- Email: [email protected]
IL-1ß and granulocyte elastase in whole gut lavage fluid predicts relapse in patients with inactive Crohn's disease
April 1998
Immunology, Microbiology, and Inflammatory Disorders A923
Crohn's disease, and acts at the colonic mucosa. Uncertainty about the precise therapeutic mechanism of the drug could be reduced if the sites of drug deposition (and therefore potential sites of action) in the colonic mucosa were more precisely defined. The aim of this study was to use confocal microscopy of living colonic mucosa to compare the uptake and retention of 5-ASA among three different cell types: lamina propria cells (including immune cells), endothelial cells of the blood vessels, and the crypt epithelial cells. Methods. Muscle-stripped mouse colonic mucosa was mounted in a perfusion chamber mounted on the stage of a Zeiss LSM410 confocal microscope. The intrinsic fluorescence of 5-ASA was excited by 351 nm laser light and emission was measured at 500-600 nm. Tissue was exposed to bilateral superfusion with physiologic medium containing 5-ASA (10 mM) for a 20 min period, followed by perfusion with medium in the absence of drug. Drug levels were measured immediately after 5-ASA removal and at 5 min. time points thereafter. To allow the accurate identification of cell types in the tissue, transmitted light images were simultaneously collected from the tissue. Results. All cell types loaded with 5-ASA to approximately equal levels: 8-10mM. However, 10 min after 5-ASA removal, the crypt cells retained 105 +- 14% of their initial 5-ASA (mean - SEM, n=12), whereas dramatically less was retained by the lamina propria ceils (53 + 8%, n=4) and endothelial cells (64 _+11%). Conclusion. The colonic epithelial cells retain 5-ASA at much higher levels than the non-epithelial cells in the colonic mucosa. Results suggest that any prolonged effects of 5-ASA therapy are more likely to be mediated by effects of the drug on epithelial cell function. This research was funded by the CCFA. • G3785 ABNORMAL INTESTINAL PERMEABILITY PREDICTS RELAPSE IN PATIENTS WITH INACTIVE CROHN'S DISEASE. IDR Arnott, S Ghosh, A Ferguson, GI Unit, University Department of Medicine, Western General Hospital, Edinburgh, EH4 2XU, UK. Background: A number of parameters predicting relapse in Crohn's disease (CD) have been proposed but none are widely used. Intestinal permeability (IP) may be abnormal in inactive CD but it is not known whether this relates to relapse. Patients and Methods: We assessed IP in 42 patients with inactive CD (CDAI<150). Disease distribution was 14 small bowel, 10 colon, 9 ileocolon and 9 post colectomy. We used 30 patients with inactive Ulcerative colitis (UC) as disease controls and 22 normal controls. IP was assessed using the differential urinary excretion of a lactulose and rhamnose test solution. The resulting urine was analysed using a direct injection HPLC system including internal and external standards. Good separation was obtained between lactose and lactulose. Patients were followed up for one year. Results: IP of CD patients was significantly higher than controls (p<0.05). Of the 42 patients with CD, 27 had normal and 14 had abnormal IP. Of those with CD and normal IP, 21 remained in remission, 1 relapsed, 4 were dependent on steroids and 1 was on azathioprine. Of those with an abnormal IP, 3 remained in remission, 4 relapsed, 3 were dependent on steroids, 2 were on azathioprine and 1 was refractory to treatment. Significantly fewer patients with inactive UC had abnormal IP (3•30) than CD (14/42) (X2=5.55, p<0.02). In patients with CD, those with a normal permeability had a significantly greater chance of remaining in remission than those with abnormal IP as depicted in the life table analysis below. Relapse Curve for IP In Crohn's Disease
,oo
l
Normal IP, nffi22
1 50-
o
1~o
1 =~o
Z2ffil 0.8, p •O.O01 RR=O.085
1 3~)0
Abnormal IP, n=7
4~o
Days to Relapse
Conclusion: Intestinal permeability is an easy to perform and non invasive method of assessing prognosis in patients with inactive Crohn's disease. The test may be of use in clinical trials of maintenance therapy for CD.
G3786 GUT LUMINAL NEUTROPHIL MIGRATION DEPENDS ON THE ANATOMICAL SITE OF CROHN'S DISEASE. IDR Arn0tt, H Dmmmond, S Ghosh, A Ferguson. Gastrointestinal Unit, Department of Medicine, Western General Hospital, Edinburgh EH4 2XU. Background: Gut luminal neutropils can be studied using whole gut lavage fluid (WGLF) obtained after bowel cleansing by a polyethylene glycolelectrolyte solution (Klean-Prep, Norgine). Cytology or an enzyme-substrate reaction to detect granulocyte elastase (GE), can be performed on unfiltered WGLF. Interleukin-8 (IL-8) is a potent neutrophil chemoattractant although other exact stimuli responsible for luminal neutrophil migration are unknown.
Methods: 121 patients with well characterised active Crohn's disease (CD) underwent whole gut lavage. The first clear specimen passed after complete gut cleansing was stored at -70°C. GE was assayed using the specific chromogenic substrate L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (Quadratech, Epsom). The lower limit of detection by this assay was 39 nkat/L. 1L-8 was assayed in unfiltered processed lavage samples using a commercially available sandwich ELISA kit (Eurogenetics, Hampton, UK) Patients were divided in to 6 groups according to the distribution of macroscopic disease determined radiologically or endoscopically. Results: 28 out of 35 patients with Crohn's colitis had detectable GE, (median 259, range <39-2742nkat/L), whereas only 4 out of 15 patients with small bowel involvement had detectable GE (median <39, range <39-266nkat/L; P<0.0001 Vs colonic disease). 28 out of 40 patients with both small bowel and colonic disease had detectable GE (median 1050, range <39-1709nkat/L; p<0.004 Vs small bowel). In the 12 patients with recurrent small bowel disease who had prior ileocaecal resection, 10 had detectable GE (median 181, range <39-1240nkat/L; p<0.003 Vs small bowel) and the concentrations were significantly higher than in those with small bowel disease but no resections (p <0.003). 10 out of 15 patients with an ileostomy had undetectable GE. 75 of the above patients in whom samples were available had IL-8 assayed in the lavage fluid. The 19 patients with colonic disease (median 2056, range 555-19200pg/ml) and the 26 patients with ileocolonic disease (median 919, range 11-15600 pg/ml) had significantly higher levels of IL-8 than 11 patients with isolated small bowel disease (median 351, range 7-1000 pg/ml, p<0.00001 and p<0.04 respectively). Conclusion: Neutrophil migration into the lumen of the gut is a feature of colonic but not small bowel Crohn's disease suggesting that neutrophil chemoattractants derived from bacterial flora may play a role in gut neutrophil migration. This hypothesis is further supported by the high GE concentrations found in recurrent small bowel disease after ileocaecal resection. High levels of IL-8 in colonic as opposed to small bowel disease are consistent with this differential neutrophil migration. G3787 IL-113 AND GRANULOCYTE ELASTASE IN WHOLE GUT LAVAGE FLUID PREDICTS RELAPSE IN PATIENTS WITH INACTIVE CROHN'S DISEASE. IDR Arnott, HE Drummond, S Ghosh, A Ferguson, GI Unit, University Department of Medicine, Western General Hospital, Edinburgh, EH4 2XU. Background and Aims: A number of parameters assessing prognosis in Crohn's disease (CD) have been proposed although none is widely used. Whole gut lavage fluid (WGLF) obtained after bowel cleansing with a polyethylene glycol electrolyte solution (Klean Prep, Norgine, UK) is an established method of studying gut inflammation and immunity. Interleukin113 (IL-113) and Granuolcyte Elastase (GE) may be present in CD patients in remission although it is not known whether this relates to relapse. Patients and Methods: 60 well characterised patients with inactive CD underwent whole gut lavage. The first clear specimen passed was collected, processed as required and stored at -70°C. Disease activity was assessed by CDAI (<150) and WGLF IgG concentration (<10tag/ml). GE was assayed by an enzyme substrate reaction using a specific chromogenic substrate L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (Quadratech, Epsom, UK). The lower limit of detection was <39 nKat/L. IL-113 was assayed in unfiltered processed lavage fluid using a commercially available sandwich ELISA kit (R&D systems, UK). The lower limit of detection was 2 pg/ml and the normal range is 0-12pg/ml. Patients were followed up for one year after whole gut lavage. Results: Of the 60 patients there were 29 patients with a normal concentration of IL-113. At follow up 22 of these patients remained in remission, 1 relapsed, 4 underwent intestinal resection and 2 were on immunosupressives. Of the 31 patients with an abnormally raised IL-lfl, 14 remained in remission, 8 relapsed, 2 underwent resection and 3 were on immunosupressives. Those with an abnormal level of IL-113 in the lavage fluid had a significantly greater chance of relapse than those with a normal level, X2=7.4, p=0.0065, relative risk 10. 50 patients had an undetectable and 10 had a detectable GE. Of those with an undetectable GE, 32 remained in remission, 6 relapsed, 6 underwent resection and 5 remained on immunosupressives. Of those with a detectable GE, 3 relapsed, 4 remained in remission and 1 patient underwent a resection. 3 patients were lost to follow up. Those with a detectable GE had a significantly greater chance of relapsing than those with an undetectable GE, 22=4.4, p<0.04, relative risk 4. Conclusion" GE and IL-113 in whole gut lavage fluid are reliable and objective methods of predicting relapse in patients with inactive CD. The presence of IL-lfl or GE in WGLF may signify continuing mucosal inflammatory events despite clinical remission. This may also be used to identify a group at high risk of relapse for clinical trials of maintenance therapy in CD.
Immunology, Microbiology, and Inflammatory Disorders A923
Crohn's disease, and acts at the colonic mucosa. Uncertainty about the precise therapeutic mechanism of the drug could be reduced if the sites of drug deposition (and therefore potential sites of action) in the colonic mucosa were more precisely defined. The aim of this study was to use confocal microscopy of living colonic mucosa to compare the uptake and retention of 5-ASA among three different cell types: lamina propria cells (including immune cells), endothelial cells of the blood vessels, and the crypt epithelial cells. Methods. Muscle-stripped mouse colonic mucosa was mounted in a perfusion chamber mounted on the stage of a Zeiss LSM410 confocal microscope. The intrinsic fluorescence of 5-ASA was excited by 351 nm laser light and emission was measured at 500-600 nm. Tissue was exposed to bilateral superfusion with physiologic medium containing 5-ASA (10 mM) for a 20 min period, followed by perfusion with medium in the absence of drug. Drug levels were measured immediately after 5-ASA removal and at 5 min. time points thereafter. To allow the accurate identification of cell types in the tissue, transmitted light images were simultaneously collected from the tissue. Results. All cell types loaded with 5-ASA to approximately equal levels: 8-10mM. However, 10 min after 5-ASA removal, the crypt cells retained 105 +- 14% of their initial 5-ASA (mean - SEM, n=12), whereas dramatically less was retained by the lamina propria ceils (53 + 8%, n=4) and endothelial cells (64 _+11%). Conclusion. The colonic epithelial cells retain 5-ASA at much higher levels than the non-epithelial cells in the colonic mucosa. Results suggest that any prolonged effects of 5-ASA therapy are more likely to be mediated by effects of the drug on epithelial cell function. This research was funded by the CCFA. • G3785 ABNORMAL INTESTINAL PERMEABILITY PREDICTS RELAPSE IN PATIENTS WITH INACTIVE CROHN'S DISEASE. IDR Arnott, S Ghosh, A Ferguson, GI Unit, University Department of Medicine, Western General Hospital, Edinburgh, EH4 2XU, UK. Background: A number of parameters predicting relapse in Crohn's disease (CD) have been proposed but none are widely used. Intestinal permeability (IP) may be abnormal in inactive CD but it is not known whether this relates to relapse. Patients and Methods: We assessed IP in 42 patients with inactive CD (CDAI<150). Disease distribution was 14 small bowel, 10 colon, 9 ileocolon and 9 post colectomy. We used 30 patients with inactive Ulcerative colitis (UC) as disease controls and 22 normal controls. IP was assessed using the differential urinary excretion of a lactulose and rhamnose test solution. The resulting urine was analysed using a direct injection HPLC system including internal and external standards. Good separation was obtained between lactose and lactulose. Patients were followed up for one year. Results: IP of CD patients was significantly higher than controls (p<0.05). Of the 42 patients with CD, 27 had normal and 14 had abnormal IP. Of those with CD and normal IP, 21 remained in remission, 1 relapsed, 4 were dependent on steroids and 1 was on azathioprine. Of those with an abnormal IP, 3 remained in remission, 4 relapsed, 3 were dependent on steroids, 2 were on azathioprine and 1 was refractory to treatment. Significantly fewer patients with inactive UC had abnormal IP (3•30) than CD (14/42) (X2=5.55, p<0.02). In patients with CD, those with a normal permeability had a significantly greater chance of remaining in remission than those with abnormal IP as depicted in the life table analysis below. Relapse Curve for IP In Crohn's Disease
,oo
l
Normal IP, nffi22
1 50-
o
1~o
1 =~o
Z2ffil 0.8, p •O.O01 RR=O.085
1 3~)0
Abnormal IP, n=7
4~o
Days to Relapse
Conclusion: Intestinal permeability is an easy to perform and non invasive method of assessing prognosis in patients with inactive Crohn's disease. The test may be of use in clinical trials of maintenance therapy for CD.
G3786 GUT LUMINAL NEUTROPHIL MIGRATION DEPENDS ON THE ANATOMICAL SITE OF CROHN'S DISEASE. IDR Arn0tt, H Dmmmond, S Ghosh, A Ferguson. Gastrointestinal Unit, Department of Medicine, Western General Hospital, Edinburgh EH4 2XU. Background: Gut luminal neutropils can be studied using whole gut lavage fluid (WGLF) obtained after bowel cleansing by a polyethylene glycolelectrolyte solution (Klean-Prep, Norgine). Cytology or an enzyme-substrate reaction to detect granulocyte elastase (GE), can be performed on unfiltered WGLF. Interleukin-8 (IL-8) is a potent neutrophil chemoattractant although other exact stimuli responsible for luminal neutrophil migration are unknown.
Methods: 121 patients with well characterised active Crohn's disease (CD) underwent whole gut lavage. The first clear specimen passed after complete gut cleansing was stored at -70°C. GE was assayed using the specific chromogenic substrate L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (Quadratech, Epsom). The lower limit of detection by this assay was 39 nkat/L. 1L-8 was assayed in unfiltered processed lavage samples using a commercially available sandwich ELISA kit (Eurogenetics, Hampton, UK) Patients were divided in to 6 groups according to the distribution of macroscopic disease determined radiologically or endoscopically. Results: 28 out of 35 patients with Crohn's colitis had detectable GE, (median 259, range <39-2742nkat/L), whereas only 4 out of 15 patients with small bowel involvement had detectable GE (median <39, range <39-266nkat/L; P<0.0001 Vs colonic disease). 28 out of 40 patients with both small bowel and colonic disease had detectable GE (median 1050, range <39-1709nkat/L; p<0.004 Vs small bowel). In the 12 patients with recurrent small bowel disease who had prior ileocaecal resection, 10 had detectable GE (median 181, range <39-1240nkat/L; p<0.003 Vs small bowel) and the concentrations were significantly higher than in those with small bowel disease but no resections (p <0.003). 10 out of 15 patients with an ileostomy had undetectable GE. 75 of the above patients in whom samples were available had IL-8 assayed in the lavage fluid. The 19 patients with colonic disease (median 2056, range 555-19200pg/ml) and the 26 patients with ileocolonic disease (median 919, range 11-15600 pg/ml) had significantly higher levels of IL-8 than 11 patients with isolated small bowel disease (median 351, range 7-1000 pg/ml, p<0.00001 and p<0.04 respectively). Conclusion: Neutrophil migration into the lumen of the gut is a feature of colonic but not small bowel Crohn's disease suggesting that neutrophil chemoattractants derived from bacterial flora may play a role in gut neutrophil migration. This hypothesis is further supported by the high GE concentrations found in recurrent small bowel disease after ileocaecal resection. High levels of IL-8 in colonic as opposed to small bowel disease are consistent with this differential neutrophil migration. G3787 IL-113 AND GRANULOCYTE ELASTASE IN WHOLE GUT LAVAGE FLUID PREDICTS RELAPSE IN PATIENTS WITH INACTIVE CROHN'S DISEASE. IDR Arnott, HE Drummond, S Ghosh, A Ferguson, GI Unit, University Department of Medicine, Western General Hospital, Edinburgh, EH4 2XU. Background and Aims: A number of parameters assessing prognosis in Crohn's disease (CD) have been proposed although none is widely used. Whole gut lavage fluid (WGLF) obtained after bowel cleansing with a polyethylene glycol electrolyte solution (Klean Prep, Norgine, UK) is an established method of studying gut inflammation and immunity. Interleukin113 (IL-113) and Granuolcyte Elastase (GE) may be present in CD patients in remission although it is not known whether this relates to relapse. Patients and Methods: 60 well characterised patients with inactive CD underwent whole gut lavage. The first clear specimen passed was collected, processed as required and stored at -70°C. Disease activity was assessed by CDAI (<150) and WGLF IgG concentration (<10tag/ml). GE was assayed by an enzyme substrate reaction using a specific chromogenic substrate L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (Quadratech, Epsom, UK). The lower limit of detection was <39 nKat/L. IL-113 was assayed in unfiltered processed lavage fluid using a commercially available sandwich ELISA kit (R&D systems, UK). The lower limit of detection was 2 pg/ml and the normal range is 0-12pg/ml. Patients were followed up for one year after whole gut lavage. Results: Of the 60 patients there were 29 patients with a normal concentration of IL-113. At follow up 22 of these patients remained in remission, 1 relapsed, 4 underwent intestinal resection and 2 were on immunosupressives. Of the 31 patients with an abnormally raised IL-lfl, 14 remained in remission, 8 relapsed, 2 underwent resection and 3 were on immunosupressives. Those with an abnormal level of IL-113 in the lavage fluid had a significantly greater chance of relapse than those with a normal level, X2=7.4, p=0.0065, relative risk 10. 50 patients had an undetectable and 10 had a detectable GE. Of those with an undetectable GE, 32 remained in remission, 6 relapsed, 6 underwent resection and 5 remained on immunosupressives. Of those with a detectable GE, 3 relapsed, 4 remained in remission and 1 patient underwent a resection. 3 patients were lost to follow up. Those with a detectable GE had a significantly greater chance of relapsing than those with an undetectable GE, 22=4.4, p<0.04, relative risk 4. Conclusion" GE and IL-113 in whole gut lavage fluid are reliable and objective methods of predicting relapse in patients with inactive CD. The presence of IL-lfl or GE in WGLF may signify continuing mucosal inflammatory events despite clinical remission. This may also be used to identify a group at high risk of relapse for clinical trials of maintenance therapy in CD.