- Email: [email protected]
Oxidation and nitration injury of key cytoskeletal proteins in colonic mucosa of inflammatory bowel disease (IBD) patients
significantly higher in active versus inactive CD, UC and GCA. Levels of AT did not differ in active versus inactive CD and UC, but were lower in active versus inactive GCA. A positive correlation was found between the Crohns DiseaseActivity Index as well as the Ulcerative Colitis Activity Index with F1,2, TAT, D-dimer, PAP and PAI-I. However,even after 12 months of follow-up, in CD and UC the mean levels of F1,2, D-dimer and PAP were significantly higher than the levels of the controls. Conclusions: Prnthrombin fragment 1 and 2, TAT, Ddimer, PAP and PAl-1 correlated with inflammatory bowel diseaseactivity. Levelsof F1,2, Ddimer and PAP were markedly raised for a long time in clinically inactive inflammatory bowel disease,underlininga chronic state of hypercoagulationand correspondinglyenhancedfibrinolysis.
ECP measurement in serum might provide a further and easy diagnostic tool to montitor clinical diseaseactivity in ulcerativecolitis, which seemsto be strongly infuenced by eosinophil granulocytes. 1432 The Clinical Implication Of Glucocorlicoid Receptor ,8 Expression In Patients With Inflammatory Bowel Disease Seung-Jae Myung, Suk-Kyun Yang, Hwoon-YongJuno, Asan Medical Ctr, Seoul South Korea; Ok-Hee Kim, Hyun-Ju Oh, Aean institute for Biomed Science, Seoul South Korea; Weon-Seon Hung, Jin-Ho Kim, Young-II Min, Asan Medical Ctr, Seoul South Korea
1435
BACKGROUND:The initial mainstay of treatment of inflammatory bowel disease (IBD) at its active stage is intravenous administration of glucocorticoids. However, there are refractory or resistant cases to the glucocorticoids treatment. It would be very useful if we can predict the responsivenessof glucocorticoids before administration. The glucocorticoid receptor fl (hGRfl) has been suggestiveto play a role as a dominant negative regulator for determining glucocorticoid response. The aim of this study was to assess whether hGR has clinical implication in patients with 180. METHODS:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 40 patients with IBDs (Ulcerative colitis (UC) 28, Crohn's disease(CD) 12) and 3 controls. RNAwas reversetranscribedand the resulting complementary DNAwas amplified using specific primers for hGRotand hGR/~.Clinicalprofiles were compared according to hGRfl positivity. RESULTS:The expression of hGRotmRNA was detected in 38 of 40 patients an(:[all 3 controls. In contrast, a hGR/3mRNA was detected in 15 patients (11 of 28 in UC, 39% and 4 of 12 in CD, 33%)and in none of controls. Clinical parameters including age, sex, and disease extent were not different between hGR,8 positive and hGR,8 negative groups. Only two patients were glucocorticoids resistant and underwent operation (1 in hGR/~positive and 1 in hGRfl negativegroup). In patients who receivedglucocorticoids therapy, the duration to initial response was significantly shorter in hGR/~ negative group compared to hGR,8 positive group (15 _+ 4 claysvs. 28 _+ 16 days, p
LMWH for the Treatment of Mild to Moderatly Active Steroid Refractory/Dependent UC - An Independent, Multicenter, Randomised, Controlled Study. L Torkvist, Dept of Gastroenterology,Karolinska Inst, Stockholm Sweden; D Stahlberg, L; L Bohman, U Sjoqvist, L Loof, G Jamerot, R Stig, R Lofberg Background: Unfractionatedheparingiven i.v. in severeulcerativecolitis (UC) has beenshown to be as effective as i.v. given glucocorticosteroids (GCS). Self-administered (s.c.) Low Molecular Weight Heparin (LMWH) has also shown promising healing results, god tolerability and saffey in moderately active UC. Heparins have low toxicity and may be of beneficial in the healing of IBD. Amis and Methods: This randomised controlled trial was designed to determine and evaluate the efficacy of adjuvant s.c. given LMWH in the treatment of outpatients with mild to moderately active UC having responding poorly on the treatment with systemic or topical GCS.The patients receieveddalteparinsodium5000 units b.i.d, or placebo s.c. for up to eight weeks as adjuvant to the present theraphy incl. SASP/5-ASAand GCS. Clinic visits were made at day O, after 4 and 8 weeks. GCS treated retreatedwith a standard dose of 30 mg prednisolone initially for one week with gradual tapering by 5 mg/week to zero. A clinical symptoms index was used in assessments of disease, flexible colonoscopy or rigid sigmoidoscopy if apropiate was carried guy at day O, 4 and 8 weeks. Labratory parameterswere assessedat visit. Results: 41 patientswere included and 35 of them finished the study and were fully evaluableat the endpoint of the trial (20 active drug and 21 placebo). No bleedingcomplicationsor adverseeventsoccuredduring the study. No statistical difference in beneficialimprovment was seen betweenthe groups (endoscopicand symptomatic improvment). Five patients in the placebo gruo vs one in active treatment withdrew from the study prematurley (P-
1433 Oxidation and Nitration Injury of Key Cytoskeletal Proteins in Colonic Mucosa of Inflammatory Bowel Disease (IBD) Patients. All Banan, Yang Zhang, Jeremy Z. Fields, Sri Komanduri, Ece Mutlu, All Keshavarzian, Rush Univ, Chicago, IL Oxidativestress & generationof peroxynitrite (ONO0-) have beenimplicated as critical factors in the pathophysiologyof IBD. Identifyingtarget proteins for oxidativestress in IBD is essential because they may provide novel targets for the development of new drug therapies. Aims: To determine 1) whether oxidation (carbonylation) & nitration (nitrotyrosination, a foot print of ONO0-) of the cytoskeletal proteins such as F-actin & microtubule (tubulin) in mucosal biopsies from IBD patients is abnormally high; 2) If mucosa from normal subjects lack these oxidative injuries. Methods: Using a novel & sensitive quantitative immunoblot we have developed, flash frozen biopsies were assessed for oxidation state (n= 9 - 16 / group). Sampleswere homogenized& processedfor PAGEfractionation & then immunoblotting using monoclonal anti-carbonyl or anti-nitrotyrosine antibodiesthat were conjugatedto peroxidase by protein cross-linking with 0.2% gluteraldehyde.Identity of bands was confirmed as actin or tubulin by use of: i) a known oxidized or nitrated actin or tubulin standard, run concurrently as a positive control, it) a specific monoclonalanti-actin or anti-tubulin antibody. For quantitation, 2-dimensionaldensitometrywas performed.Groupstested were active & inactive Ulcerative Colitis (UC), Crohn's (CC), & healthy controls. Results: Both F-actin (43 kDa) & tubulin (50 kDa) from mucosa of controls exhibited low levels of native carbonyl & nitrotyrosine immunoreactivity. In contrast, actin & tubulin from mucosa of active IBD patients showed increased levels of oxidation & nitration injury (10 & 6 fold, respectively).When IBD was in remission (i.e., inactive phase), the oxidation & nitration levels of the cytoskeleton were significantly lower, decreasingto almost normal levels. We conclude: 1) inflamed mucosa of active IBD is associatedwith abnormallyhigh levelsof oxidationand nitration of key cytoskeletal proteins; 2) Oxidation and nitration of cytoskeletal proteins may be used as a marker of disease activity in IBD; 3) oxidative damage to cytoskeletal proteins may be a useful target for developmentof novel anti-oxidativetherapies for the treatment of a wide variety oxidative GI inflammatory disorders including IBD.
1436 Treatment of Peripheral Arthrepathy in IBD with Rofecoxib, a Selective COX-2 Inhibitor. Walter Reinisch, Wolfgang Pf Miehsler, Clemens Dejaco, Peter Glauninger, Harald Vogelsang, Univ of Vienna, Vienna Austria Peripheralarthritis is the most common extraintestinal manifestation of lSO characterizedby asymmetric painor swelling of joints without bony destruction. However,treatmentof arthropathy with NSAIDs is problematic due to an increased risk to exacerbate pre-existing IBD. Selective COX-2 antagonists, by preserving the biosynthesis of protective prostaglandins are associated with reduced gastrointestinal side effects. In a prospective, non-randomized pilotstudy we evaluated the safety and efficacy of Rofecoxib (Merck Sharp & Dohme), a selective COX-2antagonist in patients with IBD suffering from peripheralarthropathy (n = 15; f/m =10/5; age: median=55 yrs, range= 32 to 67; CD=10, UC=2, IC=3). At baselineand study end, diseaseactivity of CD and UC was determined by CDAI and CAI, respectively,as well as clinical assessmentof peripheralarthropathy was evaluatedby an arthritis pain scale ranging from 0 (very good) to 4 (very poor) and a functional disability index (HAG). Rofecoxib was applied for 20 days at a initial dose of 12,5 mg/d and increased if required to 25 mg/d. Systemicallyactive steroids were not allowed as concomitant treatment, other medicationfor IBD has to be kept at stable doses throughout the study. In 2 CD patients (13%) Rofecoxib at a dose of 12,5 mg/d had to be withdrawn after 1 and 3 days respectively,due to diarrhea in one case and intestinal bleeding in the other. Both reactionsceased immediatelyafter drug discontinuation. In the other patients no adverse events could be discerned. CDAI nonsignificantly dropped in CD and IC patients from 216 (SEM-+46) to 168 (_+33; P=O.09), both UC patients remained inactive. In 11/13 patients completing the protocol peripheral arthropathy improved with an overall reduction in arthritic pain from 2.15 (SEM_+O.25)to 1.1 (_+0.21; n = 13; P = 0.0003) and functional disability from HAG 1.05 (SEM_+O.18)to 0.65 (_+0.19; P=O.O009). This is the first prospective study on the use of a selective COX-2 antagonistin patientswith peripheralarthropathyand I BD. In 13% of patientsadverseintestinal eventscould be observed,however,of only self-limiting nature not leadingto diseaseexacerbation. The anti-inflammatory activity of rofecoxib in investigated indication is promising and warrants the initiation of randomized,controlled trials.
1434 Long Term Course of Markers of Coagulation and Fibrinolysis in Relation to Clinical Disease Activity in Inflammatory Bowel Disease and in Giant Cell Arthritis Anton A. Vrij, Univ Hosp Maastricht, Maastricht Netherlands;Joop Rijken, Jan W.J. Wersch, Atrium Hosp Heerlen, Heerlen Netherlands; Reinhold W. Stockbrugger, Univ Hosp Maastricht, Maaetricht Netherlands Background: In inflammatory bowel disease (IBD), gut microvascular thrombosis as well as thromboembolic complications have repeatedly been observed. We examinedthe long term course of markersof coagulationand fibrinolysis in relationto clinical diseaseactivity. Materials and methods: In a prospectivestudy, the coagulationactivation factors prothrombin fragment 1 and 2 (F1,2) and the thrombin-anti-thrombin (TAT) complex, the coagulation inhibition factor antithrombin (AT), the fibrinolysis factor D-dimer, and the fibrinolysis inhibition factors plasmin-alpha2-antiplasmin (PAP) complex and plasminogen-activator-inhibitor-1 (PAl-l) were measured in 20 newly diagnosed patients with Crohns disease(CO), 18 with ulcerative colitis (UC), and 19 with Giant cell arteritis (GCA), during active and inactive disease(median follow-up 12 months). Included were 51 age and sex matched healthy controls for both the IBD (n=25) and GCA (n=26) group. Results: Levels of F1,2 and TAT were significantly higher in active versus inactive CD and UC, but not in GCA. D-dimer, PAP and PAl-1 were
1437 Immune Stimulation in Crohn's Disease: Safety and Efficacy of rhuGM-CSF for the Treatment of Active Crohn's Disease Joshua R. Korzenik, Brian K. Dieckgraefe,Washington Univ, St. Louis, MO Background: Multiple genetic syndromes with quantitativeor qualitative detects in neutrophil or monocyte function, such as chronic granulomatous diseaseand glycogen storage disease lb, have beendescribedin associationwith the developmentof a Crohn's disease-likeenteritis or colitis. Thesesyndromes highlight the critical importance of innate immunity in the maintenance of mucosal integrity and may provide insight into the pathogenesisof Crohn's disease (CD). Defects identified in these diseases suggest a hypothesis that initiating events in
A-277
ECP measurement in serum might provide a further and easy diagnostic tool to montitor clinical diseaseactivity in ulcerativecolitis, which seemsto be strongly infuenced by eosinophil granulocytes. 1432 The Clinical Implication Of Glucocorlicoid Receptor ,8 Expression In Patients With Inflammatory Bowel Disease Seung-Jae Myung, Suk-Kyun Yang, Hwoon-YongJuno, Asan Medical Ctr, Seoul South Korea; Ok-Hee Kim, Hyun-Ju Oh, Aean institute for Biomed Science, Seoul South Korea; Weon-Seon Hung, Jin-Ho Kim, Young-II Min, Asan Medical Ctr, Seoul South Korea
1435
BACKGROUND:The initial mainstay of treatment of inflammatory bowel disease (IBD) at its active stage is intravenous administration of glucocorticoids. However, there are refractory or resistant cases to the glucocorticoids treatment. It would be very useful if we can predict the responsivenessof glucocorticoids before administration. The glucocorticoid receptor fl (hGRfl) has been suggestiveto play a role as a dominant negative regulator for determining glucocorticoid response. The aim of this study was to assess whether hGR has clinical implication in patients with 180. METHODS:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 40 patients with IBDs (Ulcerative colitis (UC) 28, Crohn's disease(CD) 12) and 3 controls. RNAwas reversetranscribedand the resulting complementary DNAwas amplified using specific primers for hGRotand hGR/~.Clinicalprofiles were compared according to hGRfl positivity. RESULTS:The expression of hGRotmRNA was detected in 38 of 40 patients an(:[all 3 controls. In contrast, a hGR/3mRNA was detected in 15 patients (11 of 28 in UC, 39% and 4 of 12 in CD, 33%)and in none of controls. Clinical parameters including age, sex, and disease extent were not different between hGR,8 positive and hGR,8 negative groups. Only two patients were glucocorticoids resistant and underwent operation (1 in hGR/~positive and 1 in hGRfl negativegroup). In patients who receivedglucocorticoids therapy, the duration to initial response was significantly shorter in hGR/~ negative group compared to hGR,8 positive group (15 _+ 4 claysvs. 28 _+ 16 days, p
LMWH for the Treatment of Mild to Moderatly Active Steroid Refractory/Dependent UC - An Independent, Multicenter, Randomised, Controlled Study. L Torkvist, Dept of Gastroenterology,Karolinska Inst, Stockholm Sweden; D Stahlberg, L; L Bohman, U Sjoqvist, L Loof, G Jamerot, R Stig, R Lofberg Background: Unfractionatedheparingiven i.v. in severeulcerativecolitis (UC) has beenshown to be as effective as i.v. given glucocorticosteroids (GCS). Self-administered (s.c.) Low Molecular Weight Heparin (LMWH) has also shown promising healing results, god tolerability and saffey in moderately active UC. Heparins have low toxicity and may be of beneficial in the healing of IBD. Amis and Methods: This randomised controlled trial was designed to determine and evaluate the efficacy of adjuvant s.c. given LMWH in the treatment of outpatients with mild to moderately active UC having responding poorly on the treatment with systemic or topical GCS.The patients receieveddalteparinsodium5000 units b.i.d, or placebo s.c. for up to eight weeks as adjuvant to the present theraphy incl. SASP/5-ASAand GCS. Clinic visits were made at day O, after 4 and 8 weeks. GCS treated retreatedwith a standard dose of 30 mg prednisolone initially for one week with gradual tapering by 5 mg/week to zero. A clinical symptoms index was used in assessments of disease, flexible colonoscopy or rigid sigmoidoscopy if apropiate was carried guy at day O, 4 and 8 weeks. Labratory parameterswere assessedat visit. Results: 41 patientswere included and 35 of them finished the study and were fully evaluableat the endpoint of the trial (20 active drug and 21 placebo). No bleedingcomplicationsor adverseeventsoccuredduring the study. No statistical difference in beneficialimprovment was seen betweenthe groups (endoscopicand symptomatic improvment). Five patients in the placebo gruo vs one in active treatment withdrew from the study prematurley (P-
1433 Oxidation and Nitration Injury of Key Cytoskeletal Proteins in Colonic Mucosa of Inflammatory Bowel Disease (IBD) Patients. All Banan, Yang Zhang, Jeremy Z. Fields, Sri Komanduri, Ece Mutlu, All Keshavarzian, Rush Univ, Chicago, IL Oxidativestress & generationof peroxynitrite (ONO0-) have beenimplicated as critical factors in the pathophysiologyof IBD. Identifyingtarget proteins for oxidativestress in IBD is essential because they may provide novel targets for the development of new drug therapies. Aims: To determine 1) whether oxidation (carbonylation) & nitration (nitrotyrosination, a foot print of ONO0-) of the cytoskeletal proteins such as F-actin & microtubule (tubulin) in mucosal biopsies from IBD patients is abnormally high; 2) If mucosa from normal subjects lack these oxidative injuries. Methods: Using a novel & sensitive quantitative immunoblot we have developed, flash frozen biopsies were assessed for oxidation state (n= 9 - 16 / group). Sampleswere homogenized& processedfor PAGEfractionation & then immunoblotting using monoclonal anti-carbonyl or anti-nitrotyrosine antibodiesthat were conjugatedto peroxidase by protein cross-linking with 0.2% gluteraldehyde.Identity of bands was confirmed as actin or tubulin by use of: i) a known oxidized or nitrated actin or tubulin standard, run concurrently as a positive control, it) a specific monoclonalanti-actin or anti-tubulin antibody. For quantitation, 2-dimensionaldensitometrywas performed.Groupstested were active & inactive Ulcerative Colitis (UC), Crohn's (CC), & healthy controls. Results: Both F-actin (43 kDa) & tubulin (50 kDa) from mucosa of controls exhibited low levels of native carbonyl & nitrotyrosine immunoreactivity. In contrast, actin & tubulin from mucosa of active IBD patients showed increased levels of oxidation & nitration injury (10 & 6 fold, respectively).When IBD was in remission (i.e., inactive phase), the oxidation & nitration levels of the cytoskeleton were significantly lower, decreasingto almost normal levels. We conclude: 1) inflamed mucosa of active IBD is associatedwith abnormallyhigh levelsof oxidationand nitration of key cytoskeletal proteins; 2) Oxidation and nitration of cytoskeletal proteins may be used as a marker of disease activity in IBD; 3) oxidative damage to cytoskeletal proteins may be a useful target for developmentof novel anti-oxidativetherapies for the treatment of a wide variety oxidative GI inflammatory disorders including IBD.
1436 Treatment of Peripheral Arthrepathy in IBD with Rofecoxib, a Selective COX-2 Inhibitor. Walter Reinisch, Wolfgang Pf Miehsler, Clemens Dejaco, Peter Glauninger, Harald Vogelsang, Univ of Vienna, Vienna Austria Peripheralarthritis is the most common extraintestinal manifestation of lSO characterizedby asymmetric painor swelling of joints without bony destruction. However,treatmentof arthropathy with NSAIDs is problematic due to an increased risk to exacerbate pre-existing IBD. Selective COX-2 antagonists, by preserving the biosynthesis of protective prostaglandins are associated with reduced gastrointestinal side effects. In a prospective, non-randomized pilotstudy we evaluated the safety and efficacy of Rofecoxib (Merck Sharp & Dohme), a selective COX-2antagonist in patients with IBD suffering from peripheralarthropathy (n = 15; f/m =10/5; age: median=55 yrs, range= 32 to 67; CD=10, UC=2, IC=3). At baselineand study end, diseaseactivity of CD and UC was determined by CDAI and CAI, respectively,as well as clinical assessmentof peripheralarthropathy was evaluatedby an arthritis pain scale ranging from 0 (very good) to 4 (very poor) and a functional disability index (HAG). Rofecoxib was applied for 20 days at a initial dose of 12,5 mg/d and increased if required to 25 mg/d. Systemicallyactive steroids were not allowed as concomitant treatment, other medicationfor IBD has to be kept at stable doses throughout the study. In 2 CD patients (13%) Rofecoxib at a dose of 12,5 mg/d had to be withdrawn after 1 and 3 days respectively,due to diarrhea in one case and intestinal bleeding in the other. Both reactionsceased immediatelyafter drug discontinuation. In the other patients no adverse events could be discerned. CDAI nonsignificantly dropped in CD and IC patients from 216 (SEM-+46) to 168 (_+33; P=O.09), both UC patients remained inactive. In 11/13 patients completing the protocol peripheral arthropathy improved with an overall reduction in arthritic pain from 2.15 (SEM_+O.25)to 1.1 (_+0.21; n = 13; P = 0.0003) and functional disability from HAG 1.05 (SEM_+O.18)to 0.65 (_+0.19; P=O.O009). This is the first prospective study on the use of a selective COX-2 antagonistin patientswith peripheralarthropathyand I BD. In 13% of patientsadverseintestinal eventscould be observed,however,of only self-limiting nature not leadingto diseaseexacerbation. The anti-inflammatory activity of rofecoxib in investigated indication is promising and warrants the initiation of randomized,controlled trials.
1434 Long Term Course of Markers of Coagulation and Fibrinolysis in Relation to Clinical Disease Activity in Inflammatory Bowel Disease and in Giant Cell Arthritis Anton A. Vrij, Univ Hosp Maastricht, Maastricht Netherlands;Joop Rijken, Jan W.J. Wersch, Atrium Hosp Heerlen, Heerlen Netherlands; Reinhold W. Stockbrugger, Univ Hosp Maastricht, Maaetricht Netherlands Background: In inflammatory bowel disease (IBD), gut microvascular thrombosis as well as thromboembolic complications have repeatedly been observed. We examinedthe long term course of markersof coagulationand fibrinolysis in relationto clinical diseaseactivity. Materials and methods: In a prospectivestudy, the coagulationactivation factors prothrombin fragment 1 and 2 (F1,2) and the thrombin-anti-thrombin (TAT) complex, the coagulation inhibition factor antithrombin (AT), the fibrinolysis factor D-dimer, and the fibrinolysis inhibition factors plasmin-alpha2-antiplasmin (PAP) complex and plasminogen-activator-inhibitor-1 (PAl-l) were measured in 20 newly diagnosed patients with Crohns disease(CO), 18 with ulcerative colitis (UC), and 19 with Giant cell arteritis (GCA), during active and inactive disease(median follow-up 12 months). Included were 51 age and sex matched healthy controls for both the IBD (n=25) and GCA (n=26) group. Results: Levels of F1,2 and TAT were significantly higher in active versus inactive CD and UC, but not in GCA. D-dimer, PAP and PAl-1 were
1437 Immune Stimulation in Crohn's Disease: Safety and Efficacy of rhuGM-CSF for the Treatment of Active Crohn's Disease Joshua R. Korzenik, Brian K. Dieckgraefe,Washington Univ, St. Louis, MO Background: Multiple genetic syndromes with quantitativeor qualitative detects in neutrophil or monocyte function, such as chronic granulomatous diseaseand glycogen storage disease lb, have beendescribedin associationwith the developmentof a Crohn's disease-likeenteritis or colitis. Thesesyndromes highlight the critical importance of innate immunity in the maintenance of mucosal integrity and may provide insight into the pathogenesisof Crohn's disease (CD). Defects identified in these diseases suggest a hypothesis that initiating events in
A-277