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Disappearance of anti Cag-A antibodies in duodenal ulcer patients following cure for Helicobacter pylori infection
A170
•
AGA ABSTRACTS
MODULATION OF STIMULUS'SECRETION COUPLING BY V A R I O U S EXTRACELLULAR MAGNESIUM CONCENTRATIONS IN RAT PARIETAL CELLS. F.Ch.Mooren, R.Stoll, E. Spyrou, R. Schlotmann, W. Bell+ and W . D o m s c h k e , Dept. of. Medicine B, University of M~nster, and +Dept. of General Pharmakology, Hannover Medical School, 30625 Hannover, Germany Intracellular calcium [Ca]i plays a vital role in stimulus-secretion-coupling of gastric parietal cells, but not so much is known about the function of intracellular magnesium [Mg]i. We isolated single rat gastric parietal cells using everted stomach technique filled with pronase containing buffer. Cells were loaded with i0 BM Fura-2 or 5 BM Magfura-2 for the measurement of [Ca]i and [Mg]i, respectively. Measurements were performed at the single cell level with a microscope-based fluorescence system. Basal and carbachol-stimulated calcium levels were decreased at high extracellular magnesium levels (=high[Mg]ex), whereas low magnesium levels (=low[Mg]ex) had the opposite effect. Application of thapeigargin demonstrated that high[Mg]ex suppressed the release of calcium from internal stores but .also inhibited calcium influx through the plasma membrane. Acid secretion was also influenced by extracellular magnesium depending on stimulating hormon. High[Mg]ex nearly inhibited carbachol induced acid secretion, whereas low[Mg]ex enhanced acid secretion. On the other hand histamin stimulated acid secretion was inhibited by high[Mg]ex only during the first 30 minutes followed by a clear recovery.Finally we investigated the effect of secretagogues on [Mg]i. [Mg]i was decreased by carbachol, whereas histamin had no effect. These results demonstrate that magnesium plays a modulative role in calcium-dependent stimulussecretion coupling in rat parietal cells. There is also evidence that [Mg]i acts as an intracellular messenger, but this role needs further to be
Q DISAPPEARANCE OF ANTI CAG-A ANTIBODIES IN D U O D E N A L ULCER PATIENTS FOLLOWING CURE FOR EELICOBACTER PYLORI INFECTION. A.Morgando,P. Sanseverino °,C.Perotto,G.Pugliese,L.De ~Marco°,E.Cassarino,A.Ferrari,G.Verme,G.Marchiaro *, iA.Ponzetto. Gastroenterology and *Microbiology Dpt,Molinetts JHospihal,Turin,ITALY. F !Helicohactsr pylori(HP)infection correlates with presence of :duodenal ulcer.Cytotoxlc strains of HP produce a Cytotoxin[associated-gens product(Cag-A) -of 120-140 kDa.Patients :infected by such strains circulate anti Cag-A antibodies.We collected sera from 58 patients,observed in 1992;48 had duodenal ulcer (DU),5 had gastric ulcer(GU) and 5 severe gastritis.HP infection was diagnosed by histology and colture.Patients were treated with amoxicillin 500 m g q.i.d.i and metronidazole 250 m g q.i.d.for 10 days plus omeprazole 20 rag/day for 30 days.Endoscopic and serological follow-up was performed at 3,6,12 and 24 months after therapy.Sera from the entire follow-up period were tested in one single assay for presence of anti Cag-A antibodies by an immunoenzimatic assay using a purified recombinant fragment of Cag-A(gift of dr.A.Covacci,I.R.I.S.,SIENA).At diagnosis, 48/58 patients had high anti Caq-A ~itres,while low antibodies: titres were observed in the other 10 , After therapy,we divided patients into: RESPONDERS - 39 patients free from HP infection 2 years after eradication ; NON RESPONDERS - 19 patients with persistence of HF infection after 2 years;7 of them (5 D U and 2 GU) had ulcer relapse during this period.ln the RESPONDERS group,levels of IgG anti Cag-A halved in all (39/39) patients.No anti Cag-A reactivity could be detected in 31/39 at two years follow-ul~. (28/39 at one year).The remaining patients had very IoG titre,at the two years control.In the NON RESPONDERS groul~. IgG anti Cag-A showed a little decrease or remained unchanged in 18/19 patients.In one case the titre rose durin~ the follow-up.CONCLUSIONS.Specific anti Cag-A antibodie~ appear to be a marker for non invasive follow-up after HI] eradication in patients affected by DU,GU or severe gasfrlti~ and duodenitis~
GASTROENTEROLOGY, Vol. 108, No. 4
A comparison of the pathophysiological arachidonic acid metabolism in the gastric and oesophageal mucosa Gareth P Morgan and John G Williams Arachidonic acid (AA) is metabolised by two important enzyme systems. Cycleoxygenase converts AA into a series ofpmsta~,l..andins (PGs) and thromboxan~ (Tx) whilst the lipoxygenases are associated with the formation of hydroxylated derivatives (HETEs) and leukotrienes (LTs). We have previously reviewed the properties of AA metabolites in the oesophageal mucosa (Gut 1994;35:297-8) and have performed further literature surveys to increase our understanding of this important yet contentious subject. This report is the first to our knowledge that compares pathophysiological AA metabolism in the gastric and oesophageal mucosa, using the concept of PG/LT ratios. In the gastric mncosa, physiological metabolism is predominantly via the cyclooxygenase pathway such that there is a high PG/LT ratio. These PGs, especially PGF-,2 and PGI2, help to support gastric defence mechanisms by maintaining sunmncosal blood flow, stimulating bicarbonate secretion and regulating cellular turnover. In animal models of gastric injury, however,this ratio is disturbed since mncosal damage is associated with the increased production of LTs such as LTA4 and LTB4. The reduced PG/LT ratio which results may perpetuate injury since exogenous LTs can exacerbate damage, whilst lipoxygenase inhibitors confer protection in such models. Clinically, these properties can account for the ~gastrotoxic ertects or non-steroidal anti-inflammatory drags (NSAIDs). In the human gastric mucosa, NSAIDs lower the PG/LT ratio by inhibiting cycloox~fgenase and diverting the AA cascade into the lipoxygenase pathway, thereby enhancing LT formation. Misoprostil, a PGE~ analogue prevents and reverses NSAID gasrttrc damage and biochemically, this may parallel a restoration of the high PG/LT ratio. By contrast, physiologicalmetabolism in the oesophageal mucosa is predominantly via me tipoxygenase pathway such that there is a low PG/LT ratio: It appears that .these LTs, especially 12-HETE, support oesophageal defence mechanisms by mcreasmg mncosal resistance to iomc backflow. During oesophageal injury, however, this ratio is disturbed since mnc0sal damage is associated witii the increased production of PGFa. The increased PG/LT ratio may perpetuate injury, since in ,ammalmodels of oesophagitis, exogenous PGs and lipoxygenaseinhibitors c ~ accelerate injury, whilst NSAIDs prevent and reverse damage. This latter el~ect appears to result from me augmentation of 12-HETE formation and the inniuitinn of PGE2 synthesis respectively. At present, there is no satisfactory explanation for this discrepancy in such a short area of the GI tract. Differing structural, histological and physiologicalproperties may account, at least in part, for the contrasting AA metabolism between the stomacn and oesophagus, It is clear, however, that our understanding of AA metanotites in me upper GI tract is far from complete.
OXYGEN RADICAL-SCAVENGING ENZYMES IN EXTRACT OF
HELICOBACTER PYLORI M. Mori, M. Suzuki, S. Miura, and H. Ishii. Deparhnent of Internal Medicine, School of Medicine, Keio University, and Internal Medicine, Tokyo Metropolitan Hiroo General Hospital, Tokyo, Japan We have reported that toxic oxidants which are produced by activated neutrophils play a pivotal role in the development of H. pylori-induced gastric mucosal damage. (Am. J. Physiol. 263: G719, 1992, Gut 35:1375, 1994) Microorganisms are, however, characterized by the ability to produce variety of anti-oxidant enzymes. In this paper, activities of oxygen radical-scavenging enzymes (catalase and superoxide dismutase) and urease were measured in water extract of H. pylori. Methods: H. pylori (NCTC11637) was inoculated on sheep blood agar plate and harvested. Bacterial suspensions (109 cfu / ml phosphate buffer) were washed twice and incubated at 37°C for 1, 4, 12 and 24 hours or sonicated in ice. Their supernatants were obtained b y centrifugation and filtration. Superoxide dismutase (SOD) activity was spectrophotometrically measured by the cytochrome c method. Catalase activity was assayed by the fall in absorbance at 240 nm as H202 is degraded. Urease activity was determined by measuring the release of ammonia using Berthelot reaction. Results." Both activities of SOD and catalase were detected in the supematants of I hr incubation. Their activities were almost constant in 4, 12, 24 hr incubation or sonication. Urease activity was dramatically increased in proportion to the incubation period. Incubation oeriod (hr} SOD(U/ml) Catalase(U/ml) UreaseiU/ml) I 1.81 +0.20 0.52+0.15 0.086+0.03 4 1.76+0.28 0.65+0.24 0.10+0.03 12 1.78+0.34 0.52+0.21 0.19+0.08 24 1.89+0.28 0.69+0.31 0.30+0.10" sonicated 2.54+0.39 0.49+0.26 0.08+0.03 *p<0.05,compared with the value in 1 hr incubation
Conchtsions: H. pylori possesses anti-oxidant enzymes (SOD and catalase) and releases them extracellularily. However, their enzyme synthesis are limited compared with that of urease.
•
AGA ABSTRACTS
MODULATION OF STIMULUS'SECRETION COUPLING BY V A R I O U S EXTRACELLULAR MAGNESIUM CONCENTRATIONS IN RAT PARIETAL CELLS. F.Ch.Mooren, R.Stoll, E. Spyrou, R. Schlotmann, W. Bell+ and W . D o m s c h k e , Dept. of. Medicine B, University of M~nster, and +Dept. of General Pharmakology, Hannover Medical School, 30625 Hannover, Germany Intracellular calcium [Ca]i plays a vital role in stimulus-secretion-coupling of gastric parietal cells, but not so much is known about the function of intracellular magnesium [Mg]i. We isolated single rat gastric parietal cells using everted stomach technique filled with pronase containing buffer. Cells were loaded with i0 BM Fura-2 or 5 BM Magfura-2 for the measurement of [Ca]i and [Mg]i, respectively. Measurements were performed at the single cell level with a microscope-based fluorescence system. Basal and carbachol-stimulated calcium levels were decreased at high extracellular magnesium levels (=high[Mg]ex), whereas low magnesium levels (=low[Mg]ex) had the opposite effect. Application of thapeigargin demonstrated that high[Mg]ex suppressed the release of calcium from internal stores but .also inhibited calcium influx through the plasma membrane. Acid secretion was also influenced by extracellular magnesium depending on stimulating hormon. High[Mg]ex nearly inhibited carbachol induced acid secretion, whereas low[Mg]ex enhanced acid secretion. On the other hand histamin stimulated acid secretion was inhibited by high[Mg]ex only during the first 30 minutes followed by a clear recovery.Finally we investigated the effect of secretagogues on [Mg]i. [Mg]i was decreased by carbachol, whereas histamin had no effect. These results demonstrate that magnesium plays a modulative role in calcium-dependent stimulussecretion coupling in rat parietal cells. There is also evidence that [Mg]i acts as an intracellular messenger, but this role needs further to be
Q DISAPPEARANCE OF ANTI CAG-A ANTIBODIES IN D U O D E N A L ULCER PATIENTS FOLLOWING CURE FOR EELICOBACTER PYLORI INFECTION. A.Morgando,P. Sanseverino °,C.Perotto,G.Pugliese,L.De ~Marco°,E.Cassarino,A.Ferrari,G.Verme,G.Marchiaro *, iA.Ponzetto. Gastroenterology and *Microbiology Dpt,Molinetts JHospihal,Turin,ITALY. F !Helicohactsr pylori(HP)infection correlates with presence of :duodenal ulcer.Cytotoxlc strains of HP produce a Cytotoxin[associated-gens product(Cag-A) -of 120-140 kDa.Patients :infected by such strains circulate anti Cag-A antibodies.We collected sera from 58 patients,observed in 1992;48 had duodenal ulcer (DU),5 had gastric ulcer(GU) and 5 severe gastritis.HP infection was diagnosed by histology and colture.Patients were treated with amoxicillin 500 m g q.i.d.i and metronidazole 250 m g q.i.d.for 10 days plus omeprazole 20 rag/day for 30 days.Endoscopic and serological follow-up was performed at 3,6,12 and 24 months after therapy.Sera from the entire follow-up period were tested in one single assay for presence of anti Cag-A antibodies by an immunoenzimatic assay using a purified recombinant fragment of Cag-A(gift of dr.A.Covacci,I.R.I.S.,SIENA).At diagnosis, 48/58 patients had high anti Caq-A ~itres,while low antibodies: titres were observed in the other 10 , After therapy,we divided patients into: RESPONDERS - 39 patients free from HP infection 2 years after eradication ; NON RESPONDERS - 19 patients with persistence of HF infection after 2 years;7 of them (5 D U and 2 GU) had ulcer relapse during this period.ln the RESPONDERS group,levels of IgG anti Cag-A halved in all (39/39) patients.No anti Cag-A reactivity could be detected in 31/39 at two years follow-ul~. (28/39 at one year).The remaining patients had very IoG titre,at the two years control.In the NON RESPONDERS groul~. IgG anti Cag-A showed a little decrease or remained unchanged in 18/19 patients.In one case the titre rose durin~ the follow-up.CONCLUSIONS.Specific anti Cag-A antibodie~ appear to be a marker for non invasive follow-up after HI] eradication in patients affected by DU,GU or severe gasfrlti~ and duodenitis~
GASTROENTEROLOGY, Vol. 108, No. 4
A comparison of the pathophysiological arachidonic acid metabolism in the gastric and oesophageal mucosa Gareth P Morgan and John G Williams Arachidonic acid (AA) is metabolised by two important enzyme systems. Cycleoxygenase converts AA into a series ofpmsta~,l..andins (PGs) and thromboxan~ (Tx) whilst the lipoxygenases are associated with the formation of hydroxylated derivatives (HETEs) and leukotrienes (LTs). We have previously reviewed the properties of AA metabolites in the oesophageal mucosa (Gut 1994;35:297-8) and have performed further literature surveys to increase our understanding of this important yet contentious subject. This report is the first to our knowledge that compares pathophysiological AA metabolism in the gastric and oesophageal mucosa, using the concept of PG/LT ratios. In the gastric mncosa, physiological metabolism is predominantly via the cyclooxygenase pathway such that there is a high PG/LT ratio. These PGs, especially PGF-,2 and PGI2, help to support gastric defence mechanisms by maintaining sunmncosal blood flow, stimulating bicarbonate secretion and regulating cellular turnover. In animal models of gastric injury, however,this ratio is disturbed since mncosal damage is associated with the increased production of LTs such as LTA4 and LTB4. The reduced PG/LT ratio which results may perpetuate injury since exogenous LTs can exacerbate damage, whilst lipoxygenase inhibitors confer protection in such models. Clinically, these properties can account for the ~gastrotoxic ertects or non-steroidal anti-inflammatory drags (NSAIDs). In the human gastric mucosa, NSAIDs lower the PG/LT ratio by inhibiting cycloox~fgenase and diverting the AA cascade into the lipoxygenase pathway, thereby enhancing LT formation. Misoprostil, a PGE~ analogue prevents and reverses NSAID gasrttrc damage and biochemically, this may parallel a restoration of the high PG/LT ratio. By contrast, physiologicalmetabolism in the oesophageal mucosa is predominantly via me tipoxygenase pathway such that there is a low PG/LT ratio: It appears that .these LTs, especially 12-HETE, support oesophageal defence mechanisms by mcreasmg mncosal resistance to iomc backflow. During oesophageal injury, however, this ratio is disturbed since mnc0sal damage is associated witii the increased production of PGFa. The increased PG/LT ratio may perpetuate injury, since in ,ammalmodels of oesophagitis, exogenous PGs and lipoxygenaseinhibitors c ~ accelerate injury, whilst NSAIDs prevent and reverse damage. This latter el~ect appears to result from me augmentation of 12-HETE formation and the inniuitinn of PGE2 synthesis respectively. At present, there is no satisfactory explanation for this discrepancy in such a short area of the GI tract. Differing structural, histological and physiologicalproperties may account, at least in part, for the contrasting AA metabolism between the stomacn and oesophagus, It is clear, however, that our understanding of AA metanotites in me upper GI tract is far from complete.
OXYGEN RADICAL-SCAVENGING ENZYMES IN EXTRACT OF
HELICOBACTER PYLORI M. Mori, M. Suzuki, S. Miura, and H. Ishii. Deparhnent of Internal Medicine, School of Medicine, Keio University, and Internal Medicine, Tokyo Metropolitan Hiroo General Hospital, Tokyo, Japan We have reported that toxic oxidants which are produced by activated neutrophils play a pivotal role in the development of H. pylori-induced gastric mucosal damage. (Am. J. Physiol. 263: G719, 1992, Gut 35:1375, 1994) Microorganisms are, however, characterized by the ability to produce variety of anti-oxidant enzymes. In this paper, activities of oxygen radical-scavenging enzymes (catalase and superoxide dismutase) and urease were measured in water extract of H. pylori. Methods: H. pylori (NCTC11637) was inoculated on sheep blood agar plate and harvested. Bacterial suspensions (109 cfu / ml phosphate buffer) were washed twice and incubated at 37°C for 1, 4, 12 and 24 hours or sonicated in ice. Their supernatants were obtained b y centrifugation and filtration. Superoxide dismutase (SOD) activity was spectrophotometrically measured by the cytochrome c method. Catalase activity was assayed by the fall in absorbance at 240 nm as H202 is degraded. Urease activity was determined by measuring the release of ammonia using Berthelot reaction. Results." Both activities of SOD and catalase were detected in the supematants of I hr incubation. Their activities were almost constant in 4, 12, 24 hr incubation or sonication. Urease activity was dramatically increased in proportion to the incubation period. Incubation oeriod (hr} SOD(U/ml) Catalase(U/ml) UreaseiU/ml) I 1.81 +0.20 0.52+0.15 0.086+0.03 4 1.76+0.28 0.65+0.24 0.10+0.03 12 1.78+0.34 0.52+0.21 0.19+0.08 24 1.89+0.28 0.69+0.31 0.30+0.10" sonicated 2.54+0.39 0.49+0.26 0.08+0.03 *p<0.05,compared with the value in 1 hr incubation
Conchtsions: H. pylori possesses anti-oxidant enzymes (SOD and catalase) and releases them extracellularily. However, their enzyme synthesis are limited compared with that of urease.