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Experimental Biology and Medicine 232:942-949 (2007)
© 2007 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Taurocholic Acid Feeding Prevents Tumor Necrosis Factor-{alpha}–Induced Damage of Cholangiocytes by a PI3K–Mediated Pathway

Yoshiyuki Ueno*, Heather Francis{dagger}, Shannon Glaser{dagger},{ddagger}, Sharon DeMorrow{dagger}, Julie Venter{ddagger}, Antonio Benedetti§, Giammarco Fava§, Marco Marzioni§ and Gianfranco Alpini{ddagger},||,¶,1

* Department of Medicine, Tohoku University School of Medicine, Aobaku, Sendai 980-8574, Japan; {dagger} Division of Research and Education and {ddagger} Department of Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center College of Medicine, Temple, Texas 76504; § Department of Gastroenterology Universita’ Politecnica delle Marche, 60121 Ancona, Italy; and || Central Texas Veterans Health Care System and Systems Biology and Translational Medicine, Scott & White Hospital and The Texas A&M University System Health Science Center College of Medicine, Temple, Texas 76504

1 To whom requests for reprints should be addressed at Central Texas Veterans Health Care System, The Texas A&M University System Health Science Center College of Medicine, Medical Research Building, 702 S.W. H. K. Dodgen Loop, Temple, TX 76504. E-mail: galpini{at}tamu.edu or galpini{at}medicine.tamhsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cholangiopathies, such as primary biliary cirrhosis and primary sclerosis cholangitis, are characterized at the end stage by ductopenia due to increased cholangiocyte apoptosis and decreased cholangiocyte proliferation. Although cholangiocyte proliferation is associated with an increased number of intra-hepatic bile ducts and secretin-stimulated ductal secretion, ductopenia is coupled with decreased ductal mass and secretin-induced ductal secretory activity. We have shown that a single injection of actinomycin D + tumor necrosis factor-{alpha} (TNF-{alpha} ) to bile duct–ligated (BDL) rats induces cholangiocyte injury, which is characterized by loss of cholangiocyte proliferation, and secretory activity and by an increase in cholangiocyte apoptosis. We also have shown that taurocholic acid both in vivo and in vitro stimulates cholangiocyte proliferation. We hypothesize that taurocholic acid feeding protects cholangiocytes against TNF-{alpha} –induced apoptosis through a phosphatidylinositol-3-kinase (PI3K)–dependent pathway. Immediately after BDL, rats were fed taurocholic acid or control diet in the absence/presence of daily injections of wortmannin for 1 week. Seven days later, control-fed or taurocholic acid–fed rats were treated with a single intraperitoneal injection of actinomycin D + TNF-{alpha} . Twenty-four hours later we evaluated: (i) cholangiocyte apoptosis and proliferation in liver sections and (ii) basal and secretin-stimulated bile and bicarbonate secretion in bile fistula rats. Taurocholic acid feeding prevented TNF-{alpha} –induced increases in cholangiocyte apoptosis and decreases in growth and secretin-stimulated bile and bicarbonate secretion, changes that were blocked by PI3K inhibition. The PI3K survival pathway is important in bile acid protection against immune-mediated cholangiocyte injury in cholestatic liver diseases.

Key Words: apoptosis • bile flow • intrahepatic biliary epithelium • proliferation • secretin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bile is first secreted at the bile canaliculus of hepatocytes (1), and before it reaches the small intestine it is modified by cholangiocytes through a series of reabsorptive and secretory events that are modulated by several factors, including gastrointestinal hormones (e.g., secretin, somatostatin, and gastrin), neurotransmitters, and bile salts (2–8). Secretin is of particular importance, because its effects on bile secretion (4, 7, 9) and the expression of its receptor (present only in cholangiocytes in the rat liver; Ref. 10) is altered based on changes in cholangiocyte proliferation and bile duct mass observed in cholestatic liver diseases (3, 4, 10–12). For instance, in pathologic conditions associated with enhanced cholangiocyte proliferation (e.g., following bile duct ligation [BDL], partial hepatectomy or chronic bile acid feeding; Refs. 3, 4, 11) there is enhanced secretin receptor gene expression and increased secretin-stimulated ductal secretion (3, 4, 10, 11, 13). Conversely, reduced cholangiocyte growth (associated with bile duct damage, for example, after acute carbon tetrachloride [CCl4] administration) is coupled with reduced secretin receptor expression and decreased secretin-stimulated, bicarbonate-rich choleresis (12). The stimulatory effect of secretin on ductal secretion is mediated by its interaction with basolateral receptors and an increase in intracellular cAMP levels (13, 14). Increased cAMP levels (13, 14) leads to opening of the Cl– channel, cystic fibrosis transmembrane regulator (15), and activation of the Cl– /HCO3– exchanger (16), with subsequent secretion of bicarbonate into bile (4).

Impaired bile secretion (during cholestasis) is due to functional damage of either hepatocytes or cholangiocytes (9, 17). Chronic cholestasis is a characteristic of several chronic liver diseases, including biliary strictures (18), primary sclerosing cholangitis (PSC; Ref. 19), and cholangiocarcinoma (20). Studies have demonstrated the role of impaired canalicular membrane transport function as well as the effects of impaired bile flow and bile salt accumulation on hepatocyte damage (21–24). Although there is growing information on the role of cholangiocytes in the development of cholestasis (12, 25, 26), the mechanisms of cholangiocyte injury during chronic cholestasis remain unclear.

Tumor necrosis factor-{alpha} (TNF-{alpha} ) regulates epithelial cell injury as well as immune-mediated cholangiocyte damage (27). Immune-mediated cholangiocyte injury has been suggested to play a role in the pathogenesis of cholangiopathies, including primary biliary cirrhosis (PBC) and PSC (9, 28). Furthermore, systemic levels of TNF-{alpha} are enhanced following biliary obstruction in mice (29). In vitro studies have shown that TNF-{alpha} , in combination with interleukin-1 (IL-1), IL-6, and interferon-{gamma}, inhibits cAMP-dependent ductal secretion (30). Although we have shown (26) that TNF-{alpha} , when administered in combination with actinomycin D, induces cholangiocyte damage and loss of ductal secretion in BDL rats, information on the role of TNF-{alpha} and death receptors in the modulation of cholangiocyte function during chronic cholestasis is limited.

Bile acids regulate the apoptotic, proliferative, and secretory activities of the intrahepatic biliary epithelium (2, 3, 9, 31–34) by entering cholangiocytes by a specific transporter, the Na+-dependent apical bile acid transporter, ASBT (35). Studies have shown that bile acids (taurocholic acid and ursodeoxycholic acid) protect intrahepatic bile ducts against injury induced by parasympathetic (32, 34) or sympathetic (36) denervation or CCl4 administration (33) by a phosphatidylinositol-3-kinase (PI3K)–dependent pathway (37), a major determinant of cell growth and survival (33, 34). No information exists regarding either (i) the protective effect of taurocholic acid against TNF-{alpha} –induced changes in cholangiocyte apoptosis and proliferation and secretin-stimulated ductal secretion, and (ii) role of the PI3K system in taurocholic acid prevention of TNF-{alpha} –induced changes in cholangiocyte functions. Thus, we posed the following questions: (i) Does taurocholic acid feeding protect the intrahepatic biliary epithelium from the damage induced by the administration of actinomycin + TNF-{alpha} administration to BDL rats (26)? and (ii) Is taurocholic acid prevention of TNF-{alpha} –induced changes in cholangiocyte apoptosis and proliferation blocked by the in vivo administration of the PI3K inhibitor, wortmannin (34)?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.
Reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Rat chow (containing AIN 76, control diet, or 1% taurocholic acid in AIN 76) was prepared by Dyets Inc. (Bethlehem, PA). Porcine secretin was purchased from Peninsula Laboratories (Belmont, CA). The monoclonal mouse antibody against proliferating cellular nuclear antigen (PCNA) was purchased from DAKO (Kyoto, Japan). The mouse anti–cytokeratin-19 (anti–CK-19) antibody was purchased from Amersham (Arlington Heights, IL). Recombinant TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN).

Animal Models.
Male Fisher 344 rats (175–200 g) were purchased from Charles River (Wilmington, MA), maintained in a temperature-controlled environment (20° C–22° C) with a 12:12-hr light:dark cycle, and fed ad libitum standard rat chow. Rats had free access to drinking water. The studies were performed in two models. The first included BDL (for liver block collection) or bile duct–incannulated (BDI, for bile collection) rats that, immediately after BDL (4) or BDI (4), were fed control diet (AIN 76) for 1 week, followed by a single intraperitoneal injection of 0.9% NaCl or actinomycin D (100 µ g/kg body wt) + TNF-{alpha} (50 ng/kg body wt; Ref. 26). Twenty-four hours later, the animals were sacrificed for the selected experiments. The second model included BDL or BDI rats that, immediately after BDL or BDI (4), were fed 1% taurocholic acid (which represents an approximate dose of 275 µ mol/day; Ref. 3) for 1 week in the presence of daily injections of 0.9% NaCl or wortmannin (0.7 mg/kg body wt; Ref. 34) dissolved in dimethyl sulfoxide (DMSO), followed by a single intra-peritoneal injection of actinomycin D (100 µ g/kg body wt) + TNF-{alpha} (50 ng/kg body wt; Ref. 26). Twenty-four hours later, the animals were sacrificed for the selected experiments. BDL and BDI were performed as described (4). The groups of animals used in the study are summarized in Table 1Go. Since we have previously shown that daily injections of DMSO or wortmannin to BDL rats do not affect cholangiocyte survival, proliferation, and functional activity (36, 38), and that taurocholic acid feeding to BDL rats does not alter cholangiocyte apoptosis and increase cholangiocyte growth (32), these groups of animals were not included in the study. Before each experimental procedure, the animals were anesthetized intraperitoneally with pentobarbital sodium (50 mg/kg body wt) according to the regulations of the panel on euthanasia of the American Veterinarian Medical Association.


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  		Table 1. Animal Modelsa
 
Measurement of Cholangiocyte Apoptosis, Proliferation, and Secretion.
We performed studies to evaluate whether: (i) in vivo administration of actinomycin D + TNF-{alpha} to BDL rats induces damage of intrahepatic bile ducts, leading to enhanced apoptosis and loss of proliferative and secretory activity of cholangiocytes; (ii) taurocholic acid feeding prevents actinomycin D + TNF-{alpha} –induced duct damage and loss of cholangiocyte proliferation and secretion (26); and (iii) consistent with the concept that PI3K plays a role in taurocholic acid prevention of actinomycin D + TNF-{alpha} duct damage, chronic daily intraperitoneal injections of wortmannin block taurocholic acid prevention of actinomycin D + TNF-{alpha} –induced changes in cholangiocyte apoptosis, proliferation, and secretion.

Cholangiocyte apoptosis was evaluated by TUNEL analysis in liver sections using a commercially available kit (Wako Chemicals, Tokyo, Japan). Following counterstaining with hematoxylin solution, sections (4 µ m thick; three slides evaluated per group of animals) were examined by light microscopy with an Olympus BX-40 (Melville, NY) microscope equipped with a CCD camera. Approximately 200 cells per slide were counted in a coded fashion in seven nonoverlapping fields.

Cholangiocyte proliferation was measured by quantitative evaluation of the number of PCNA- and CK-19–positive cholangiocytes in liver sections (5 µ m thick; three slides evaluated per group of animals) from the selected group of animals. Immunohistochemistry for PCNA or CK- 19 was performed in paraffin-embedded sections (5 µ m; three slides analyzed for each group) as described (11, 25). Sections were counterstained with hematoxylin and examined with a microscope (U-PMTVC; Olympus Optical Co., Tokyo, Japan). More than 100 cholangiocytes were counted in a random, blinded fashion in three different fields for each group of animals. Data were expressed as number of PCNA-or CK-19–positive cholangiocytes per 100 cholangiocytes.

Following anesthesia with sodium pentobarbital, the selected rats were surgically prepared for bile collection as described (4). When steady-state bile flow was reached (60–70 mins from the infusion of Krebs Ringer Henseleit [KRH]), the selected animal was infused with secretin (100 nM) for 30 mins and a final infusion of KRH for 30 mins. Bile was collected every 10 mins in preweighed tubes and immediately stored at –70° C before determination of bicarbonate concentration. Bicarbonate concentration (measured as total CO2) in the selected bile samples was determined by an ABL 520 Blood Gas System (Radiometer Medical A/S, Copenhagen, Denmark).

Statistical Analysis.
All data are expressed as mean ± SEM. Differences between groups were analyzed by the Student’s unpaired t test when two groups were analyzed and analysis of variance (ANOVA) when more than two groups were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Measurement of Cholangiocyte Apoptosis, Proliferation, and Secretion.
In agreement with previous studies (26), a single injection of actinomycin D +TNF-{alpha} to 1-week BDL rats significantly increased the number of apoptotic cholangiocytes compared with liver sections from BDL rats treated with a single injection of NaCl (Fig. 1Go). Taurocholic acid feeding partially prevented the actino-mycin D + TNF-{alpha} –induced increase in cholangiocyte apoptosis (Fig. 1Go). Consistent with the concept that the PI3K system plays a role in taurocholic acid protective effects on bile duct injury, wortmannin intraperitoneal injections blocked taurocholic acid prevention of actino-mycin D + TNF-{alpha} –induced increase in cholangiocyte apoptosis (Fig. 1Go). A single injection of actinomycin D + TNF-{alpha} decreased the number of PCNA- and CK-19–positive cholangiocytes compared with liver sections from BDL rats treated with a single injection of NaCl (Figs. 2Go and 3Go). Taurocholic acid prevention of actinomycin D + TNF-{alpha} changes in cholangiocyte proliferation was blocked by wortmannin (Figs. 2Go and 3Go).


Figure 1
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  		Figure 1. Measurement of cholangiocyte apoptosis by TUNEL analysis in liver sections from the selected group of animals. A single injection of actinomycin D + TNF-{alpha} to BDL rats significantly increased the number of apoptotic cholangiocytes compared with liver sections from BDL rats treated with a single injection of NaCl. Taurocholic acid feeding prevented actinomycin D + TNF-{alpha} –induced increase in cholangiocyte apoptosis. Wortmannin blocked taurocholic acid prevention of actinomycin D + TNF-{alpha} –induced increase in cholangiocyte apoptosis. Data are mean ± SEM of seven cumulative values from 10 randomly selected portal areas. *P < 0.05 versus cholangiocyte apoptosis from BDL rats treated with a single injection of NaCl. #P < 0.05 versus cholangiocyte apoptosis from: (i) 1-week BDL rats treated with a single injection of actinomycin D +TNF-{alpha} and (ii) rats that immediately after BDL were fed taurocholic acid for 1 week and simultaneously treated by intraperitoneal injections of wortmannin before being treated with a single injection of actino-mycin D + TNF-{alpha} .

 

Figure 2
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  		Figure 2. Measurement of PCNA-positive cholangiocytes in liver sections from the selected group of animals. A single injection of actinomycin D + TNF-{alpha} decreased the number of PCNA-positive cholangiocytes compared with liver sections from BDL rats treated with a single injection of NaCl. Taurocholic acid prevention of actinomycin D + TNF-{alpha} decrease in cholangiocyte proliferation was blocked by wortmannin. *P < 0.05 versus cholangiocyte apoptosis from BDL rats treated with a single injection of NaCl. Data are mean ± SEM of seven cumulative values from 10 randomly selected portal areas. #P < 0.05 versus cholangiocyte apoptosis from: (i) BDL rats treated with a single injection of actinomycin D + TNF-{alpha} and (ii) rats that immediately after BDL were fed taurocholic acid for 1 week and simultaneously treated by intraperitoneal injections of wortmannin before being treated with a single injection of actinomycin D + TNF-{alpha} . Bar = 100 µ m.

 

Figure 3
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  		Figure 3. Measurement of CK-19–positive cholangiocytes in liver sections from the selected group of animals. A single injection of actinomycin D +TNF-{alpha} decreased the number of CK-19–positive cholangiocytes compared with liver sections from BDL rats treated with a single injection of NaCl. Taurocholic acid prevention of actinomycin D + TNF-{alpha} decrease in the number of CK-19–positive cholangiocytes was blocked by wortmannin. Data are mean ± SEM of seven cumulative values from 10 randomly selected portal areas. *P < 0.05 versus cholangiocyte apoptosis from (i) BDL rats treated with a single injection of NaCl; and (ii) rats that immediately after BDL were fed taurocholic acid for 1 week and simultaneously treated by intraperitoneal injections of wortmannin before being treated with a single injection of actinomycin D +TNF-{alpha} . Bar = 100 µ m.

 
Basal and secretin-stimulated bile flow, bicarbonate concentration, and secretion of BDI rats treated with a single injection of NaCl were similar to those of previous studies (Table 2Go; Refs. 4, 26). In agreement with our previous findings (26), in BDI rats treated with a single injection of actinomycin D + TNF-{alpha} , secretin did not increase bile flow and bicarbonate concentration and secretion compared with BDI rats treated with a single injection of NaCl (Table 2Go). Taurocholic acid feeding to BDI rats (treated with a single injection of actinomycin D + TNF-{alpha} ) prevented TNF-{alpha} inhibition of secretin-stimulated bicarbonate-rich choleresis, which was similar to that of BDI rats treated with NaCl (Table 2Go). Consistent with the concept that the PI3K system regulates bile acid effects on cholangiocyte functions, chronic administration of wortmannin to taurocholic acid–fed BDI rats (treated with a single injection of actinomycin D +TNF-{alpha} ) blocked the protective effect of taurocholic acid on secretin-stimulated, bicarbonate-rich choleresis (Table 2Go). In this group of animals, secretin did not increase bile flow and bicarbonate concentration and secretion (Table 2Go).


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  		Table 2. Measurement of Basal and Secretin-Stimulated Bile Flow and Bicarbonate Concentration and Secretiona
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings of this study relate to the protective role of taurocholic acid feeding versus TNF-{alpha} –induced changes in cholangiocyte apoptosis and proliferation and secretin-stimulated ductal secretion. We have shown that taurocholic acid feeding prevented TNF-{alpha} –induced increases in cholangiocyte apoptosis and decreases in biliary growth and secretin-stimulated ductal secretion, changes that were blocked by the simultaneous administration of the PI3K inhibitor, wortmannin (34).

Although cholangiopathies have different etiologies, they are characterized by chronic damage of different portions of the biliary tree, with consequent dysregulation of the balance between survival and death of cholangiocytes (9, 28, 39), a pathologic condition leading to vanishing of bile ducts and liver failure (9, 28, 39). This sequence of pathologic events seems to be paced by the progressive loss of bile ducts to adequately provide a compensatory proliferative response to chronic liver injury (28). Unfortunately, the knowledge of the factors modulating cholangiocyte proliferative response to liver injury is limited. Our study demonstrates that the balance between apoptosis and proliferation of cholangiocytes and their functional activity in the course of cholestasis (following extrahepatic bile duct obstruction) are likely modulated by the PI3K system (sensitivity to wortmannin) by the cooperative action of bile acids and TNF-{alpha} .

Studies suggest that TNF-{alpha} (a key mediator in inducing and processing inflammatory events; Refs. 40, 41) may play a role in the development of immune-mediated liver diseases (27). For example, during intrahepatic cholestasis there is enhanced formation of basal and endotoxin-induced TNF-{alpha} , which has been linked with the severity of liver damage (41). In experimental models of cholestasis (41) as well as in patients with PBC (42), there is increased expression of TNF-{alpha} and related receptors in cholangiocytes (41, 42). Moreover, TNF-{alpha} (when administered in combination with actinomycin D) induces cholangiocyte damage, decreased cholangiocyte proliferation, and loss of ductal secretion in BDL rats (26). Although oral bile acid replacement inhibits endotoxin-induced TNF-{alpha} production in cholestatic rats (41), no information exists regarding the coordinated role of bile acids and TNF-{alpha} in the regulation of cholangiocyte functions.

We first evaluated the effect of taurocholic acid feeding on TNF-{alpha} –induced changes in cholangiocyte functions and showed that taurocholic acid feeding prevents both the increase in cholangiocyte apoptosis and the decrease in cholangiocyte growth and secretin-stimulated, bicarbonate-rich choleresis induced by TNF-{alpha} . Our data support the concept that bile acids modulate the balance between cholangiocyte damage and proliferation. In support of this notion, although some bile acids (e.g., chenodeoxycholic and deoxycholic acids) are cytotoxic (27, 43, 44), other bile salts (e.g., taurochenodeoxycholic, ursodeoxycholic, tauroursodeoxycholic, and taurocholic acids) are cytoprotective in a number of different cells, including cholangiocytes (27, 32–34, 43, 45–48). Taurocholic acid, the most predominant "tauro-conjugated" bile acid in human bile (49), plays a key role in the regulation of the balance between cholangiocyte apoptosis and growth (3, 32, 33, 50). In support of this concept, prolonged bile depletion reduced the endogenous bile acid pool, cholangiocyte growth, and secretin-stimulated ductal secretion of BDL rats (50). In addition, taurocholic acid both in vivo and in vitro protects BDL cholangiocytes from a CCl4-induced increase in apoptosis and loss of proliferative and secretory activity (33).

We next performed experiments to demonstrate that the protective effects of taurocholic acid on TNF-{alpha} –induced changes in cholangiocyte apoptosis, proliferation, and secretin-stimulated ductal secretion occur through a PI3K-dependent pathway (sensitivity to wortmannin) that plays a role in the modulation of cell growth and survival (33, 34). Consistent with this concept, taurocholic acid prevention of TNF-{alpha} effects on cholangiocyte apoptosis, proliferation, and secretin-stimulated ductal secretion were ablated by wortmannin. Our studies are consistent with the concept that PI3K plays an important role in bile acid modulation of cholangiocyte functions. The regulatory protein PI3K is involved in many different signaling pathways and regulates key functions of a number of cells, including cholangiocytes (32–34, 50–52). PI3K is considered a key intracellular factor that is responsible for the regulation of antiapoptotic signals and modulation of the balance between cell loss/growth (32–34, 53, 54). Overexpression of PI3K in cells is accompanied by a marked antiapoptotic effect and a significant increase in cell survival following radiation (19, 24). In support of our findings, the hydrophobic bile salt, taurochenodeoxycholate, activates PI3K-dependent survival mechanisms in hepatocytes, an effect preventing the cytotoxicity of this bile acid (43). In addition, cytoprotection against bile acid–induced apoptosis in rat hepatocytes is dependent on the PI3K pathway (55, 56).

We have shown recently that both ursodeoxycholic and taurocholic acids exert similar prosurvival effects on cholangiocytes (32–34). Specifically, in models where cholestasis and induction of cholangiocyte death are simultaneously recreated, the administration of ursodeoxycholic and taurocholic acids prevents cholangiocyte apoptosis and maintains bile duct mass and functional activity to a similar extent. Moreover, we treated tauroursodeoxycholic acid (TUDCA)–fed BDL rats with a single intraperitoneal injection of actinomycin D +TNF-{alpha} , and we observed1 that, similar to taurocholic acid, the treatment with TUDCA resulted in the prevention of the reduction of cholangiocyte growth and of the increase of cholangiocyte apoptosis.

In summary, our study demonstrates that taurocholic acid feeding prevents (through a PI3K-dependent pathway) cholangiocyte loss and the lack of proliferative and functional response to extrahepatic bile duct obstruction due to TNF-{alpha} . The present studies allow a better understanding of the mechanisms by which bile ducts are damaged and how certain bile acids protect against immune-mediated cholangiocyte injury in cholestatic liver diseases, including PBC and PSC (9, 28). Failure of bile acids to activate specific cytoprotective mechanisms in cholangiocytes may promote apoptosis and loss of cholangiocytes in these diseases.


   Acknowledgments
 
We would like to thank Meg Chrisler and Glen Cryer, Division of Communication, Scott & White, for their assistance in editing the manuscript.


   Footnotes
 
This work was supported by a Veterans’ Affairs Merit Award, a Veterans’ Affairs Research Scholar Award, the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Scott & White, by a MIUR grant 2003060137_004 to the Department of Gastroenterology, by a grant from Health and Labor Sciences Research Grants for the Research on Measures for Intractable Diseases (from the Ministry of Health, Labor, and Welfare of Japan) and by Grant-in Aid for Scientific Research C (16590573) from Japan Society for the Promotion of Science to Y.U.; and by MIUR grant 2005067975_004 to M.M.

1 Alpini, unpublished data, 2007 Back

Received for publication January 22, 2007. Accepted for publication March 19, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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