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Ischaemic colitis is not just a disease of elderly arteriopaths
Intestinal Disorders A349
April 1998
G1422 TEMPORAL CHANGES IN INFLAMMATORY MEDIATOR LEVELS AND COLONIC VASCULAR ARCHITECTURE IN AN ANIMAL MODEL OF COLITIS. CB Appleyard, LJ Hanson & WH Percy, Department of Physiology & Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069.
• G1424 PROLONGED VS. TRANSIENT HISTONE HYPERACETYLATION: DIFFERENTIAL EFFECTS ON ENTEROCYTE GROWTH AND DIFFERENTIATION. S. Archer. J. Johnson, S. Meng, P. Grable, J. B. Matthews, R. A. Hodin. Dept. of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.
In a rabbit trlnitrubanzesulfonic acid (TNBS) model of colitis there are significant increases in blood flow to both the colonic muscularis proprla and mucosa/muscularls mucosae within 1 hr (Gastrn. 1997; 112: A345). The aim of this study was to investigate the possible temporal relationship between levels of vasoactive inflammatory mediators and changes in both number and diameter of colonic blood vessels in this model. METHODS: Colitis was induced in rabbits by intracvlonic administration of TNBS in 50% ethanol. Animals were euthanized 1, 6 and 48 hr after induction and the distal 10cm of colon removed and scored for macroscopic damage. The muscle layers were separated from the mucosa/muscularis mucosae before measurement of mediator levels by radioimmunoassay. Blood vessels were visualized using a modified NADPH-diaphorase stain and vessel diameter in both muscle and mucosa was measured. The number of mucosal vessels per mm2 was also counted. A scoring system of 0---~3 (normal--->extensive)for epithelial damage or TNBS penetration was used to determine the integrity of the mucosa and the extent of TNBS movement through the lamina propria. RESULTS: TNBS caused significant colonic damage at all time points compared to control (untreated) or ethanol vehicle. Mediator levels (where LTC4+=LTCfl)4/E~/F4) were:
As small intestinal epithelial ceils migrate up the crypt-villus axis, they express a differentiated phenotype and undergo cell cycle arrest. The molecular mechanisms responsible for these changes are poorly understood. It is known that an alteration in chromatin structure by histone hyperacetylation is associated with decreased cell growth and increased gene expression. Therefore, in these studies, we sought to examine the role of histone hyperacetylation in enterocyte differentiation and its associated growth arrest. METHODS: HT-29 cells were grown in standard media and treated with the histone hyperacetylating agents, sodium butyrate (NaBu, 5mM) or trichostatin A (TSA, 0.3 laM), both inhibitors of the histone deacetylase enzyme. Transient histone hyperacetylation was accomplished by treating cells with a single dose of TSA, a reversible inhibitor of histone deacetylase. Prolonged histone hyperacetylation was accomplished by treating cells with a single dose of NaBu or repeated doses of TSA (every 8 hrs.). Northern analyses were performed using cDNA probes specific for the enterocyte differentiation markers, intestinal alkaline phosphatase (lAP) and villin, the Na/K/2CL cotransporter (NKCC, which decreases in the differentiated phenotype), the cell cycle inhibitor, p21, and the actin control. 3H-thymidine incorporation assays were performed using standard protocol. Histone Acetvlation Assays: Nuclear histones from NaBu- or TSA-treated cells were extracted by acid/acetone precipitation, electrophoresed on an acid/urea/triton gel and stained with coomassie blue. RESULTS: Treatment of ceils with a single dose of TSA resulted in a rapid increase in the level of histone H4 acetylation by 0.5 hrs., with a peak by 4 hrs. and a return to the basal hypoacetylated state by 24 hrs. p21 mRNA levels paralleled these changes in histone hyperacetylation, peaking at 4 hrs. (15-fold increase, p < 0.001), but returning to basal levels at 24 hrs. These changes were associated with growth arrest (50% decrease in 3H-thymidine incorporation, p < 0.001), but no change in the IAP, villin, nor NKCC mRNAs. In dramatic contrast, prolonged histone H4 hyperacetylation was seen in response to either NaBu or repeated TSA treatment, beginning by 0.5 hrs. and remaining elevated up to 24 hrs. As with transient histone hyperacetylation, there was decreased cell growth (50% decrease in 3H-thymidine incorporation, p < 0.00l). However, there were now persistent increases in p21 mRNA expression and cellular differentiation was evidenced by increases in lAP and villin mRNAs (10- and 2.5 fold respectively, p < 0.001), and decreases in NKCC mRNA (10-fold, p < 0.001), at 24 hrs. CONCLUSIONS: In HT-29 cells, transient histone hyperacetylation is sufficient to induce growth arrest, but prolonged histone hyperacetylation is required for differentiation to occur.
Control TNBS Iht TNBS6hr TNBS48hr muscle TXB2 22.9±7.7 74.4± I4.3a 165.4±41.08 177.8± 48.11~ mucosa TXB2 24.8±4.9 205.9±43.58 284.7±45.38 240.3±68.98 muscle LTB4 315.9±49.4 220.6±67.1 449.7±114.5 651.4±162.2 mucosa LTB4 240.6±46.4 180.1±44.6 902.6±164.78 753.9±121.2I~ muscle LTC4+ 642.4± 148.3 382.3± 77.3 1095.9± 364.3 961.3± 136.4 mucosa LTC4+ 910.2±157.5 850.9±211.1 2377.1±623.8tt 1473.4±214.5 t~p< 0.05.I~p< 0.01,8p< 0.001comparedto control;pg/mgprotein± s.c.;n >6
El'OH48hr 32.9±12.2 72.0±27.3 481.4±198.1 500.8±146.3 495.1± 80.1 777.0±133.8
There were no significant differences in PGE2 levels in any layer at any time. A loss of mucosal integrity and an increase in TNBS penetration through the lamina propria occurred over time (2.4 ± 0.2, p < 0.01 and 2.2 ± 0.4, p < 0.05 respectively at 48hr post-TNBS, n=5). Neither of these parameters was temporally-related to mediator levels. There was no significant change from control in the diameter or number of blood vessels in either the muscle (longitudinally oriented vessels, diameter 2.9 ± 0.91am; circularly oriented vessels, diameter 24.0±0.0tam) or mucosal layer (vessel diameter 1.7±0.11am & 144.2±24.0 vessels/ram2, each 20 observations from 2 rabbits). CONCLUSIONS: 1. TNBS-induced colonic damage is accompanied by time-dependent changes in tissue levels of inflammatory mediators. 2. Changes in mediator levels occur as a result of the inflammatory process and not damage p e r se as TNBS penetration continues to increase after mediator levels have peaked. 3. Acute changes in blood flow occur independently of these inflammatory mediator levels and are not associated with altered vascular architecture. Supported by the Crohn's and Colitis Foundation of America & DK 49803. • G1423 IMPAIRED NA÷-K÷ PUMP ACTIVITY IN THE ERYTHROCYTES OF CHILDREN WITH TREATED COELIAC DISEASE. A. Arat6. M. Dobos, B. V~is~helyi, H. Bod~nszky, T. Tulassay. First Department of Paediatrics, Semmelweis University of Medicine, Budapest, Hungary
Inherited or acquired changes in the epithelial cell-surface membranes may contribute to the pathogenesis of coeliac disease (CD). The Na+-K+ATPase is an ubiquitous enzyme, which is an integral part of the membranes. To find out whether a primary change in the activity of the Na+-K+ATPase occurs in this disorder, the activity of this enzyme, as well as, the intracellular sodium and potassium concentrations were studied in the erythrocytes of symptomless children with CD on a strict gluten-free diet. We examined the erythrocytes (EC) of 19 children with CD on a gluten free diet (mean age: 9.5 years, range: 4.6-15.6 years) and of 19 age matched controls. The Na+-K+ATPase activity was measured in detergent pretreated EC and in haemoglobin-free red blood cell membranes (i.e. ghosts). The sodium and potassium concentrations were determined in washed then haemolysed EC with emission spectrophotometry. The Na÷-K+ATPase activity was significantly lower in the detergent pretreated EC of patients (1860 ± 500 lamol PilL EC/hour, mean ± SD) than in controls (3181 ± 754) (p < 0.001) but in ghosts the activity of this enzyme was similar to that of the controls (359 ± 159 lamol Pi/g protein/hour vs 394 ± 96). The Na÷ concentration of EC was significantly higher in children with CD (7.52 ± 1.92 mmol/L) than in controls (6.09 - 1.69) (p < 0.02) The K+concentration of the EC was similar in the patients and controls (92.6 ± 7.0 vs. 91.5 ± 12.3). The increase of intracellular sodium concentration and the decreased Na÷-K+ATPase activity in the EC of children with CD raise the possibility that a primary disturbance of membrane functions exists in this disorder as the detected changes occured in symptomless patients on gluten free diet. It is possible that in CD an unidentified component is bound to the cell-surfaces membranes which inhibits the Na÷-K+ pump. This factor is probably loose-bound and not an integral part of the membrane because in ghosts the pump activity was not decreased.
G1425 ISCHAEMIC COLITIS IS NOT JUST A DISEASE OF ELDERLY ARTERIOPATHS. IDR Amott, S Ghosh, A Ferguson, G1 Unit, University Department of Medicine, Western General Hospital, Edinburgh, EH4 2XU. Background: Ischaemic colitis is generally considered a disease of the elderly with a background of cardiovascular disease. The "watershed" area around the splenic flexure is considered to be typically involved. Aim: We aimed to assess the influence that age, co-morbidity, and clinical presentation had on the type, severity and anatomy of ischaemic colitis and the proportion of patients in whom there was a specific clotting abnormality. Patients and Methods: We identified 24 patients (16 females, mean age 64) with ischaemic colitis. The case notes were obtained and, if still alive, they were invited to attend a follow up clinic where they were clinically reassessed, disease details verified and blood taken for a thrombophilia screen. Results: Five patients (21%) were below the age of 45 and 7 out of 24 were found to have died by the time of follow up. Four of these had died of ischaemic colitis during the acute episode. The 4 patients that died of ischaemic colitis had a more extensive and more severe type of the disease and presented with worse clinical features. The main predisposing factors were ischaemic heart disease in 12 (50%) and malignancy in 5 (21%). Unusually 6/24 cases (25%) had right sided lesions and this distribution conferred a good prognosis, 6 had disease around the splenic flexure, 8 has rectosigmoid disease and 4 had an extensive pattern. Shock, peritonism, extensive disease, uncontrolled atrial fibrillation and occurrence as an inpatient were all poor prognostic factors. Blood results showed that those with more extensive disease tended to have a lower haemoglobin (p=0.023). Clotting factor abnormalities could be detected in 3 out of 9 patients despite a time lapse between the assay and the episode of ischaemic colitis. Conclusion: Ischaemic colitis appears to have two patterns of severity, and the anatomical site of involvement is more variable than a developmental explanation of the vascular supply. A significant proportion of the patients may be young. Clotting abnormalities may be detected in a minority even on retrospective testing.
April 1998
G1422 TEMPORAL CHANGES IN INFLAMMATORY MEDIATOR LEVELS AND COLONIC VASCULAR ARCHITECTURE IN AN ANIMAL MODEL OF COLITIS. CB Appleyard, LJ Hanson & WH Percy, Department of Physiology & Pharmacology, School of Medicine, University of South Dakota, Vermillion, SD 57069.
• G1424 PROLONGED VS. TRANSIENT HISTONE HYPERACETYLATION: DIFFERENTIAL EFFECTS ON ENTEROCYTE GROWTH AND DIFFERENTIATION. S. Archer. J. Johnson, S. Meng, P. Grable, J. B. Matthews, R. A. Hodin. Dept. of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.
In a rabbit trlnitrubanzesulfonic acid (TNBS) model of colitis there are significant increases in blood flow to both the colonic muscularis proprla and mucosa/muscularls mucosae within 1 hr (Gastrn. 1997; 112: A345). The aim of this study was to investigate the possible temporal relationship between levels of vasoactive inflammatory mediators and changes in both number and diameter of colonic blood vessels in this model. METHODS: Colitis was induced in rabbits by intracvlonic administration of TNBS in 50% ethanol. Animals were euthanized 1, 6 and 48 hr after induction and the distal 10cm of colon removed and scored for macroscopic damage. The muscle layers were separated from the mucosa/muscularis mucosae before measurement of mediator levels by radioimmunoassay. Blood vessels were visualized using a modified NADPH-diaphorase stain and vessel diameter in both muscle and mucosa was measured. The number of mucosal vessels per mm2 was also counted. A scoring system of 0---~3 (normal--->extensive)for epithelial damage or TNBS penetration was used to determine the integrity of the mucosa and the extent of TNBS movement through the lamina propria. RESULTS: TNBS caused significant colonic damage at all time points compared to control (untreated) or ethanol vehicle. Mediator levels (where LTC4+=LTCfl)4/E~/F4) were:
As small intestinal epithelial ceils migrate up the crypt-villus axis, they express a differentiated phenotype and undergo cell cycle arrest. The molecular mechanisms responsible for these changes are poorly understood. It is known that an alteration in chromatin structure by histone hyperacetylation is associated with decreased cell growth and increased gene expression. Therefore, in these studies, we sought to examine the role of histone hyperacetylation in enterocyte differentiation and its associated growth arrest. METHODS: HT-29 cells were grown in standard media and treated with the histone hyperacetylating agents, sodium butyrate (NaBu, 5mM) or trichostatin A (TSA, 0.3 laM), both inhibitors of the histone deacetylase enzyme. Transient histone hyperacetylation was accomplished by treating cells with a single dose of TSA, a reversible inhibitor of histone deacetylase. Prolonged histone hyperacetylation was accomplished by treating cells with a single dose of NaBu or repeated doses of TSA (every 8 hrs.). Northern analyses were performed using cDNA probes specific for the enterocyte differentiation markers, intestinal alkaline phosphatase (lAP) and villin, the Na/K/2CL cotransporter (NKCC, which decreases in the differentiated phenotype), the cell cycle inhibitor, p21, and the actin control. 3H-thymidine incorporation assays were performed using standard protocol. Histone Acetvlation Assays: Nuclear histones from NaBu- or TSA-treated cells were extracted by acid/acetone precipitation, electrophoresed on an acid/urea/triton gel and stained with coomassie blue. RESULTS: Treatment of ceils with a single dose of TSA resulted in a rapid increase in the level of histone H4 acetylation by 0.5 hrs., with a peak by 4 hrs. and a return to the basal hypoacetylated state by 24 hrs. p21 mRNA levels paralleled these changes in histone hyperacetylation, peaking at 4 hrs. (15-fold increase, p < 0.001), but returning to basal levels at 24 hrs. These changes were associated with growth arrest (50% decrease in 3H-thymidine incorporation, p < 0.001), but no change in the IAP, villin, nor NKCC mRNAs. In dramatic contrast, prolonged histone H4 hyperacetylation was seen in response to either NaBu or repeated TSA treatment, beginning by 0.5 hrs. and remaining elevated up to 24 hrs. As with transient histone hyperacetylation, there was decreased cell growth (50% decrease in 3H-thymidine incorporation, p < 0.00l). However, there were now persistent increases in p21 mRNA expression and cellular differentiation was evidenced by increases in lAP and villin mRNAs (10- and 2.5 fold respectively, p < 0.001), and decreases in NKCC mRNA (10-fold, p < 0.001), at 24 hrs. CONCLUSIONS: In HT-29 cells, transient histone hyperacetylation is sufficient to induce growth arrest, but prolonged histone hyperacetylation is required for differentiation to occur.
Control TNBS Iht TNBS6hr TNBS48hr muscle TXB2 22.9±7.7 74.4± I4.3a 165.4±41.08 177.8± 48.11~ mucosa TXB2 24.8±4.9 205.9±43.58 284.7±45.38 240.3±68.98 muscle LTB4 315.9±49.4 220.6±67.1 449.7±114.5 651.4±162.2 mucosa LTB4 240.6±46.4 180.1±44.6 902.6±164.78 753.9±121.2I~ muscle LTC4+ 642.4± 148.3 382.3± 77.3 1095.9± 364.3 961.3± 136.4 mucosa LTC4+ 910.2±157.5 850.9±211.1 2377.1±623.8tt 1473.4±214.5 t~p< 0.05.I~p< 0.01,8p< 0.001comparedto control;pg/mgprotein± s.c.;n >6
El'OH48hr 32.9±12.2 72.0±27.3 481.4±198.1 500.8±146.3 495.1± 80.1 777.0±133.8
There were no significant differences in PGE2 levels in any layer at any time. A loss of mucosal integrity and an increase in TNBS penetration through the lamina propria occurred over time (2.4 ± 0.2, p < 0.01 and 2.2 ± 0.4, p < 0.05 respectively at 48hr post-TNBS, n=5). Neither of these parameters was temporally-related to mediator levels. There was no significant change from control in the diameter or number of blood vessels in either the muscle (longitudinally oriented vessels, diameter 2.9 ± 0.91am; circularly oriented vessels, diameter 24.0±0.0tam) or mucosal layer (vessel diameter 1.7±0.11am & 144.2±24.0 vessels/ram2, each 20 observations from 2 rabbits). CONCLUSIONS: 1. TNBS-induced colonic damage is accompanied by time-dependent changes in tissue levels of inflammatory mediators. 2. Changes in mediator levels occur as a result of the inflammatory process and not damage p e r se as TNBS penetration continues to increase after mediator levels have peaked. 3. Acute changes in blood flow occur independently of these inflammatory mediator levels and are not associated with altered vascular architecture. Supported by the Crohn's and Colitis Foundation of America & DK 49803. • G1423 IMPAIRED NA÷-K÷ PUMP ACTIVITY IN THE ERYTHROCYTES OF CHILDREN WITH TREATED COELIAC DISEASE. A. Arat6. M. Dobos, B. V~is~helyi, H. Bod~nszky, T. Tulassay. First Department of Paediatrics, Semmelweis University of Medicine, Budapest, Hungary
Inherited or acquired changes in the epithelial cell-surface membranes may contribute to the pathogenesis of coeliac disease (CD). The Na+-K+ATPase is an ubiquitous enzyme, which is an integral part of the membranes. To find out whether a primary change in the activity of the Na+-K+ATPase occurs in this disorder, the activity of this enzyme, as well as, the intracellular sodium and potassium concentrations were studied in the erythrocytes of symptomless children with CD on a strict gluten-free diet. We examined the erythrocytes (EC) of 19 children with CD on a gluten free diet (mean age: 9.5 years, range: 4.6-15.6 years) and of 19 age matched controls. The Na+-K+ATPase activity was measured in detergent pretreated EC and in haemoglobin-free red blood cell membranes (i.e. ghosts). The sodium and potassium concentrations were determined in washed then haemolysed EC with emission spectrophotometry. The Na÷-K+ATPase activity was significantly lower in the detergent pretreated EC of patients (1860 ± 500 lamol PilL EC/hour, mean ± SD) than in controls (3181 ± 754) (p < 0.001) but in ghosts the activity of this enzyme was similar to that of the controls (359 ± 159 lamol Pi/g protein/hour vs 394 ± 96). The Na÷ concentration of EC was significantly higher in children with CD (7.52 ± 1.92 mmol/L) than in controls (6.09 - 1.69) (p < 0.02) The K+concentration of the EC was similar in the patients and controls (92.6 ± 7.0 vs. 91.5 ± 12.3). The increase of intracellular sodium concentration and the decreased Na÷-K+ATPase activity in the EC of children with CD raise the possibility that a primary disturbance of membrane functions exists in this disorder as the detected changes occured in symptomless patients on gluten free diet. It is possible that in CD an unidentified component is bound to the cell-surfaces membranes which inhibits the Na÷-K+ pump. This factor is probably loose-bound and not an integral part of the membrane because in ghosts the pump activity was not decreased.
G1425 ISCHAEMIC COLITIS IS NOT JUST A DISEASE OF ELDERLY ARTERIOPATHS. IDR Amott, S Ghosh, A Ferguson, G1 Unit, University Department of Medicine, Western General Hospital, Edinburgh, EH4 2XU. Background: Ischaemic colitis is generally considered a disease of the elderly with a background of cardiovascular disease. The "watershed" area around the splenic flexure is considered to be typically involved. Aim: We aimed to assess the influence that age, co-morbidity, and clinical presentation had on the type, severity and anatomy of ischaemic colitis and the proportion of patients in whom there was a specific clotting abnormality. Patients and Methods: We identified 24 patients (16 females, mean age 64) with ischaemic colitis. The case notes were obtained and, if still alive, they were invited to attend a follow up clinic where they were clinically reassessed, disease details verified and blood taken for a thrombophilia screen. Results: Five patients (21%) were below the age of 45 and 7 out of 24 were found to have died by the time of follow up. Four of these had died of ischaemic colitis during the acute episode. The 4 patients that died of ischaemic colitis had a more extensive and more severe type of the disease and presented with worse clinical features. The main predisposing factors were ischaemic heart disease in 12 (50%) and malignancy in 5 (21%). Unusually 6/24 cases (25%) had right sided lesions and this distribution conferred a good prognosis, 6 had disease around the splenic flexure, 8 has rectosigmoid disease and 4 had an extensive pattern. Shock, peritonism, extensive disease, uncontrolled atrial fibrillation and occurrence as an inpatient were all poor prognostic factors. Blood results showed that those with more extensive disease tended to have a lower haemoglobin (p=0.023). Clotting factor abnormalities could be detected in 3 out of 9 patients despite a time lapse between the assay and the episode of ischaemic colitis. Conclusion: Ischaemic colitis appears to have two patterns of severity, and the anatomical site of involvement is more variable than a developmental explanation of the vascular supply. A significant proportion of the patients may be young. Clotting abnormalities may be detected in a minority even on retrospective testing.