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Eosinophilic esophagitis: Is histology specific for the clinico-pathological syndrome
GASTROENTEROLOGY Vol. 114, No. 4
A62 AGA ABSTRACTS G0249
EOSINOPHILIC ESOPHAGITIS: IS HISTOLOGY SPECIFIC F O R THE CLINICO-PATHOLOGICAL SYNDROME? Ziad T. Awad, Thomas C. Smyrk, Robert E. Marsh, Tetsuya Tomonaga, Yutaka Shiino, Charles J. Filipi. Creighton University School of Medicine, Omaha, NE INTRODUCTION: Since intraepithelial eosinophils (IEE) are a common finding in reflux esophagitis, and may be quite common in some cases, it is usually said that the diagnosis of eosinophilic esophagitis requires a combination of clinical and pathological features. In contrast, we hypothesized that the triad of IEE, spongiosis, and a thickened basal zone forms a unique histologic picture that allows identification of eosinophilic esophagitis without reference to the clinical feature. METHOD: We performed a retrospective review to test this hypothesis. The study set included a consecutive series of 200 patients who had endoscopy, biopsy, and 24 hour pH monitoring, and 18 patients listed in the computerized pathology files with a diagnosis of eosinophilic esophagitis, Biopsies were reviewed and graded without knowledge of clinical findings, lEE were quantified as IEE/high power microscopic field. Spongiosis was graded 0-4 semiquantitively as follows: 0- no intercellular edema; 1- slight separation of cells with prominence of intercellular bridges; 2- diffuse, obvious separation with intercellular bridges intact; 3- separation of cells with focal disruption of intercellular bridges; 4- extensive disruption of intercellular bridges with formation of intraepithelial lakes. Basal zone thickness was evaluated in welloriented areas and was expressed as the percentage of total epithelial thickness occupied by the cells whose nuclei were separated by less than one nuclear diameter. Biopsies with > 20 IEE/hpf and spongiosis > grade 2 and basal zone thickness > 20% was classified as eosinophilic esophagitis. RESULT: Biopsies from 33 patients fulfilled all 3 criteria. There were 27 males and 6 females. The average age was 30.8 years (range 11-53). 29 patients presented with a chief complaint of dysphagia (88%), 3 patients complained of heartburn (9%), and 1 patient of chest pain (3%). The average duration of symptom was 69 months (range 1-360). The 24 hour pH monitoring was abnormal in 13 patients (39.3%); 9=positive alkaline reflux (pH > 15.1), l=positive acid reflux (pH > 14.8), and 3=positive for both acid and alkaline reflux. 8 patients (24%) had stricture (esophageal diameter less than 12mm) as diagnosed by esophagram and endoscopy; of those patients, 5 had alkaline reflux, 1 had both acid and alkaline reflux, 1 was normal, and 1 was not tested. CONCLUSION: The triad of lEE, spongiosis, and a thickened basal zone is a specific histologic marker for the chief complaint of dysphagia (88%). Contrary to our expectation, the histologic triad did not select for normal endoscopy and normal 24 hour pH monitoring, since stricture (24%) and alkaline reflux (36% of patients tested) were both common. It may be more than one condition can produce the histology described here. Alternatively, it may be that undetected alkaline reflux is responsible for the clinicopathologic syndrome of eosinophilic esophagitis with dysphagia. • G0250
ACUTE AND CHRONIC STRESS-INDUCED OXIDATIVE G A S T R O I N T E S T I N A L MUCOSAL INJURY IN RATS, AND PROTECTION BY BISMUTH SUBSALICYLATE (BSS). D. Bagchi, C.B. Williams, M. Milnes, R.L. Krohn, M. Bagchi, J. Balmoori, X. Ye, M.X. Tran, S.J. Stohs, Creighton University School of Pharmacy & Allied Health Professions, Omaha, NE 68178; O.R. Carryl, S. Mitra, Health Care Research Center, The Procter & Gamble Company, Mason, OH 45040. Reactive oxygen species (ROS) are implicated in the pathogenesis of stressinduced gastrointestinal (GI) injury. In the present study, we have investigated the effects of acute and chronic stress on the enhanced production of ROS including superoxide anion [SA; as determined by cytochrome c reduction assay (CCR)] and hydroxyl radicals (OH), and correlated the enhanced production of these free radicals with increased DNA fragmentation (DF), lipid peroxidation (LP) and membrane microviscosity (MV), indices of oxidative membrane and DNA damage, in the gastric (GM) and intestinal mucosa (IM) of female Sprague-Dawley rats. Furthermore, we determined the protective ability of BSS against the GI mucosai injury induced by acute and chronic stress. Acute stress was induced by waterimmersion for 90 min, while chronic stress was induced by water-immersion for 15 rain/day for 15 consecutive days. Half of the animals exposed to waterimmersion acute stress were pretreated orally with 15 mg BSS/kg 30 min prior to water-immersion. Similarly, half of the animals exposed to waterimmersion chronic stress were pretreated orally with 7.5 nag BSS/kg/day for 15 consecutive days 30 rain prior to water-immersion. Results: Acute stress produced maximal injury to both GM and IM as compared to chronic stress. Acute stress increased CCR and OH production by 10.0- and 14.3-fold, respectively, in the GM, and 10.4- and 17.0-fold in the IM. Pretreatment with BSS prevented the acute stress-induced increase in CCR and OH production. Acute stress increased LP, DF and MV by 3.6-, 4.0- and ll.6-fold respectively, in the GM, and 4.1-, 5.0- and 16.2-fold increases in IM. BSS decreased acute stress-induced LP, DF and MV by approx. 26%, 35% and 30%, respectively, in the GM, and by 20%, 36% and 30%, respectively, in the IM. BSS administration provided significant protection against chronic stressinduced nose bleeding in rats. Chronic stress increased CCR and OH production by 4.8- and 6.3-fold, respectively, in the GM, and 4.6- and 6.9fold in the IM. Chronic stress increased LP and DF by 2.9- and 3.3-fold, respectively, in the GM, and 3.3- and 4.2-fold increases in the IM. Daily
administration of BSS provided greater protection against chronic stressinduced oxidative gastrointestinal injury as compared to the acute stress. Conclusion: These results demonstrate that acute and chronic stress can induce gastrointestinal oxidative stress and mucosal injury through enhanced production of ROS, and that BSS can significantly protect against GI injury by scavenging these ROS. This research was funded by the Procter & Gamble Company, Mason, OH G0251 MECHANISM OF GASTROPROTECTION BY BISMUTH SUBSALICYLATE AGAINST CHEMICALLY-INDUCED OXIDATIVE INJURY IN HUMAN GASTRIC MUCOSAL CELLS. D. Bagchi, T.R. McGinn, X. Ye, J. Balmoori, M. Bagchi, S.J. Stohs, Creighton University Health Sciences Center, Omaha, NE; C. Kuszynski, University of Nebraska Medical Center, Omaha, NE; O.R. Carryl, S. Mitra, The Procter & Gamble Company, Mason, OH. Oxygen free radicals (OFR) are implicated in the pathogenesis of chemicallyinduced gastric mucosal injury. We have investigated the effects of ethanol (EtOH), hydrochloric acid (HCI) and sodium hydroxide (NaOH) on the (i) enhanced production of OFR including superoxide anion and hydroxyl radicals (OH), (ii) modulation of intracellular oxidized states by laser scanning confocal microscopy (LSMC) and (iii) DNA fragmentation (DNAF), indices of oxidative tissue and DNA damage in gastric mucosal cells (GC). GC from Helicobacter pylori-negative endoscopic biopsies were isolated and cultured from human subjects. The induction of OFR and DNA damage in GC following exposure to EtOH (15%), HC1 (150 raM) and NaOH (150 mM) were assessed by cytochrorne c reduction (superoxide anion production), OH production, changes in intracellular oxidized states by LSCM and DNA-F. Furthermore, the protective ability of BSS was assessed at the concentrations of 25, 50 and 100 mg/liter. Incubation of GC with EtOH, HC1 and NaOH increased superoxide anion production by approx. 8.0-, 6.1- and 7.1-fold, respectively, and increased OH production by 13.3-, 9.6- and 8.9-fold, respectively, as compared to untreated GC. Incubation of GC with EtOH, HCI and NaOH increased DNA-F by approx. 6.7-, 4.3- and 4.8-fold, respectively. Approx. 20.3-, 17.5- and 13.1-fold increases in fluorescence intensities were observed following incubation of GC with EtOH, HC1 and NaOH, respectively, demonstrating dramatic changes in the intracellular oxidized states of GC following exposure to these necrotizing agaents. Preincubation of GC with 25, 50 and 100 mg/liter of BSS decreased EtOH-induced increases in intracellular oxidized states in GC by 36%, 56% and 66%, respectively, demonstrating a concentration-dependent protective ability by BSS. Similar results were observed with respect to BSS in terms of superoxide anion production, OH production and DNA damage. The present study demonstrates that EtOH, HCI and NaOH induce oxidative stress and DNA damage in GC, and BSS can significantly attenuate gastric injury by scavenging the OFR. This research was funded by the Procter & Gamble Company, Mason, OH. G0252
TRANSIENT ABERRANT PHOSPHORYLATION AND NUCLEAR LOCALISATION OF CATENINS EARLY IN T H E P R O G R E S S I O N OF BARRETT'S NEOPLASIA. T.A. Bailey 2, L.R. Biddlestone 2, C.E. May 3, R. Bruton 1, D. R. Grand 1, D.S.A. Sanders l, H. Barr2, N.A. Shepherd 2, P.L Warner3, LA.Z. Jankowski I. qnstitute for Cancer Studies University of Birmingham, Birmingham, B15 2TJ; 2Gloucester Gastroenterology Group & Cranfield Medical Institute, Gloucester, GL1 3NN Barrett's dysplasia and its related cancer arises in the oesophagus as a result of chronic gastro-esophageal reflux which transforms stratified squamous epithelium to mucin secreting epithelium termed Barrett's mucosa. Understanding the sequence of molecular events may aid future management of this disease. Reductions in the cell adhesion molecule E-cadherin have been noted to occur in severely dysplastic and neoplastic tissue. Consequently, we investigated the related adhesion molecules; [3 and T catenins which bind to cadherins and desmosomes and modulate adhesion and the APC gene product which regulates the intracellular catenin levels. In the majority of low grade dysplastic tissue we confirm that there APC downregulation occurs (n = 17, p < 0.05). As the disease progresses to high grade dysplasia a significant proportion have 13 or y catenins accumulating in the nucleus ( > 50% of cases with high grade dysplasia, p < 0.05) and were hyperphosphorylated on tyrosine residues (n = 10, p<0.05). This phenomenon of nuclear localisation and hyperphosphorylation of catenins is transient as it decreases in adjacent invasive cancers (n = 30, p < 0.05). We found no structural alterations in the catenin genes which is consistent with this phenomenon. 13 catenin was unable to co-immunoprecipitate E-cadherin and 7 catenin was unable to co-immunoprecipitate desmogleins (desmosomespecific cadherins), in dysplasia, even when they were co-expressed (n = 10, p < 0.05). As a result E-cadherirgcatenin and desmoglein/catenin complexes are decreased, especially in high grade dysplasia (n = 7, p < 0.05). These results suggest that APC downregulation and hyperphosphorylation of catenins by increased kinase activity occur in high grade dysplasia. In addition the transient nuclear localisation of catenins may have implications for transcriptional regulation early in Barrett's neoplasia.
A62 AGA ABSTRACTS G0249
EOSINOPHILIC ESOPHAGITIS: IS HISTOLOGY SPECIFIC F O R THE CLINICO-PATHOLOGICAL SYNDROME? Ziad T. Awad, Thomas C. Smyrk, Robert E. Marsh, Tetsuya Tomonaga, Yutaka Shiino, Charles J. Filipi. Creighton University School of Medicine, Omaha, NE INTRODUCTION: Since intraepithelial eosinophils (IEE) are a common finding in reflux esophagitis, and may be quite common in some cases, it is usually said that the diagnosis of eosinophilic esophagitis requires a combination of clinical and pathological features. In contrast, we hypothesized that the triad of IEE, spongiosis, and a thickened basal zone forms a unique histologic picture that allows identification of eosinophilic esophagitis without reference to the clinical feature. METHOD: We performed a retrospective review to test this hypothesis. The study set included a consecutive series of 200 patients who had endoscopy, biopsy, and 24 hour pH monitoring, and 18 patients listed in the computerized pathology files with a diagnosis of eosinophilic esophagitis, Biopsies were reviewed and graded without knowledge of clinical findings, lEE were quantified as IEE/high power microscopic field. Spongiosis was graded 0-4 semiquantitively as follows: 0- no intercellular edema; 1- slight separation of cells with prominence of intercellular bridges; 2- diffuse, obvious separation with intercellular bridges intact; 3- separation of cells with focal disruption of intercellular bridges; 4- extensive disruption of intercellular bridges with formation of intraepithelial lakes. Basal zone thickness was evaluated in welloriented areas and was expressed as the percentage of total epithelial thickness occupied by the cells whose nuclei were separated by less than one nuclear diameter. Biopsies with > 20 IEE/hpf and spongiosis > grade 2 and basal zone thickness > 20% was classified as eosinophilic esophagitis. RESULT: Biopsies from 33 patients fulfilled all 3 criteria. There were 27 males and 6 females. The average age was 30.8 years (range 11-53). 29 patients presented with a chief complaint of dysphagia (88%), 3 patients complained of heartburn (9%), and 1 patient of chest pain (3%). The average duration of symptom was 69 months (range 1-360). The 24 hour pH monitoring was abnormal in 13 patients (39.3%); 9=positive alkaline reflux (pH > 15.1), l=positive acid reflux (pH > 14.8), and 3=positive for both acid and alkaline reflux. 8 patients (24%) had stricture (esophageal diameter less than 12mm) as diagnosed by esophagram and endoscopy; of those patients, 5 had alkaline reflux, 1 had both acid and alkaline reflux, 1 was normal, and 1 was not tested. CONCLUSION: The triad of lEE, spongiosis, and a thickened basal zone is a specific histologic marker for the chief complaint of dysphagia (88%). Contrary to our expectation, the histologic triad did not select for normal endoscopy and normal 24 hour pH monitoring, since stricture (24%) and alkaline reflux (36% of patients tested) were both common. It may be more than one condition can produce the histology described here. Alternatively, it may be that undetected alkaline reflux is responsible for the clinicopathologic syndrome of eosinophilic esophagitis with dysphagia. • G0250
ACUTE AND CHRONIC STRESS-INDUCED OXIDATIVE G A S T R O I N T E S T I N A L MUCOSAL INJURY IN RATS, AND PROTECTION BY BISMUTH SUBSALICYLATE (BSS). D. Bagchi, C.B. Williams, M. Milnes, R.L. Krohn, M. Bagchi, J. Balmoori, X. Ye, M.X. Tran, S.J. Stohs, Creighton University School of Pharmacy & Allied Health Professions, Omaha, NE 68178; O.R. Carryl, S. Mitra, Health Care Research Center, The Procter & Gamble Company, Mason, OH 45040. Reactive oxygen species (ROS) are implicated in the pathogenesis of stressinduced gastrointestinal (GI) injury. In the present study, we have investigated the effects of acute and chronic stress on the enhanced production of ROS including superoxide anion [SA; as determined by cytochrome c reduction assay (CCR)] and hydroxyl radicals (OH), and correlated the enhanced production of these free radicals with increased DNA fragmentation (DF), lipid peroxidation (LP) and membrane microviscosity (MV), indices of oxidative membrane and DNA damage, in the gastric (GM) and intestinal mucosa (IM) of female Sprague-Dawley rats. Furthermore, we determined the protective ability of BSS against the GI mucosai injury induced by acute and chronic stress. Acute stress was induced by waterimmersion for 90 min, while chronic stress was induced by water-immersion for 15 rain/day for 15 consecutive days. Half of the animals exposed to waterimmersion acute stress were pretreated orally with 15 mg BSS/kg 30 min prior to water-immersion. Similarly, half of the animals exposed to waterimmersion chronic stress were pretreated orally with 7.5 nag BSS/kg/day for 15 consecutive days 30 rain prior to water-immersion. Results: Acute stress produced maximal injury to both GM and IM as compared to chronic stress. Acute stress increased CCR and OH production by 10.0- and 14.3-fold, respectively, in the GM, and 10.4- and 17.0-fold in the IM. Pretreatment with BSS prevented the acute stress-induced increase in CCR and OH production. Acute stress increased LP, DF and MV by 3.6-, 4.0- and ll.6-fold respectively, in the GM, and 4.1-, 5.0- and 16.2-fold increases in IM. BSS decreased acute stress-induced LP, DF and MV by approx. 26%, 35% and 30%, respectively, in the GM, and by 20%, 36% and 30%, respectively, in the IM. BSS administration provided significant protection against chronic stressinduced nose bleeding in rats. Chronic stress increased CCR and OH production by 4.8- and 6.3-fold, respectively, in the GM, and 4.6- and 6.9fold in the IM. Chronic stress increased LP and DF by 2.9- and 3.3-fold, respectively, in the GM, and 3.3- and 4.2-fold increases in the IM. Daily
administration of BSS provided greater protection against chronic stressinduced oxidative gastrointestinal injury as compared to the acute stress. Conclusion: These results demonstrate that acute and chronic stress can induce gastrointestinal oxidative stress and mucosal injury through enhanced production of ROS, and that BSS can significantly protect against GI injury by scavenging these ROS. This research was funded by the Procter & Gamble Company, Mason, OH G0251 MECHANISM OF GASTROPROTECTION BY BISMUTH SUBSALICYLATE AGAINST CHEMICALLY-INDUCED OXIDATIVE INJURY IN HUMAN GASTRIC MUCOSAL CELLS. D. Bagchi, T.R. McGinn, X. Ye, J. Balmoori, M. Bagchi, S.J. Stohs, Creighton University Health Sciences Center, Omaha, NE; C. Kuszynski, University of Nebraska Medical Center, Omaha, NE; O.R. Carryl, S. Mitra, The Procter & Gamble Company, Mason, OH. Oxygen free radicals (OFR) are implicated in the pathogenesis of chemicallyinduced gastric mucosal injury. We have investigated the effects of ethanol (EtOH), hydrochloric acid (HCI) and sodium hydroxide (NaOH) on the (i) enhanced production of OFR including superoxide anion and hydroxyl radicals (OH), (ii) modulation of intracellular oxidized states by laser scanning confocal microscopy (LSMC) and (iii) DNA fragmentation (DNAF), indices of oxidative tissue and DNA damage in gastric mucosal cells (GC). GC from Helicobacter pylori-negative endoscopic biopsies were isolated and cultured from human subjects. The induction of OFR and DNA damage in GC following exposure to EtOH (15%), HC1 (150 raM) and NaOH (150 mM) were assessed by cytochrorne c reduction (superoxide anion production), OH production, changes in intracellular oxidized states by LSCM and DNA-F. Furthermore, the protective ability of BSS was assessed at the concentrations of 25, 50 and 100 mg/liter. Incubation of GC with EtOH, HC1 and NaOH increased superoxide anion production by approx. 8.0-, 6.1- and 7.1-fold, respectively, and increased OH production by 13.3-, 9.6- and 8.9-fold, respectively, as compared to untreated GC. Incubation of GC with EtOH, HCI and NaOH increased DNA-F by approx. 6.7-, 4.3- and 4.8-fold, respectively. Approx. 20.3-, 17.5- and 13.1-fold increases in fluorescence intensities were observed following incubation of GC with EtOH, HC1 and NaOH, respectively, demonstrating dramatic changes in the intracellular oxidized states of GC following exposure to these necrotizing agaents. Preincubation of GC with 25, 50 and 100 mg/liter of BSS decreased EtOH-induced increases in intracellular oxidized states in GC by 36%, 56% and 66%, respectively, demonstrating a concentration-dependent protective ability by BSS. Similar results were observed with respect to BSS in terms of superoxide anion production, OH production and DNA damage. The present study demonstrates that EtOH, HCI and NaOH induce oxidative stress and DNA damage in GC, and BSS can significantly attenuate gastric injury by scavenging the OFR. This research was funded by the Procter & Gamble Company, Mason, OH. G0252
TRANSIENT ABERRANT PHOSPHORYLATION AND NUCLEAR LOCALISATION OF CATENINS EARLY IN T H E P R O G R E S S I O N OF BARRETT'S NEOPLASIA. T.A. Bailey 2, L.R. Biddlestone 2, C.E. May 3, R. Bruton 1, D. R. Grand 1, D.S.A. Sanders l, H. Barr2, N.A. Shepherd 2, P.L Warner3, LA.Z. Jankowski I. qnstitute for Cancer Studies University of Birmingham, Birmingham, B15 2TJ; 2Gloucester Gastroenterology Group & Cranfield Medical Institute, Gloucester, GL1 3NN Barrett's dysplasia and its related cancer arises in the oesophagus as a result of chronic gastro-esophageal reflux which transforms stratified squamous epithelium to mucin secreting epithelium termed Barrett's mucosa. Understanding the sequence of molecular events may aid future management of this disease. Reductions in the cell adhesion molecule E-cadherin have been noted to occur in severely dysplastic and neoplastic tissue. Consequently, we investigated the related adhesion molecules; [3 and T catenins which bind to cadherins and desmosomes and modulate adhesion and the APC gene product which regulates the intracellular catenin levels. In the majority of low grade dysplastic tissue we confirm that there APC downregulation occurs (n = 17, p < 0.05). As the disease progresses to high grade dysplasia a significant proportion have 13 or y catenins accumulating in the nucleus ( > 50% of cases with high grade dysplasia, p < 0.05) and were hyperphosphorylated on tyrosine residues (n = 10, p<0.05). This phenomenon of nuclear localisation and hyperphosphorylation of catenins is transient as it decreases in adjacent invasive cancers (n = 30, p < 0.05). We found no structural alterations in the catenin genes which is consistent with this phenomenon. 13 catenin was unable to co-immunoprecipitate E-cadherin and 7 catenin was unable to co-immunoprecipitate desmogleins (desmosomespecific cadherins), in dysplasia, even when they were co-expressed (n = 10, p < 0.05). As a result E-cadherirgcatenin and desmoglein/catenin complexes are decreased, especially in high grade dysplasia (n = 7, p < 0.05). These results suggest that APC downregulation and hyperphosphorylation of catenins by increased kinase activity occur in high grade dysplasia. In addition the transient nuclear localisation of catenins may have implications for transcriptional regulation early in Barrett's neoplasia.