- Email: [email protected]
Reduction of rat liver P450 enzyme function and glycogen content after induction of TNB-colitis
Al112 AGA ABSTRACTS • G4548
GASTROENTEROLOGY Vol. 114, No. 4 G4550
INCREASE OF BONE MINERAL DENSITY WITH SODIUM FLUORIDE IN PATIENTS WITH CROHN'S DISEASE. C.v. Timitz, J. Klaus, J. Briickel, A. Rieber, B.O. B~Shm, G. Adler, M. Reinshagen. Department of Medicine I, University of Ulm, Germany.
SULFASALAZINE IS A POTENT AND SPECIFIC INHIBITOR OF NUCLEAR FACTOR KAPPA B ACTIVATION. C. Wahl, R.M. Schmid, G. Adler, S. Livtav*. Departments of Internal Medicine I and Pediatrics*, University Ulm, 89070 Ulm, Germany.
Introduction: Crohn's disease is frequently accompanied by osteopenia,
Introduction: Transcription factors of the NF-KB/Rel family are critical for the inducible expression of multiple genes involved in inflammatory responses. Sulfasalazine, and its salicylate moiety, 5-aminosalicylic acid (5-ASA) are among the most effective agents for the treatment and prevention of inflammatory bowel disease. In addition, sulfasalazine has become established for the treatment of rheumatoid arthritis. However, the mode of action of these drugs remains unclear. Methods: SW620 cell were activated with TNF and LPS. NF-~:B/Rel binding activity was analyzed in electrophoretic mobility shift and supershift assays. NF-~:B dependent transcriptional activity was quantitated in transient transfection assays, mRNA levels of NF-~:B subunits were detected using Northern analysis. Kinetics of IKBc~degradation and nuclear translocation of NF-~:B/Rel were detected using Western blot analysis and confocal laser microscopy. Phosphorylation of IKBc¢ was detected in the presence of proteasome inhibitors. Results: Treatment of SW620 colon cells with sulfasalazine inhibited TNF~ or LPS induced NF-KB activation in a dose dependent fashion. NF-~=B dependent transcription was inhibited by sulfasalazine at micro- to millimolar concentrations. In contrast, 5-ASA or sulfapyridine did not block NF-~B activation at all doses tested. TNFc~ induced nuclear translocation of NF-~:B/Rel was prevented by sulfasalazine through inhibition of I~:B~ degradation. When blocking proteasome mediated degradation of IKBc~, we could demonstrate that sulfasalazine interfered with IKBcc phosphorylation, suggesting a direct effect on an IKBc~kinase or on another upstream signal. The inhibition of NF~:B activation seems to be specific since other DNA binding activities such as AP1 were not affected. Conclusion: These results demonstrate that sulfasalazine is a potent and specific inhibitor of NF-KB activation and thus may explain some of the known biological properties of sulfasalazine.
which can lead to osteoporosis and vertebral fractures. The optimal strategy to prevent bone loss in these patients is not established. The effect of sodium fluoride treatment on bone mineral density in Crohn's disease patients has not been studied before. Methods: We carried out a one-year prospective clinical trial in 33 patients with chronic active Crohn's disease who were randomly assigned to receive either calcium (500 mg b.i.d.) and 1000 IU vitamin D3, or additionally retarded release sodium fluoride (25 mg t.i.d.). The diagnosis of Crohn's disease was established for at least two years and all patients had received systemic high-does steroid therapy during the previous year. 11 of 15 patients who received calcium/vitamin D and 15 of 18 patients who received additionally sodium fluoride completed the study. Primary endpoint of the study was the increase of bone mineral density, measured by dual energy X-ray absorptiometry (DXA) after one year of treatment. Bone alkaline specific phosphatase (BAP) and osteocalcin were used as markers for bone turnover. Results: In the calcium/vitamin D treated group, bone density was not significantly changed after one year of treatment whereas in the calcium / vitamin D / fluoride group bone density of the lumbar spine was significantly increased from 84,88 % +/- 3,38 (Z-score, mean -+ SEM) before therapy to 93,93% +/- 2,73 after therapy. Increase of bone density was positively correlated to the osteoblastic markers BAP (r=0,65) and osteocalcin (r=0,72). Conclusion: Sodium fluoride in combination with vitamin D and calcium is an effective, well tolerated and inexpensive treatment to increase lumbar bone density in patients with chronic active Crohn's disease and osteopenia. G4549
SYSTEMIC AVAILABILITY AND PHARMACODYNAMIC EFFECTS AFTER MULTIPLE RECTAL ADMINISTRATION OF BUDESONIDE FOAM IN HEALTHY VOLUNTEERS. M. Wagner 1, H.W.MNlmann 1, A.Tromml, P. Froehlich 2, A.C.M~511mann2, S.Homrighausen 1, J.Barth 1, M.Krieg l, G.Hochhans 2. 1University of Bochum, Germany, 2University of Florida, Gainesville, FL. In the therapy of active distal Crohn's disease and ulcerative colitis the therapeutic success of budesonide is based on its high local anti-inflammatory potency and low systemic bioavailability. For local delivery, enemas and foams for the rectal application of budesonide have been developed. It has been observed that enemas are sometimes not convenient for the patients because of their large volume, while foams are easier to retain. Aim of the study: To describe the pharmacokinetics and cumulative pharmacodynamic effects after multiple rectal application of budesonide foam in different doses and volumes. Methods: 16 male and 16 female healthy volunteers (average age: 29 -+4 years) received budesonide foam (Budenofalk) 3 times daily in 8 hour intervals in 3 different doses (3xl mg in 50ml, 3x2 mg in 50ml, 3x3 mg in 50ml, 3x2 mg in 100ml, 3x3 mg in 150ml). Budesonide plasma levels were measured by RIA and HPLC/RIA. Lymphocyte and granulocyte counts were monitored over 24 hours. Results: Budesonide concentration time profiles were analyzed by noncompartmental analysis. After application of foam, Budesonide was rapidly absorbed with a tmax of 1,5 hours. For foam preparations delivered with the same volume (50 ml), pharmacokinetics were linear as indicated by the lack of statistical differences between dose corrected Cmax and AUC-values. The mean residence time was determined to be 4 hours. When budesonide was delivered in increasing volumes (50, 100, 150 ml) dose adjusted AUC estimates decreased, indicating a reduced bioavailability. The effects on lymphocytes and granulocytes after administration of budesonide foam showed no dosedependency and were significantly smaller than those after administration of the same doses of methylprednisolone. Conclusion: A rapid absorption of budesonide was observed after rectal administration of budesonide foam in different doses. The resulting plasma levels were similar to those observed after application of identical doses of tablets. The volume of the budesonide preparation applied affected plasma level profiles. After multiple dosing no signs of accumulation were observed. The systemic bioavailability was low which explains the rather insignificant degree of side effects. These results further support the strategy of using budesonide as a topically highly active, high clearance drug for the side specific therapy of Crohn's disease. Therefore, the budesonide foam preparation represents an important advantage in the effective treatment of inflammatory bowel diseases in patients who require steroid therapy. Supported by Dr. Falk Pharma GmbH, Freiburg, Germany
G4551
CLOSTRIDIUM DIFFICILE TOXIN-INDUCED IL-8 RELEASE BY MONOCYTES FOLLOWS TYROSINE KINASE, NF-~:B AND NF-IL6 ACTIVATION. M Warny, AC Keates, S Keates, C Pothoulakis, CP Kelly. Department of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Clostridium difficile toxin-induced colitis is characterized by neutrophil activation and infiltration of the colonic mucosa. We have reported that C. difficile toxins A and B activate the transcription factor NF-~:B and potently upregulate IL-8 mRNA and protein production in human monocytes. The aims of this study were: 1) to determine whether tyrusine kinase activation is required for toxin-induced IL-8 up-regulation and, 2) to determine the transcription factors which regulate IL-8 gene expression in toxin-treated THP-1 monocytic cells. Methods: THP-1 cells were preincubated for 2 hours with the tyrosine kinase inhibitors herbimycin A (10 ~M) or genistein (100 ~M) and stimulated for 4 hours with LPS-free toxins A or B (10 to 100 nM). IL-8 production was measured by ELISA. LPS (100 ng/ml) and IL-113 (10 ng/ml) were used as controls. Tyrnsine-phosphorylated proteins were detected by immunoblotting of whole cell lysates. Regulation of IL-8 gene expression was examined by transfecting THP-1 cells with luciferase reporter gene constructs carrying either the "full length" 5' promoter region of the human IL-8 gene or constructs containing targeted deletions of its NF-~:B, NF-IL6 or AP-1 binding sites. Results: Both genistein and herbimycin A blocked toxin-induced IL-8 upregulation (by 70% and 100% respectively) but had marginal effects on LPS-stimulated IL-8 production (0% and 20% inhibition). Toxins A and B induced tyrosine phosphorylation of a 120 kDa protein. Phosphorylation of a 70 kDa protein was increased by toxin B alone. IL-8 reporter gene activity in THP-1 cells was increased 8-fold 4 hours after toxin A stimulation. Deletion of the NF-KB or NF-IL6 binding sites reduced toxin A-induced luciferase activity by 75% and 45% respectively, while AP-1 site deletion had little effect. Conclusion: IL-8 production by human monocytes exposed to C. difl~cile toxin A or toxin B requires activation of tyrosine kinases. These kinases may be responsible for the observed activation of the transcription factor NF-~:B. Activated NF-~:B then binds the KB site of the IL-8 gene promotor and upregulates IL-8 gene expression in co-operation with activated NF-IL6. Back~,round and Aims:
G4552
REDUCTION OF RAT LIVER P450 ENZYME FUNCTION AND GLYCOGEN CONTENT AFTER INDUCTION OF TNB-COLITIS. H. Weidenbach S. Leiz, M. Bachem #, N. Dikoponlos, M. Reinshagen, R.M Schmid, G. Adler. Department of Internal Medicine I and #Department of Clinical Chemistry, University of Ulm, Ulm, Germany. Introduction: Inflammatory bowel diseases (IBD) are often associated with liver disease of unknown cause. Mediators released by inflammatory cells in
Immunology,Microbiology, and InflammatoryDisorders Al113
April 1998 the mucosa may contribute to the liver disorder. The aim of the study was to study the global function of the liver P450 enzyme system and the acute phase reaction during the time course of the TNB-colitis in Wistar rats. Methods: Colitis was induced by single intrarectal application of the hapten, trinitrobenzene sulphonic acid (TNB) in 50% ethanol. Endotoxin and 6-ketoprostaglandin-F1, ~ levels in portal blood were analyzed. Liver glycogen content was determined after hydrolyzing with amyloglucosidase. Myeloperoxidase activity was used as a marker for neutrophil infiltration in liver and colon. P450 enzyme function was studied by determination lidocaine metabolism to monoethylglycinexylidide (MEG-X). By determination of ct2maeroglobulin the liver acute phase reaction was studied. In addition, the binding activity of the transcription factor NF-t:B was analyzed by electrophoretic mobility shifts. Results: The myeloperoxidase measurement and the histological data reflect a marked neutrophil infiltration in colon mucosa. In contrast myeloperoxidase was not detectable in the liver and histology was negative for leucocytic infiltration over the whole observation period. 1 day after induction of colitis, lidocaine metabolism was significantly decreased (cont. 1697-+ 100 to 1086 -+ 228 ng/ml) and reached control level after 14 days. Simultaneously, glycogen content decreased (199.9 -+ 25 to 15.6 -+6.1 pmol/u-glucosyl/g) and reached control level at day 14 after TNB-colitis induction. Endotoxin (portal vein), NF-t~B binding activity (liver) and alpha 2 makroglobulin (systemic) were not detectable at any time point. However, the levels of 6-ketoprostaglandin-Fla in portal vein blood increased significant (cont. 416 -+74 pg/ml to 3071 - 528 pg/ml [day 1] and 6554 -+ 1323 pg/ml [day 2]). Conclusions: Experimental TNB-colitis is associated with a reduction in liver P450 function and a decrease in glycogen content. The present data favour a role of prostaglandins synthesized inflamed mucosa contributing to these observations. In contrast the acute phase reaction appears not to be involved in the reduction of P450 function and liver glycogen content during the time course of the TNB-colitis. • G4553 THE ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF DUODENAL ULCER INDUCED BY CYSTEAMINE IN RATS. ZF Wen, CH Cho, KC Lai, SK Lam. Departments of Medicine and Pharmacology, The University of Hong Kong, Hong Kong, China. Background and Aims: Nitric oxide plays an important role in the inflammatory processes. The purpose of this study is to investigate whether nitric oxide also participates in the pathogenesis of experimental duodenal ulcer. Methods: SD male rats were divided into four groups: 1) normal saline (NS) + NS; 2) NS+Cysteamine-HCL (Cys), 3) NG-nitro-L-arginine methyl ester (L-NAME) 10mg/kg+Cys; 4) L-NAME 20mg/kg+Cys (n=8-12). Duodenal ulcer was induced by two subcutaneous injections of Cys 200 mg/kg. L-NAME was given orally 30 min before each injection of Cys. Rats were killed 24 hr after the last injection of Cys. The amount of nitric oxide produced was measured by electron paramagnetic resonance (EPR) using DETC-Fe as a trapper. Inducible nitric oxide synthase (NOS) expression in the duodenum was studied by LSAB immnunohistoehemical staining. Results: There was no inducible NOS expression in the mucosa of normal duodenum but a strong positive expression at the duodenal ulcer base and margin was noted. Nitric oxide signal detected by EPR in the duodenum was significantly increased after treatment with Cys. A NOS inhibitor, L-NAME could significantly prevent the development of duodenal ulcer induced by Cys. NS+NS NS+Cys L-NAME(10 mg.kg) L_NAME(20mg/kg) Cys Cys 0 9.76 -+2.01 1.31 ± 1.21" 0.83 ± 0.47*
Ulcer area (mm2) Ulcer score 0 1.69 ± 0.18 0.38 ± 0.26* *P < 0.01 compared with NS+Cys group.
0.50 ± 0.16"
Conclusion: This study suggests that endogenous nitric oxide derived from inducible NOS may play an important role in the development of duodenal ulcer induced by Cys. • G4554 MOUSE
STRAINS
RESISTANT
AND
SUSCEPTIBLE
TO
HELICOBACTER HEPATICUS-RELATED HEPATITIS DIFFER IN SYSTEMIC IMMUNE RESPONSE TO EXPERIMENTAL INFECTION. M. T. Wharv and J.G. Fox, Massachusetts Institute of Technology, Cambridge, MA 02139 USA. Infection of the hepatic biliary system with Helicobacter hepaticus in the A/JCr mouse results in chronic active hepatitis and an increased risk for hepatic tumors. The propensity for the A/JCr mouse to develop inflammatory lesions in the liver in comparison to more resistant strains such as C57BL/6 mice, suggests that differences in the immune response to H. hepaticus may be a major determinant in lesion development. This study examined the immune response of A/JCr and C57BI_/6 mice to H. hepaticus during a 6 month experimental infection. The proliferative response of splenic mononuclear ceils and the humoral response (serum IgG and secretory IgA) to H. hepaticus antigens were compared. Spleen cells isolated from infected A/JCr mice developed a significant antigenic response (p<0.01) to
H. hepaticus while the response of cells isolated from infected C57BL/6 mice did not differ from control responses (p=0.3). Infected AJ/Cr mice also developed a higher serum IgG titer sooner than infected C57BL/6 mice (p<0.01). Both strains of mice produced predominantly IgG2a serum antibodies to H. hepaticus which is consistent with a Thl immune response, as reported for humans infected with H. pylori and mice with H. felis. Significant levels of secretory IgA developed early post-infection in both mouse strains and although there was a trend for the C57BL/6 mice to produce more IgA against H. hepaticus, the difference was not statistically significant. These results suggest that the extent of lesion development in response to H. hepaticus may be correlated with the magnitude of the systemic humoral and Thl immune response. The greater immune response of the susceptible A/JCr mouse to H. hepaticus may contribute to lesion development or may be enhanced secondary to increased antigenic stimulation during the host inflammatory response. • G4555 MEDICAL THERAPY, NOT SURGERY, IS THE APPROPRIATE FIRST LINE TREATMENT FOR CROHN'S ENTEROVESICAL FISTULA. SC Wheeler, JF Marion, DH Present. Division of Gastroenterology, Dept. of Medicine, Mount Sinai School of Medicine, New York, NY, 10029. Purpose: Enterovesical (EV) fistula is a major complication of Crohn's disease. Surgery has long been considered the first line of therapy for these patients. To test the validity of this approach we reviewed our experience with EV fistula treated medically. Methods: We reviewed the charts of 31 patients with EV fistulae in one private practice from 1969-1997, each confirmed radiographically and/or surgically. Collected data included standard demographics, presenting symptoms, predisposing factors, (prior therapy, site of disease), disease activity, types of therapy and long term outcome (maintenance of response, and subsequent need for surgery). Results: 25/31 (81%) patients with EV fistula were male and 6 (19%) were female. All were treated using a standard therapeutic regimen including 5-ASA, chronic antibiotics, and 6-mercaptopurine to allow steroid tapering. Clinical response was noted in 18/31 (58%) with complete fistula closure in 13/31 (42%). 12/18 responders (67%) have done well (avoided surgery or maintained their response over a mean follow-up of 8.1 years (0.6-17.4 years). Six patients recurred when therapy was lowered or stopped. Four of six reclosed their fistula after resuming or increasing medication. Steroid use within one year of fistula diagnosis was associated with a poor prognosis for closure of EV fistula. 85% of failures had received prior steroids while only 50% of responders had received prior steroids (p<0.05). There were no deaths, ascending urinary tract infections or significant drug toxicity during 136.8 patient/years of treatment and follow-up. Conclusion: Medical therapy is effective and safe in the treatment of EV fistula(early response 58% and long term response 39%). Surgery should be deferred in the majority of patients until after a trial of intensive medical management with steroid discontinuation. • G4556 ANALYSIS OF INTERLEUKIN 10 GENE EXPRESSION IN AN IN VITRO MODEL OF ENTEROCYTE DIFFERENTIATION USING CaCo-2 CELLS. G.E, Wild, J. Hasan, M.J. Ropeleski, J. McCann. And A.B.R. Thomson. McGill University Inflammatory Bowel Disease Research Program. Montreal, Quebec and the Department of Gastroenterology, University of Alberta, Edmonton. CANADA. There is a growing body of experimental evidence which suggests that intestinal epithelial ceils synthesize an array of cytokines which participate in the mucosal immune response to antigen. In view of the clinical importance of Th2 cell derived cytokine, Interleukin -10, we sought to characterize the expression of this anti-inflammatory cytokine in an enteroeyte model using the well established Caco-2 cell. Methods: Caco-2 cells were cultured according to standard techniques and exposed to INFv (100U/ml) for 24hr. Interleukin-10 protein levels were measured by ELISA and IL-10 protein was localized by immunostaining. The level of II-10 mRNA abundance was determined by slot-blot and quantitative RT-PCR. Interleukin 10 transcripts were localized by in situ hybridization. Results: Exposure to INFy resulted in a 5 fold decrease of immunodetectable IL-10. This down regulation of IL-10 protein expression was confirmed by immunostaining. While I1-10 protein was detectable at all stages of cell culture, protein levels were most abundant in the post-confluent, differentiated state. Northern analysis disclosed a 4 kb transcript. IL-10 mRNA abundance was decreased in the presence of INFv. This decrease was confirmed by RT-PCR and in situ hybridization studies. I1-10 mRNA abundance was greatest in post confluent, differentiated Caco-2 cells. Conclusions: This is the first report of the expression of the antiinflammatory cytokine, Interleukin-10, in a model of the intestinal epithelium. The response of enterocyte IL-10 to INFy is similar to that which is observed in Th2 subsets of CD4 lymphocytes. The results presented here underscore the importance of the intestinal epithelium in mucosal immune function. Supported by Les Fonds de la Recherche en Sante du Quebec and the Crohn's and Colitis Foundation of Canada.
GASTROENTEROLOGY Vol. 114, No. 4 G4550
INCREASE OF BONE MINERAL DENSITY WITH SODIUM FLUORIDE IN PATIENTS WITH CROHN'S DISEASE. C.v. Timitz, J. Klaus, J. Briickel, A. Rieber, B.O. B~Shm, G. Adler, M. Reinshagen. Department of Medicine I, University of Ulm, Germany.
SULFASALAZINE IS A POTENT AND SPECIFIC INHIBITOR OF NUCLEAR FACTOR KAPPA B ACTIVATION. C. Wahl, R.M. Schmid, G. Adler, S. Livtav*. Departments of Internal Medicine I and Pediatrics*, University Ulm, 89070 Ulm, Germany.
Introduction: Crohn's disease is frequently accompanied by osteopenia,
Introduction: Transcription factors of the NF-KB/Rel family are critical for the inducible expression of multiple genes involved in inflammatory responses. Sulfasalazine, and its salicylate moiety, 5-aminosalicylic acid (5-ASA) are among the most effective agents for the treatment and prevention of inflammatory bowel disease. In addition, sulfasalazine has become established for the treatment of rheumatoid arthritis. However, the mode of action of these drugs remains unclear. Methods: SW620 cell were activated with TNF and LPS. NF-~:B/Rel binding activity was analyzed in electrophoretic mobility shift and supershift assays. NF-~:B dependent transcriptional activity was quantitated in transient transfection assays, mRNA levels of NF-~:B subunits were detected using Northern analysis. Kinetics of IKBc~degradation and nuclear translocation of NF-~:B/Rel were detected using Western blot analysis and confocal laser microscopy. Phosphorylation of IKBc¢ was detected in the presence of proteasome inhibitors. Results: Treatment of SW620 colon cells with sulfasalazine inhibited TNF~ or LPS induced NF-KB activation in a dose dependent fashion. NF-~=B dependent transcription was inhibited by sulfasalazine at micro- to millimolar concentrations. In contrast, 5-ASA or sulfapyridine did not block NF-~B activation at all doses tested. TNFc~ induced nuclear translocation of NF-~:B/Rel was prevented by sulfasalazine through inhibition of I~:B~ degradation. When blocking proteasome mediated degradation of IKBc~, we could demonstrate that sulfasalazine interfered with IKBcc phosphorylation, suggesting a direct effect on an IKBc~kinase or on another upstream signal. The inhibition of NF~:B activation seems to be specific since other DNA binding activities such as AP1 were not affected. Conclusion: These results demonstrate that sulfasalazine is a potent and specific inhibitor of NF-KB activation and thus may explain some of the known biological properties of sulfasalazine.
which can lead to osteoporosis and vertebral fractures. The optimal strategy to prevent bone loss in these patients is not established. The effect of sodium fluoride treatment on bone mineral density in Crohn's disease patients has not been studied before. Methods: We carried out a one-year prospective clinical trial in 33 patients with chronic active Crohn's disease who were randomly assigned to receive either calcium (500 mg b.i.d.) and 1000 IU vitamin D3, or additionally retarded release sodium fluoride (25 mg t.i.d.). The diagnosis of Crohn's disease was established for at least two years and all patients had received systemic high-does steroid therapy during the previous year. 11 of 15 patients who received calcium/vitamin D and 15 of 18 patients who received additionally sodium fluoride completed the study. Primary endpoint of the study was the increase of bone mineral density, measured by dual energy X-ray absorptiometry (DXA) after one year of treatment. Bone alkaline specific phosphatase (BAP) and osteocalcin were used as markers for bone turnover. Results: In the calcium/vitamin D treated group, bone density was not significantly changed after one year of treatment whereas in the calcium / vitamin D / fluoride group bone density of the lumbar spine was significantly increased from 84,88 % +/- 3,38 (Z-score, mean -+ SEM) before therapy to 93,93% +/- 2,73 after therapy. Increase of bone density was positively correlated to the osteoblastic markers BAP (r=0,65) and osteocalcin (r=0,72). Conclusion: Sodium fluoride in combination with vitamin D and calcium is an effective, well tolerated and inexpensive treatment to increase lumbar bone density in patients with chronic active Crohn's disease and osteopenia. G4549
SYSTEMIC AVAILABILITY AND PHARMACODYNAMIC EFFECTS AFTER MULTIPLE RECTAL ADMINISTRATION OF BUDESONIDE FOAM IN HEALTHY VOLUNTEERS. M. Wagner 1, H.W.MNlmann 1, A.Tromml, P. Froehlich 2, A.C.M~511mann2, S.Homrighausen 1, J.Barth 1, M.Krieg l, G.Hochhans 2. 1University of Bochum, Germany, 2University of Florida, Gainesville, FL. In the therapy of active distal Crohn's disease and ulcerative colitis the therapeutic success of budesonide is based on its high local anti-inflammatory potency and low systemic bioavailability. For local delivery, enemas and foams for the rectal application of budesonide have been developed. It has been observed that enemas are sometimes not convenient for the patients because of their large volume, while foams are easier to retain. Aim of the study: To describe the pharmacokinetics and cumulative pharmacodynamic effects after multiple rectal application of budesonide foam in different doses and volumes. Methods: 16 male and 16 female healthy volunteers (average age: 29 -+4 years) received budesonide foam (Budenofalk) 3 times daily in 8 hour intervals in 3 different doses (3xl mg in 50ml, 3x2 mg in 50ml, 3x3 mg in 50ml, 3x2 mg in 100ml, 3x3 mg in 150ml). Budesonide plasma levels were measured by RIA and HPLC/RIA. Lymphocyte and granulocyte counts were monitored over 24 hours. Results: Budesonide concentration time profiles were analyzed by noncompartmental analysis. After application of foam, Budesonide was rapidly absorbed with a tmax of 1,5 hours. For foam preparations delivered with the same volume (50 ml), pharmacokinetics were linear as indicated by the lack of statistical differences between dose corrected Cmax and AUC-values. The mean residence time was determined to be 4 hours. When budesonide was delivered in increasing volumes (50, 100, 150 ml) dose adjusted AUC estimates decreased, indicating a reduced bioavailability. The effects on lymphocytes and granulocytes after administration of budesonide foam showed no dosedependency and were significantly smaller than those after administration of the same doses of methylprednisolone. Conclusion: A rapid absorption of budesonide was observed after rectal administration of budesonide foam in different doses. The resulting plasma levels were similar to those observed after application of identical doses of tablets. The volume of the budesonide preparation applied affected plasma level profiles. After multiple dosing no signs of accumulation were observed. The systemic bioavailability was low which explains the rather insignificant degree of side effects. These results further support the strategy of using budesonide as a topically highly active, high clearance drug for the side specific therapy of Crohn's disease. Therefore, the budesonide foam preparation represents an important advantage in the effective treatment of inflammatory bowel diseases in patients who require steroid therapy. Supported by Dr. Falk Pharma GmbH, Freiburg, Germany
G4551
CLOSTRIDIUM DIFFICILE TOXIN-INDUCED IL-8 RELEASE BY MONOCYTES FOLLOWS TYROSINE KINASE, NF-~:B AND NF-IL6 ACTIVATION. M Warny, AC Keates, S Keates, C Pothoulakis, CP Kelly. Department of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Clostridium difficile toxin-induced colitis is characterized by neutrophil activation and infiltration of the colonic mucosa. We have reported that C. difficile toxins A and B activate the transcription factor NF-~:B and potently upregulate IL-8 mRNA and protein production in human monocytes. The aims of this study were: 1) to determine whether tyrusine kinase activation is required for toxin-induced IL-8 up-regulation and, 2) to determine the transcription factors which regulate IL-8 gene expression in toxin-treated THP-1 monocytic cells. Methods: THP-1 cells were preincubated for 2 hours with the tyrosine kinase inhibitors herbimycin A (10 ~M) or genistein (100 ~M) and stimulated for 4 hours with LPS-free toxins A or B (10 to 100 nM). IL-8 production was measured by ELISA. LPS (100 ng/ml) and IL-113 (10 ng/ml) were used as controls. Tyrnsine-phosphorylated proteins were detected by immunoblotting of whole cell lysates. Regulation of IL-8 gene expression was examined by transfecting THP-1 cells with luciferase reporter gene constructs carrying either the "full length" 5' promoter region of the human IL-8 gene or constructs containing targeted deletions of its NF-~:B, NF-IL6 or AP-1 binding sites. Results: Both genistein and herbimycin A blocked toxin-induced IL-8 upregulation (by 70% and 100% respectively) but had marginal effects on LPS-stimulated IL-8 production (0% and 20% inhibition). Toxins A and B induced tyrosine phosphorylation of a 120 kDa protein. Phosphorylation of a 70 kDa protein was increased by toxin B alone. IL-8 reporter gene activity in THP-1 cells was increased 8-fold 4 hours after toxin A stimulation. Deletion of the NF-KB or NF-IL6 binding sites reduced toxin A-induced luciferase activity by 75% and 45% respectively, while AP-1 site deletion had little effect. Conclusion: IL-8 production by human monocytes exposed to C. difl~cile toxin A or toxin B requires activation of tyrosine kinases. These kinases may be responsible for the observed activation of the transcription factor NF-~:B. Activated NF-~:B then binds the KB site of the IL-8 gene promotor and upregulates IL-8 gene expression in co-operation with activated NF-IL6. Back~,round and Aims:
G4552
REDUCTION OF RAT LIVER P450 ENZYME FUNCTION AND GLYCOGEN CONTENT AFTER INDUCTION OF TNB-COLITIS. H. Weidenbach S. Leiz, M. Bachem #, N. Dikoponlos, M. Reinshagen, R.M Schmid, G. Adler. Department of Internal Medicine I and #Department of Clinical Chemistry, University of Ulm, Ulm, Germany. Introduction: Inflammatory bowel diseases (IBD) are often associated with liver disease of unknown cause. Mediators released by inflammatory cells in
Immunology,Microbiology, and InflammatoryDisorders Al113
April 1998 the mucosa may contribute to the liver disorder. The aim of the study was to study the global function of the liver P450 enzyme system and the acute phase reaction during the time course of the TNB-colitis in Wistar rats. Methods: Colitis was induced by single intrarectal application of the hapten, trinitrobenzene sulphonic acid (TNB) in 50% ethanol. Endotoxin and 6-ketoprostaglandin-F1, ~ levels in portal blood were analyzed. Liver glycogen content was determined after hydrolyzing with amyloglucosidase. Myeloperoxidase activity was used as a marker for neutrophil infiltration in liver and colon. P450 enzyme function was studied by determination lidocaine metabolism to monoethylglycinexylidide (MEG-X). By determination of ct2maeroglobulin the liver acute phase reaction was studied. In addition, the binding activity of the transcription factor NF-t:B was analyzed by electrophoretic mobility shifts. Results: The myeloperoxidase measurement and the histological data reflect a marked neutrophil infiltration in colon mucosa. In contrast myeloperoxidase was not detectable in the liver and histology was negative for leucocytic infiltration over the whole observation period. 1 day after induction of colitis, lidocaine metabolism was significantly decreased (cont. 1697-+ 100 to 1086 -+ 228 ng/ml) and reached control level after 14 days. Simultaneously, glycogen content decreased (199.9 -+ 25 to 15.6 -+6.1 pmol/u-glucosyl/g) and reached control level at day 14 after TNB-colitis induction. Endotoxin (portal vein), NF-t~B binding activity (liver) and alpha 2 makroglobulin (systemic) were not detectable at any time point. However, the levels of 6-ketoprostaglandin-Fla in portal vein blood increased significant (cont. 416 -+74 pg/ml to 3071 - 528 pg/ml [day 1] and 6554 -+ 1323 pg/ml [day 2]). Conclusions: Experimental TNB-colitis is associated with a reduction in liver P450 function and a decrease in glycogen content. The present data favour a role of prostaglandins synthesized inflamed mucosa contributing to these observations. In contrast the acute phase reaction appears not to be involved in the reduction of P450 function and liver glycogen content during the time course of the TNB-colitis. • G4553 THE ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF DUODENAL ULCER INDUCED BY CYSTEAMINE IN RATS. ZF Wen, CH Cho, KC Lai, SK Lam. Departments of Medicine and Pharmacology, The University of Hong Kong, Hong Kong, China. Background and Aims: Nitric oxide plays an important role in the inflammatory processes. The purpose of this study is to investigate whether nitric oxide also participates in the pathogenesis of experimental duodenal ulcer. Methods: SD male rats were divided into four groups: 1) normal saline (NS) + NS; 2) NS+Cysteamine-HCL (Cys), 3) NG-nitro-L-arginine methyl ester (L-NAME) 10mg/kg+Cys; 4) L-NAME 20mg/kg+Cys (n=8-12). Duodenal ulcer was induced by two subcutaneous injections of Cys 200 mg/kg. L-NAME was given orally 30 min before each injection of Cys. Rats were killed 24 hr after the last injection of Cys. The amount of nitric oxide produced was measured by electron paramagnetic resonance (EPR) using DETC-Fe as a trapper. Inducible nitric oxide synthase (NOS) expression in the duodenum was studied by LSAB immnunohistoehemical staining. Results: There was no inducible NOS expression in the mucosa of normal duodenum but a strong positive expression at the duodenal ulcer base and margin was noted. Nitric oxide signal detected by EPR in the duodenum was significantly increased after treatment with Cys. A NOS inhibitor, L-NAME could significantly prevent the development of duodenal ulcer induced by Cys. NS+NS NS+Cys L-NAME(10 mg.kg) L_NAME(20mg/kg) Cys Cys 0 9.76 -+2.01 1.31 ± 1.21" 0.83 ± 0.47*
Ulcer area (mm2) Ulcer score 0 1.69 ± 0.18 0.38 ± 0.26* *P < 0.01 compared with NS+Cys group.
0.50 ± 0.16"
Conclusion: This study suggests that endogenous nitric oxide derived from inducible NOS may play an important role in the development of duodenal ulcer induced by Cys. • G4554 MOUSE
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HELICOBACTER HEPATICUS-RELATED HEPATITIS DIFFER IN SYSTEMIC IMMUNE RESPONSE TO EXPERIMENTAL INFECTION. M. T. Wharv and J.G. Fox, Massachusetts Institute of Technology, Cambridge, MA 02139 USA. Infection of the hepatic biliary system with Helicobacter hepaticus in the A/JCr mouse results in chronic active hepatitis and an increased risk for hepatic tumors. The propensity for the A/JCr mouse to develop inflammatory lesions in the liver in comparison to more resistant strains such as C57BL/6 mice, suggests that differences in the immune response to H. hepaticus may be a major determinant in lesion development. This study examined the immune response of A/JCr and C57BI_/6 mice to H. hepaticus during a 6 month experimental infection. The proliferative response of splenic mononuclear ceils and the humoral response (serum IgG and secretory IgA) to H. hepaticus antigens were compared. Spleen cells isolated from infected A/JCr mice developed a significant antigenic response (p<0.01) to
H. hepaticus while the response of cells isolated from infected C57BL/6 mice did not differ from control responses (p=0.3). Infected AJ/Cr mice also developed a higher serum IgG titer sooner than infected C57BL/6 mice (p<0.01). Both strains of mice produced predominantly IgG2a serum antibodies to H. hepaticus which is consistent with a Thl immune response, as reported for humans infected with H. pylori and mice with H. felis. Significant levels of secretory IgA developed early post-infection in both mouse strains and although there was a trend for the C57BL/6 mice to produce more IgA against H. hepaticus, the difference was not statistically significant. These results suggest that the extent of lesion development in response to H. hepaticus may be correlated with the magnitude of the systemic humoral and Thl immune response. The greater immune response of the susceptible A/JCr mouse to H. hepaticus may contribute to lesion development or may be enhanced secondary to increased antigenic stimulation during the host inflammatory response. • G4555 MEDICAL THERAPY, NOT SURGERY, IS THE APPROPRIATE FIRST LINE TREATMENT FOR CROHN'S ENTEROVESICAL FISTULA. SC Wheeler, JF Marion, DH Present. Division of Gastroenterology, Dept. of Medicine, Mount Sinai School of Medicine, New York, NY, 10029. Purpose: Enterovesical (EV) fistula is a major complication of Crohn's disease. Surgery has long been considered the first line of therapy for these patients. To test the validity of this approach we reviewed our experience with EV fistula treated medically. Methods: We reviewed the charts of 31 patients with EV fistulae in one private practice from 1969-1997, each confirmed radiographically and/or surgically. Collected data included standard demographics, presenting symptoms, predisposing factors, (prior therapy, site of disease), disease activity, types of therapy and long term outcome (maintenance of response, and subsequent need for surgery). Results: 25/31 (81%) patients with EV fistula were male and 6 (19%) were female. All were treated using a standard therapeutic regimen including 5-ASA, chronic antibiotics, and 6-mercaptopurine to allow steroid tapering. Clinical response was noted in 18/31 (58%) with complete fistula closure in 13/31 (42%). 12/18 responders (67%) have done well (avoided surgery or maintained their response over a mean follow-up of 8.1 years (0.6-17.4 years). Six patients recurred when therapy was lowered or stopped. Four of six reclosed their fistula after resuming or increasing medication. Steroid use within one year of fistula diagnosis was associated with a poor prognosis for closure of EV fistula. 85% of failures had received prior steroids while only 50% of responders had received prior steroids (p<0.05). There were no deaths, ascending urinary tract infections or significant drug toxicity during 136.8 patient/years of treatment and follow-up. Conclusion: Medical therapy is effective and safe in the treatment of EV fistula(early response 58% and long term response 39%). Surgery should be deferred in the majority of patients until after a trial of intensive medical management with steroid discontinuation. • G4556 ANALYSIS OF INTERLEUKIN 10 GENE EXPRESSION IN AN IN VITRO MODEL OF ENTEROCYTE DIFFERENTIATION USING CaCo-2 CELLS. G.E, Wild, J. Hasan, M.J. Ropeleski, J. McCann. And A.B.R. Thomson. McGill University Inflammatory Bowel Disease Research Program. Montreal, Quebec and the Department of Gastroenterology, University of Alberta, Edmonton. CANADA. There is a growing body of experimental evidence which suggests that intestinal epithelial ceils synthesize an array of cytokines which participate in the mucosal immune response to antigen. In view of the clinical importance of Th2 cell derived cytokine, Interleukin -10, we sought to characterize the expression of this anti-inflammatory cytokine in an enteroeyte model using the well established Caco-2 cell. Methods: Caco-2 cells were cultured according to standard techniques and exposed to INFv (100U/ml) for 24hr. Interleukin-10 protein levels were measured by ELISA and IL-10 protein was localized by immunostaining. The level of II-10 mRNA abundance was determined by slot-blot and quantitative RT-PCR. Interleukin 10 transcripts were localized by in situ hybridization. Results: Exposure to INFy resulted in a 5 fold decrease of immunodetectable IL-10. This down regulation of IL-10 protein expression was confirmed by immunostaining. While I1-10 protein was detectable at all stages of cell culture, protein levels were most abundant in the post-confluent, differentiated state. Northern analysis disclosed a 4 kb transcript. IL-10 mRNA abundance was decreased in the presence of INFv. This decrease was confirmed by RT-PCR and in situ hybridization studies. I1-10 mRNA abundance was greatest in post confluent, differentiated Caco-2 cells. Conclusions: This is the first report of the expression of the antiinflammatory cytokine, Interleukin-10, in a model of the intestinal epithelium. The response of enterocyte IL-10 to INFy is similar to that which is observed in Th2 subsets of CD4 lymphocytes. The results presented here underscore the importance of the intestinal epithelium in mucosal immune function. Supported by Les Fonds de la Recherche en Sante du Quebec and the Crohn's and Colitis Foundation of Canada.