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Is “refractory” coeliac disease a homogeneous entity?
AGAA1l33
April 2000
5220 DOWN REGULATED IN ADENOMA (DRA) DOES NOT ENCODE THE MAJOR COMPONENT OF CL-HCO) EXCHANGE IN RAT DISTAL COLON. Vazhaikkurichi M. Rajendran, Henry J. Binder, Yale Univ, New Haven, CT. Congenital chloride diarrhea (chloridorrhea) is due to a defect in colonic and ileal CI-HC0 3 exchange and recently has been shown to be secondary to an alteration in DRA gene. In vitro expression of DRA cDNA in HEK cells revealed CI-HC0 3 exchange. In rat distal colon Na-dependent CI absorption is due to an apical membrane CI-HC0 3 exchange while anion exchange (AE) isoforms AEI and AE2 specific mRNA are expressed in colonic epithelial cells. We have reported that Na-depletion substantially inhibits both Na-dependent CI absorption (73%) and CI-HC0 3 exchange (67%). Thus, we predicted that the mRNA that encodes the CI-HC0 3 exchange in rat distal colon should be reduced in Na-depleted animals. Therefore, DRA, AEI and AE2 specific mRNA abundance were determined in normal and Na-depleted rats. DRA and AE2 mRNA abundances were not altered in Na-depleted animals. In contrast, AEI mRNA abundance was reduced by 86%. We conclude that in the rat distal colon AEI mRNA encodes the major component of CI-HC0 3 exchange (i.e. aldosterone-sensitive component) and that DRA only encodes a small fraction of CI-HC0 3 exchange (i.e. the aldosterone-insensitive fraction). Although evidence exists that DRA encodes CI-HC0 3 exchange in human colon, not all colonic apical membrane CI-HC0 3 exchanges are encoded by DRA and the relative contribution of DRA and AEI may be species and tissue specific.
5221 DELTA OPIOID RECEPTORS ON ENTEROCYTES OF MURINE SMALL INTESTINE. Rhoda A. Reddix, Joseph Chiban, LSU Health Sci Ctr, New Orleans, LA. It has been shown that delta opioid receptors are the primary opioid receptors in human intestinal mucosal epithelia. These receptors have also been well characterized in rabbit,rat and guinea pig intestine. However, very little is known concerning the opioid receptor subtype mediating the proabsorptive effects of endogenous opioids in murine intestine. Previous results from our laboratory and those of others have shown that the delta opioid receptor analog, [D-Pen 2,D-Pen s]enkephalin (DPDPE,lOuM) evoked a dose-dependent increase in ion absorption in murine small intestine. To determine whether the proabsorptive effects were paritally mediated by epithelial delta opioid receptors, enterocytes were isolated from the small intestine of BALB/C mice. The cells (106 cells/ml) were subjected to flow cytometry to monitor the displacement of naloxone-FITC by DPDPE as an index of delta opioid receptor binding. As a positive control for these studies, a concentration-response curve for naltrexoneFITC was generated in NG I08 neuroblastoma cells, in the absence and presence of 10uM DPDPE. All data were expressed as the percent reduction in the maximal percentage of cells binding either naltrexone-FITC or naloxone-FITC. Pretreatment with 10uM DPDPE attenuated O.luM, luM and 10 uM naltrexone-FITC flourescence by 93.5, 90.8, 78.1%, respectively. Similary, IuM DPDPE reduced 0.1 nM naloxone-FITC flourescence by 38.5% in enterocytes (10 6 cells/ml) isolated from murine small intestine. This is the first to demonstrate delta opioid receptor binding in mucosal epithelium of murine small intestine. Hence, the murine small intestine may by used as a model to study delta opioid receptor function under physiological and pathophysiological conditions. Supported by a LSU Health Sciences Center Bridge Grant.
5222 IS "REFRACTORY" COELIAC DISEASE A HOMOGENEOUS ENTITY? Christopher Rodgriues, P. G. Isaacson, Parveen J. Kumar, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom; Royal Free and Univ Coli Med Sch, London, United Kingdom. "Refractory" coeliac disease has a dreadful prognosis and treatment is unhelpful. 5 patients with "refractory" coeliac disease, defined as subtotal villous atrophy unresponsive to a strict gluten free diet, (4 F, age range 35 - 68 yrs) presented with steatorrhoea or diarrhoea, weight loss, abdominal pain, vomiting, ankle swelling and lethargy. All were severely malnourished and had hypoalbuminaemia, anaemia and hypocalcaemia Small bowel mucosa showed subtotal villous atrophy with crypt hyperplasia, and an increase in intraepithelial lymphocytes (IELS)but not the hypoplastic Marsh Type 4 lesion. 3 patients also developed small bowel ulceration: I had multiple ulcers in the duodenum and proximal jejunum, 1 developed ulcerative jejunitis with multiple strictures and the third extensive ulcerative jejunoileitis. IgA endomysial antibodies were absent in 4 patients. Contrast radiology and CT scans showed small bowel dilatation with wall thickening, effacement of the fold pattern and prolonged orocaecal transit. A scalloped mucosal pattern and/or multiple jejunal ulcers were seen in 3 patients in whom enteroscopy was carried out. Immunohistochemistry and molecular genetic analysis of affected small bowel was carried out in 2 patients. One showed that 44% of the intraepithelial lymphocytes (IELS) were CD3 +, CD8- but PCR for TCR gamma showed a polyclonal pattern. The second had normal IEL phenotype i.e. CD3 +, CD8 + but PCR showed 2 distinct dominant bands. The latter patient later developed a lymphoma. All patients received steroids, 4 Azathioprine, 3 an elemental diet, 2 TPN
and 2 antibiotics for small bowel bacterial overgrowth. I patient had a diagnostic laparotomy for suspected lymphoma and 2 underwent surgery for strictures/perforations. Two patients died from pulmonary and small bowel T-cell lymphomas 12 and 10 yrs respectively, after the onset of disease. Two patients remain severely malnourished with chronic diarrhoea but I is asymptomatic, with persistent villous atrophy. Patients with subtotal villus atrophy refractory to a gluten-free diet are a heterogeneous group and do not have classical coeliac disease. Patients have an inevitable downhill progression and most develop T cell lymphoma.
5223 CHANGES IN THE EXPRESSION OF INTESTINAL PROTEINS DURING EXPERIMENTAL STRESS. Ricardo Rodriguez, Esteban Saperas, Francisco Guarner, Juan-Ramon Malagelada, Digest System Research Unit Hosp Gen Vall d'Hebron, Barcelona, Spain. The molecular mechanisms involved in the responses elicited by stress in the gut are poorly understood. Stress induces changes in gene transcription in various tissues and these changes facilitate adaptative phenotypic alterations. Gene regulation is accomplished by different mechanisms that induce or repress specific genes. AIM: To investigate the effect of acute and chronic stress on protein expression and to elucidate the nuclear transcriptional factors involved. METHODS: Groups of male SpragueDawley rats (n =6) were submitted to water avoidance stress or sham stress for I hour, for I (acute) or 5 (chronic) consecutive days.Fecal pellet output and body weight were monitored daily. Trunk blood was collected 6h after I or 5-day stress for measurement of plasma corticosterone levels, and segments of jejunum were removed and homogenized. Cytosolic proteins were separated by high resolution two-dimensional polyacrilamide gels (2D-PAGE) (pI: 4.0-8.5, MW: 1O-120KD). Nuclear transcription factors SPI, API, AP2, NFkappaB, OCT-I and CREB were measured by EMSA from crude nuclear fractions. RESULTS: Acute and chronic stress induced colorectal and adrenal activation and slowed body weight gain. About 1000 spots on average were recognized in each gel. Acute and chronic stress dramatically changed the pattern of protein expression: 37% of jejunal protein in stress could not be matched with sham. In acute stress gels, 23% of proteins expressed in sham were not observed and 14% were newly synthetized or overexpressed. In contrast, in 5-day chronic stress 15% of proteins in sham were not observed whereas a 22% were newly synthetized. Comparison between acute and chronic gels demonstrated different protein pattern. Parallel to these differences on protein expression, nuclear transcription factors were also modified differentially. Acute stress enhanced levels of nuclear NFkappaB while chronic stress decreased the content of this nuclear factor. CREB levels were only lightly increased by chronic stress, whereas API factor was lightly enhanced by both the acute and chronic stress. SPI, AP2 and OCT-I factors were unchanged. CONCLUSION: Changes in the activation of nuclear transcription factors occur in response to stress but a different pattern in gut gene expression occurs depending on the duration of the stress stimulus.
5224 PATHOPHYSIOLOGY OF CANINE NON-SPECIFIC DIETARY SENSITIVITY. Vivien E. Rolfe, Colleen A. Adams, Virpi V. Smith, Richard M. Butterwick, Roger M. Batt, Waltham Ctr for Pet Nutrition, Leicestershire, United Kingdom; Great Ormond Street Hosp, London, United Kingdom. Introduction Dogs which produce poor faecal consistency when fed certain diets are described to have non-specific dietary sensitivity (NSDS), but the underlying pathophysiological mechanisms are unknown. We investigated whether there were colonic structural and absorptive abnormalities in NSDS dogs. Methods A group of NSDS dogs (n=12) and control dogs (n = 10) of various breed, age and sex were compared during a 28 day trial. Colonic samples were subjected to histological analysis by an independent histopathologist. Colonic electrolyte absorption was measured in vivo by inserting dialysis bags (Kreb's saline, 6 mls) into the distal colon for 30 minutes. Colonic crypt water transport was measured in vitro using digital confocal microscopy by incubating a mucosal sample in Kreb's saline containing fluorescein isothiocyanate dextran (FITC, mol. wt. 9000, I roM) at 37 degrees. Results The histological appearance of colonic mucosa in control dogs was healthy, while in NSDS dogs the mucosa displayed a Iymphocytic-plasmacytic infiltrate with crypt disruption. Na+ absorption in vivo was significantly reduced (p<0.05, ANOVA) in NSDS dogs (-39.2 :t 6.4 mM.hr) compared to controls (-52.3 :t 6.76 mM.hr), as was CI- (NSDS -48.2 :t 4.64 versus controls -59.8 :t 7.1 mM.hr, p<0.05). In NSDS dogs the FITC fluorescence intensity within the crypt lumen was reduced compared to control dogs (control 62.6 :t 7.5 versus NSDS 27.4 :t 3.6 Ll intensity.30 mins, p<0.05 ANOVA), indicating a reduction in crypt water uptake. Conclusions Canine NSDS is associated with a mild Iymphocytic-plasmacytic infiltrate. These microscopic structural changes may account for the reduction in colonic water and electrolyte absorptive function, which in turn leads to diarrhoea.
April 2000
5220 DOWN REGULATED IN ADENOMA (DRA) DOES NOT ENCODE THE MAJOR COMPONENT OF CL-HCO) EXCHANGE IN RAT DISTAL COLON. Vazhaikkurichi M. Rajendran, Henry J. Binder, Yale Univ, New Haven, CT. Congenital chloride diarrhea (chloridorrhea) is due to a defect in colonic and ileal CI-HC0 3 exchange and recently has been shown to be secondary to an alteration in DRA gene. In vitro expression of DRA cDNA in HEK cells revealed CI-HC0 3 exchange. In rat distal colon Na-dependent CI absorption is due to an apical membrane CI-HC0 3 exchange while anion exchange (AE) isoforms AEI and AE2 specific mRNA are expressed in colonic epithelial cells. We have reported that Na-depletion substantially inhibits both Na-dependent CI absorption (73%) and CI-HC0 3 exchange (67%). Thus, we predicted that the mRNA that encodes the CI-HC0 3 exchange in rat distal colon should be reduced in Na-depleted animals. Therefore, DRA, AEI and AE2 specific mRNA abundance were determined in normal and Na-depleted rats. DRA and AE2 mRNA abundances were not altered in Na-depleted animals. In contrast, AEI mRNA abundance was reduced by 86%. We conclude that in the rat distal colon AEI mRNA encodes the major component of CI-HC0 3 exchange (i.e. aldosterone-sensitive component) and that DRA only encodes a small fraction of CI-HC0 3 exchange (i.e. the aldosterone-insensitive fraction). Although evidence exists that DRA encodes CI-HC0 3 exchange in human colon, not all colonic apical membrane CI-HC0 3 exchanges are encoded by DRA and the relative contribution of DRA and AEI may be species and tissue specific.
5221 DELTA OPIOID RECEPTORS ON ENTEROCYTES OF MURINE SMALL INTESTINE. Rhoda A. Reddix, Joseph Chiban, LSU Health Sci Ctr, New Orleans, LA. It has been shown that delta opioid receptors are the primary opioid receptors in human intestinal mucosal epithelia. These receptors have also been well characterized in rabbit,rat and guinea pig intestine. However, very little is known concerning the opioid receptor subtype mediating the proabsorptive effects of endogenous opioids in murine intestine. Previous results from our laboratory and those of others have shown that the delta opioid receptor analog, [D-Pen 2,D-Pen s]enkephalin (DPDPE,lOuM) evoked a dose-dependent increase in ion absorption in murine small intestine. To determine whether the proabsorptive effects were paritally mediated by epithelial delta opioid receptors, enterocytes were isolated from the small intestine of BALB/C mice. The cells (106 cells/ml) were subjected to flow cytometry to monitor the displacement of naloxone-FITC by DPDPE as an index of delta opioid receptor binding. As a positive control for these studies, a concentration-response curve for naltrexoneFITC was generated in NG I08 neuroblastoma cells, in the absence and presence of 10uM DPDPE. All data were expressed as the percent reduction in the maximal percentage of cells binding either naltrexone-FITC or naloxone-FITC. Pretreatment with 10uM DPDPE attenuated O.luM, luM and 10 uM naltrexone-FITC flourescence by 93.5, 90.8, 78.1%, respectively. Similary, IuM DPDPE reduced 0.1 nM naloxone-FITC flourescence by 38.5% in enterocytes (10 6 cells/ml) isolated from murine small intestine. This is the first to demonstrate delta opioid receptor binding in mucosal epithelium of murine small intestine. Hence, the murine small intestine may by used as a model to study delta opioid receptor function under physiological and pathophysiological conditions. Supported by a LSU Health Sciences Center Bridge Grant.
5222 IS "REFRACTORY" COELIAC DISEASE A HOMOGENEOUS ENTITY? Christopher Rodgriues, P. G. Isaacson, Parveen J. Kumar, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom; Royal Free and Univ Coli Med Sch, London, United Kingdom. "Refractory" coeliac disease has a dreadful prognosis and treatment is unhelpful. 5 patients with "refractory" coeliac disease, defined as subtotal villous atrophy unresponsive to a strict gluten free diet, (4 F, age range 35 - 68 yrs) presented with steatorrhoea or diarrhoea, weight loss, abdominal pain, vomiting, ankle swelling and lethargy. All were severely malnourished and had hypoalbuminaemia, anaemia and hypocalcaemia Small bowel mucosa showed subtotal villous atrophy with crypt hyperplasia, and an increase in intraepithelial lymphocytes (IELS)but not the hypoplastic Marsh Type 4 lesion. 3 patients also developed small bowel ulceration: I had multiple ulcers in the duodenum and proximal jejunum, 1 developed ulcerative jejunitis with multiple strictures and the third extensive ulcerative jejunoileitis. IgA endomysial antibodies were absent in 4 patients. Contrast radiology and CT scans showed small bowel dilatation with wall thickening, effacement of the fold pattern and prolonged orocaecal transit. A scalloped mucosal pattern and/or multiple jejunal ulcers were seen in 3 patients in whom enteroscopy was carried out. Immunohistochemistry and molecular genetic analysis of affected small bowel was carried out in 2 patients. One showed that 44% of the intraepithelial lymphocytes (IELS) were CD3 +, CD8- but PCR for TCR gamma showed a polyclonal pattern. The second had normal IEL phenotype i.e. CD3 +, CD8 + but PCR showed 2 distinct dominant bands. The latter patient later developed a lymphoma. All patients received steroids, 4 Azathioprine, 3 an elemental diet, 2 TPN
and 2 antibiotics for small bowel bacterial overgrowth. I patient had a diagnostic laparotomy for suspected lymphoma and 2 underwent surgery for strictures/perforations. Two patients died from pulmonary and small bowel T-cell lymphomas 12 and 10 yrs respectively, after the onset of disease. Two patients remain severely malnourished with chronic diarrhoea but I is asymptomatic, with persistent villous atrophy. Patients with subtotal villus atrophy refractory to a gluten-free diet are a heterogeneous group and do not have classical coeliac disease. Patients have an inevitable downhill progression and most develop T cell lymphoma.
5223 CHANGES IN THE EXPRESSION OF INTESTINAL PROTEINS DURING EXPERIMENTAL STRESS. Ricardo Rodriguez, Esteban Saperas, Francisco Guarner, Juan-Ramon Malagelada, Digest System Research Unit Hosp Gen Vall d'Hebron, Barcelona, Spain. The molecular mechanisms involved in the responses elicited by stress in the gut are poorly understood. Stress induces changes in gene transcription in various tissues and these changes facilitate adaptative phenotypic alterations. Gene regulation is accomplished by different mechanisms that induce or repress specific genes. AIM: To investigate the effect of acute and chronic stress on protein expression and to elucidate the nuclear transcriptional factors involved. METHODS: Groups of male SpragueDawley rats (n =6) were submitted to water avoidance stress or sham stress for I hour, for I (acute) or 5 (chronic) consecutive days.Fecal pellet output and body weight were monitored daily. Trunk blood was collected 6h after I or 5-day stress for measurement of plasma corticosterone levels, and segments of jejunum were removed and homogenized. Cytosolic proteins were separated by high resolution two-dimensional polyacrilamide gels (2D-PAGE) (pI: 4.0-8.5, MW: 1O-120KD). Nuclear transcription factors SPI, API, AP2, NFkappaB, OCT-I and CREB were measured by EMSA from crude nuclear fractions. RESULTS: Acute and chronic stress induced colorectal and adrenal activation and slowed body weight gain. About 1000 spots on average were recognized in each gel. Acute and chronic stress dramatically changed the pattern of protein expression: 37% of jejunal protein in stress could not be matched with sham. In acute stress gels, 23% of proteins expressed in sham were not observed and 14% were newly synthetized or overexpressed. In contrast, in 5-day chronic stress 15% of proteins in sham were not observed whereas a 22% were newly synthetized. Comparison between acute and chronic gels demonstrated different protein pattern. Parallel to these differences on protein expression, nuclear transcription factors were also modified differentially. Acute stress enhanced levels of nuclear NFkappaB while chronic stress decreased the content of this nuclear factor. CREB levels were only lightly increased by chronic stress, whereas API factor was lightly enhanced by both the acute and chronic stress. SPI, AP2 and OCT-I factors were unchanged. CONCLUSION: Changes in the activation of nuclear transcription factors occur in response to stress but a different pattern in gut gene expression occurs depending on the duration of the stress stimulus.
5224 PATHOPHYSIOLOGY OF CANINE NON-SPECIFIC DIETARY SENSITIVITY. Vivien E. Rolfe, Colleen A. Adams, Virpi V. Smith, Richard M. Butterwick, Roger M. Batt, Waltham Ctr for Pet Nutrition, Leicestershire, United Kingdom; Great Ormond Street Hosp, London, United Kingdom. Introduction Dogs which produce poor faecal consistency when fed certain diets are described to have non-specific dietary sensitivity (NSDS), but the underlying pathophysiological mechanisms are unknown. We investigated whether there were colonic structural and absorptive abnormalities in NSDS dogs. Methods A group of NSDS dogs (n=12) and control dogs (n = 10) of various breed, age and sex were compared during a 28 day trial. Colonic samples were subjected to histological analysis by an independent histopathologist. Colonic electrolyte absorption was measured in vivo by inserting dialysis bags (Kreb's saline, 6 mls) into the distal colon for 30 minutes. Colonic crypt water transport was measured in vitro using digital confocal microscopy by incubating a mucosal sample in Kreb's saline containing fluorescein isothiocyanate dextran (FITC, mol. wt. 9000, I roM) at 37 degrees. Results The histological appearance of colonic mucosa in control dogs was healthy, while in NSDS dogs the mucosa displayed a Iymphocytic-plasmacytic infiltrate with crypt disruption. Na+ absorption in vivo was significantly reduced (p<0.05, ANOVA) in NSDS dogs (-39.2 :t 6.4 mM.hr) compared to controls (-52.3 :t 6.76 mM.hr), as was CI- (NSDS -48.2 :t 4.64 versus controls -59.8 :t 7.1 mM.hr, p<0.05). In NSDS dogs the FITC fluorescence intensity within the crypt lumen was reduced compared to control dogs (control 62.6 :t 7.5 versus NSDS 27.4 :t 3.6 Ll intensity.30 mins, p<0.05 ANOVA), indicating a reduction in crypt water uptake. Conclusions Canine NSDS is associated with a mild Iymphocytic-plasmacytic infiltrate. These microscopic structural changes may account for the reduction in colonic water and electrolyte absorptive function, which in turn leads to diarrhoea.