrimethylammonium hydroxide. The MPO activity was determined by measuring the hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine and expressed as units per gram wet weight of pancreas.

Measurement of plasma corticosterone levels. Plasma corticosterone levels in the placebo (n = 7), low-dose (n = 6), and high-dose (n = 6) groups were determined as described previously (34).

Flow cytometric analysis. Thymocytes and peripheral lymphocytes in the placebo (n = 4) and high dose of tacrolimus (n = 8) groups were prepared and stained with both fluorescein isothiocyanate-conjugated mouse anti rat-CD4 and R-phycoerythrin-conjugated mouse anti rat-CD8a MAbs (Becton-Dickinson, Mountain View, CA) as described previously (15). Freshly stained cells were analyzed by single-color or double-color fluorescence distribution in a FACScan flow cytometer (Becton-Dickinson).

Statistics. Data are expressed as means ± SE. Statistical differences among groups were identified using one-way ANOVA. Multiple comparisons were performed using the least significant difference method. Differences were analyzed by the Student's t-test.


    RESULTS
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INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

Spatial association of T cells with apoptotic acinar cells during chronic pancreatitis. At the age of 10 wk, there were no appreciable histological changes in the pancreas. Infiltration of CD8-positive cells was negligible (Fig. 1A). At 15 wk, inflammation had developed but was uneven among the lobules. In the inflamed portions, a large number of CD8-positive cells infiltrated in the interlobular connective tissue and further invaded the parenchyma, making contact with acinar cells (Fig. 1B). At 20 wk, the acinar tissue was destroyed, leaving ductular structures embedded in fibrous tissue, in which CD8-positive cells were still abundant (Fig. 1C).


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Fig. 1.   Immunohistochemistry of CD8 in pancreas tissue at the age of 10 wk (A), 15 wk (B), and 20 wk (C). A: no appreciable infiltration of positive cells is evident. L, islet of Langerhans; V, blood vessels. B: CD8-positive cells have infiltrated the interlobular connective tissue (asterisks) and also invaded the parenchyma (arrows). The portion within the square is enlarged in Fig. 3F. C: the parenchyma has disappeared after destruction, and ductular structures (D) alone remain in the fibrous tissue. Positive cells are distributed in and around the fibrous tissue. Magnification, ×160.

Serial section analysis of H-E-stained (Fig. 2A) and immunostained sections (Fig. 2, B-D) at 15 wk demonstrated that the cellular infiltrates consist of F4/80-positive cells (monocytes/macrophages) (Fig. 2B), CD8-positive cells (Fig. 2C), and CD4-positive cells (Fig. 2D). All showed similar distributions. However, CD8-positive cells in particular invaded the parenchyma. In H-E-stained sections, neutrophils were also rich in cellular infiltrates.


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Fig. 2.   Serial sections of pancreas at the age of 15 wk stained with hematoxylin and eosin (H-E; A) and immunostained for F4/80 (B), CD8 (C), and CD4 (D). A: cell infiltration is seen in the interlobular connective tissue (asterisks) and in the parenchyma. The acini are decreased in volume, and the interacinar spaces are wider than normal. B-D: F4/80-positive cells (B), CD8-positive cells (C), and CD4-positive cells (D) are distributed in similar patterns. Some have invaded the parenchyma, as indicated by arrows. The portion in the square in Fig. 2C is enlarged in Fig. 3E. Magnification, ×170.

Apoptotic acinar cells as demonstrated by the TUNEL method were more numerous in inflamed (Fig. 3B) than in noninflamed lobules (Fig. 3A) at 15 wk. In the inflamed portions, infiltrating cells were also positive in interlobular connective tissue (Fig. 3B). Acinar cells exhibited various sizes of apoptotic bodies with round cytoplasm and condensed nuclear chromatin (Fig. 3C). Halos were usually present around apoptotic bodies, which were densely stained with the TUNEL method (Fig. 3D). In immunostained sections, CD8-positive cells were attached to acinar cells, some of which exhibited apoptotic features (Fig. 3E).


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Fig. 3.   Apoptotic cells in the pancreas at the age of 15 wk. A and B: frequency of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells in noninflamed (A) and inflamed (B) portions of the same pancreas. In the former portion, neither inflammatory cell infiltration nor positive cells are found, whereas in the latter positive staining is frequent in the acinar cells and interlobular connective tissue (asterisks). C: apoptotic acinar cells in an H-E-stained section. Apoptotic bodies (arrowheads) of various sizes are round and often contain chromatin-dense nuclear fragments. A, acinar cells; C, centroacinar cells. D: TUNEL-positive acinar cells or apoptotic bodies (arrowheads) are seen in the acini. Lymphocytes (small arrows) infiltrate between the acini. Cp, capillaries. E and F: immunohistochemistry for CD8. CD8-positive cells (large arrows) are adherent to acini, in which apoptotic bodies (arrowheads) are evident. Magnification: A and B, ×80; C, ×1,000; D, ×320; E and F, ×1,100.

Preventive effect of 10-wk treatment with tacrolimus on acinar cell apoptosis and the development of pancreatitis. There was a small but significant decrease in body weight in the rats treated with the high dose (0.1 mg · kg-1 · day-1) of tacrolimus compared with those in placebo and low-dose groups (Table 1). The pancreas weight, on the other hand, showed a significant increase. In H-E-stained sections, inflammatory cell infiltration, destruction of acini, and proliferation of connective tissue were evident in the placebo group (Fig. 4A). In contrast, no inflammatory changes or injuries were apparent in any lobules of the pancreas treated with the high dose of tacrolimus (Fig. 4C). Histological scores revealed that tacrolimus dose-dependently suppressed the development of chronic pancreatitis (Table 1). Consistent with this, pancreatic MPO activity was significantly decreased by the high dose of tacrolimus (Table 1). Histologically, TUNEL-positive acinar cells were apparent in the placebo group, whereas these positive cells were extremely rare in the high-dose group (Fig. 4B and 4D). The effect was also confirmed by numerical analyses (Table 1).

                              
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Table 1.   Effects of tacrolimus on the body weight, pancreas weight, histological scores, pancreatic myeloperoxidase activity, and number of apoptotic acinar cells in 20-wk-old male WBN/Kob rats



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Fig. 4.   A: H-E-stained pancreas tissue of a 20-wk-old WBN/Kob rat treated with the placebo. Inflammatory cell infiltration, destruction of acini, and proliferation of connective tissue are evident. B: TUNEL-positive acinar cells are seen in a 20-wk-old WBN/Kob rat treated with the placebo. C: no inflammatory changes or injuries are apparent in any lobules of the pancreas treated with the high dose of tacrolimus. D: no TUNEL-positive acinar cells are apparent in a 20-wk-old WBN/Kob rat given high dose of tacrolimus. Magnification, ×120.

Effect of tacrolimus treatment on the plasma corticosterone levels. Plasma corticosterone levels did not significantly differ among the placebo, low-dose, and high-dose groups (187.8 ± 5.0, 172.8 ± 9.4, and 221.6 ± 16.8 ng/ml, respectively).

Effects of tacrolimus treatment on the infiltrated T cells in the pancreas and T cell subsets in the thymus and peripheral blood. In the immunostained pancreatic sections, both CD4- and CD8-positive T cells were evident in the placebo group, whereas the positive cells were almost absent in the high-dose tacrolimus group. Quantitative analyses of CD4- and CD8-positive cells revealed that a high dose of tacrolimus significantly decreased the numbers of both CD4- and CD8-positive cells compared with the placebo (CD4, 0.03 ± 0.01 vs. 3.73 ± 1.13; CD8, 0.04 ± 0.02 vs. 5.50 ± 1.24; P < 0.05 for both). Flow cytometric analyses revealed that a high dose of tacrolimus did not alter the proportions of CD4- and CD8-positive cells in the thymus and peripheral blood compared with the placebo treatment (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have demonstrated here that T cells, in particular CD8-positive cells, infiltrated deeply into the pancreatic parenchyma in aging male WBN/Kob rats and made a close association with apoptotic acinar cells, whereas F4/80-positive cells (monocytes/macrophages) and CD4-positive cells, although abundant like CD8-positive cells, less frequently invaded the acini. Because cytotoxic T cells bear Fas ligands that participate in apoptotic cell death (16, 30), CD8-positive cells infiltrating into the acini are most probably involved in the observed acinar cell apoptosis. This interpretation was supported by the present finding that tacrolimus, a potent suppressant of T cell proliferation, cytotoxic T cell generation, and T cell-derived cytokine production (18, 19), completely prevented the occurrence of acinar cell apoptosis, infiltration of both CD4- and CD8-positive cells, and development of chronic pancreatitis.

It has been documented that cell-mediated autoimmune mechanisms are involved in the development of chronic pancreatitis in humans (2, 7), and Bedossa et al. (3) demonstrated large numbers of CD8-positive cells infiltrating areas of inflammation in human chronic pancreatitis. It was described that 82 and 66% of a series of 93 patients with chronic pancreatitis, respectively, expressed major histocompatibility complex class I and class II immunoreactivity in exocrine epithelial cells, suggesting that an unknown antigen may trigger a cell-mediated autoimmune reaction (3, 14). More recently, Okazaki et al. (29) reported that CD8- and CD4-positive cells were increased in the peripheral blood with a Th1-type immune response involvement in autoimmune chronic pancreatitis in humans (29). Consistent with these findings, we confirmed that CD8- and CD4-positive cells contribute to autoimmunity in the present model.

Neonatal thymectomy or intrathymic injection of islet tissue prevents spontaneous diabetes mellitus in BioBreeding/Worcester rats and NOD mice, suggesting that thymus-dependent, cell-mediated autoimmune destruction of pancreatic beta -cells is responsible for their development of diabetes mellitus (8, 20, 21). Indeed, diabetes requires both CD4- and CD8-positive cells, homing of the latter to the pancreas being mediated by CD4-positive cell-dependent processes in NOD mice (23, 33). CD8-positive cells cause the initial beta -cell injury that sheds and loads beta -cell autoantigens onto antigen-presenting cells, thus activating diabetogenic autoreactive CD4-positive cells (1). Furthermore, Fas mediation was demonstrated for beta -cell injury in NOD mice by Chervonsky et al. (5). We could not analyze the expression of Fas antigen and Fas ligand in the pancreas because antibodies against them are not commercially available for rats. The mechanism of acinar cell apoptosis induced by T cells in the present model remains to be elucidated.

The immunosuppressant tacrolimus, isolated from the fermentation of a strain of Streptomyces tsukubaensis (18, 19), has been demonstrated to attenuate the development of several kinds of autoimmune disease such as diabetes (4, 27), myocarditis (10), and glomerulonephritis (25) in experimental animals. This agent is already applied in clinics for the treatment of inflammatory bowel diseases (6). However, tacrolimus has not been tried in the treatment of the animal model of chronic pancreatitis. The high dose used here, 0.1 mg · kg-1 · day-1, is equivalent to that employed for immunosuppressive therapy in humans (22) and gave satisfactory results in the present model of chronic pancreatitis as demonstrated by marked improvement in histological scores. In addition, treatment with tacrolimus not only prevented infiltration of macrophages and neutrophils but also prevented infiltration of T cells, as demonstrated by pancreatic MPO activity and immunohistochemistry.

To elucidate another possible mechanism by which tacrolimus prevented acinar cell apoptosis, we focused our attention on the plasma corticosterone levels, since a decrease in endogenous corticosterone proved to be responsible for the induction of acinar cell apoptosis in the present model (11). We confirmed that the plasma corticosterone levels were not altered by tacrolimus treatment, suggesting that prevention of acinar cell apoptosis by tacrolimus may not be related to endogenous corticosterone levels. We further investigated the effect of tacrolimus on the T cell subsets in the thymus and peripheral blood, since it was reported that tacrolimus (1 mg/kg) alters the thymocyte populations in Lewis rats (36). However, we found that there were no differences in the T cell subsets between placebo and high dose of tacrolimus groups. This discrepancy may be explained by the differences of dosage and animals. On the other hand, tacrolimus was reported to suppress activations of nuclear factor-kappa B and intracellular adhesion molecule-1, resulting in the reduced accumulation of leukocytes in ischemia-reperfusion injury of the rat heart (32). One might speculate that tacrolimus exerted protection by the same mechanism. However, we do not believe that this is the case for this model since CD8-positive cells frequently invaded the acini and closely adhered to the apoptotic acinar cells, suggesting that CD8-positive cells play a key role in the induction of acinar cell apoptosis. We speculate that prevention of T cell infiltration by tacrolimus resulted in reduced acinar cell apoptosis, leading to disappearance of monocytes/macrophages or neutrophils.

The present study demonstrated the efficacy of tacrolimus for suppression of chronic pancreatitis in male WBN/Kob rats and also raised the possibility of clinical use for chronic pancreatitis. Caution is necessary, however, in this context, because a combination of an innocuous dose of caerulein and tacrolimus was reported to induce pancreatic injury (12). The model of spontaneously occurring chronic pancreatitis employed here, at least in part, reflects autoimmune chronic pancreatitis in humans and thus is useful for exploration of the pathogenesis and effective maneuvers for this disease.


    FOOTNOTES

Address for reprint requests and other correspondence: T. Yamada, First Dept. of Internal Medicine, Nagoya City Univ. Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan (E-mail: yamtmaki{at}med.nagoya-cu.ac.jp).

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 15 December 2000; accepted in final form 10 September 2001.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(6):G1397-G1404
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