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Correlation of surface pH versus mucus gel thickness by in vivo confocal microscopy of rat gastric mucosa
Esophageal, Gastric, and Duodenal Disorders A93
April 1998 and HM-CAP, but the FLEX PACK assay failed to detect two (33%). Conclusions: (1) The Chiron lysate and HM-CAP are equally sensitive in detecting HP antibody but the lysate assay is twice as specific; (2) the FLEX PACK assay is significantly less sensitive than the other two assays; (3) 29% of pediatric patients with abdominal pain have antibodies to CagA or VacA. These patients are accurately detected by Chiron lysate or HM-CAP assays, but not by FLEX PACK. This study was funded in part by a grant from EPI. • G0377 CORRELATION OF SURFACE pH VERSUS MUCUS GEL THICKNESS BY I N VIVO CONFOCAL MICROSCOPY OF RAT GASTRIC MUCOSA. S. Chu, S. Tanaka, J.D. Kannitz and M.H. Montrose. Depts. Of Medicine, Johns Hopkins Univ., West LA VAMC & UCLA, Los Angeles CA and Baltimore MD The mucus gel layer on the gastric surface is believed to protect the epithelium from secreted gastric acid by sequestering bicarbonate secreted from the surface epithelium. The aim of our study was to measure mucus gel thickness in our preparation and to use the spatial resolution of confocal microscopy to test if surface pH (pHs) was protected by gastric mucus. Methods. Anesthetized rats were supine on a Zeiss LSM410 confocal microscope stage, and the exposed gastric mucosal surface (Peptides 17:155, 1996) was imaged in living animals while superfused continuously with 37°C Kreb's solution containing 10pM CI-NERF (pH-sensitive dye) and 0.5mM Lucifer yellow (reference dye). pHs was calculated from emission ratios of dye 0-25pm from the surface. Mucus gel was visualized by endogenous suspended particles in the gel, and the thickness of mucus reported by this method correlated precisely with the thickness detected by placing latex microspheres on the surface. Results. Mucus gel thickness was calculated from 24 rats under nonstimulated conditions. Gel thickness was highly variable (range 0-270 pm) in local regions of the gastric surface ( < lmm2), so a maximum and a minimum thickness were recorded for each image. All values are plotted in the frequency histogram. Average gel thickness was 70 +- 39 mm (SD, n=78), pHs was independent of overlying gel thickness when the superfusate was pH 3 or pH 5 (pHs~4 in all conditions). However, after stimulation of bicarbonate secretion (10pg/ml dimethyl-PGE2), surface pH was 4.5 under thin (0-25 pm) gel layers but was pH 5.5 under thicker gels ( > 50 pm). Conclusions. In unstimulated tissue, mucus gel thickness is similar to that observed by others, but does not have significant effect on pHs. Only thicker mucus gels enhance the gastric barrier after stimulation of bicarbonate secretion. 3O 25 20 15 10 5 0 ~
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• G0378 SURFACE pH OF RAT STOMACH IS REGULATED BY LUMINAL pH AND HORMONAL AGONISTS. S. Chu, S. Tanaka, LD. Kaunitz and M.H. Montrose. Depts. Of Medicine, Johns Hopkins Univ., West LA VAMC & UCLA, Los Angeles, CA and Baltimore, MD It is believed that secreted mucus and bicarbonate form an alkaline juxtamucosal mucus gel layer which protects the gastric surface from secreted acid. However, in response to luminal pH 5, a value which approximates the luminal pH of the fed rat stomach (pH 4.5 -+0.2, mean -+SEM n=10), in vivo confocal microscopy reported a gastric surface pH (pHs) of ~pH 3.7. The aim of this study was to measure pHs when the gastric mucosa was exposed to pH values which approximate the luminal pH of the rat stomach after an overnight fast (pH 2.7 + 0.2, n=4), and to examine whether a stimulator of bicarbonate secretion (PGE2) affected pHs. Methods. Gastric mucosa was exposed in anesthetized rats as described (Peptides 17:155, 1996). Rats were supine on a Zeiss LSM 410 confocal microscope stage, and the gastric mucosal surface was imaged in living animals while superfused continuously with 37°C Kreb's solution. Superfusates contained 10 laM CI-NERF (pH-sensitive dye) and 0.5 mM Lucifer yellow (non pH-sensitive reference dye). Extracelfular pH was calculated from emission ratios calibrated for pH 2-6, and pHs was defined as pH measured 0-25 lam adjacent to the surface. Results. Under pH 5 superfusion, pHs was 3.76 _ 0.1 (n=5) and 50 pg/kg i.v. pentagastrin acidified pHs by 0.5 pH unit. When superfusates were switched between pH 5 and 3, it took ~30 min to re-establish a stable pHs. With pH3 superfusion, steady state pHs was relatively alkaline; pH 4.5 _+0.2 (n=4). In
some areas pHs was higher than pH 6.5, beyond calibration range. If pH 3 superfusion was stopped transiently ( < 2 rain) a wave of alkaline secretion emanated from the tissue: implying continuous secretion of base equivalents. Under pH 3 superfusion, topical dimethyl-PGE 2 (10 pg/ml) further alkalinized pHs by 1 pH unit, and accelerated base secretion. Dimethyl-PGE 2 was not able to reverse the acidic surface observed in the presence of gastrin with pH 5 superfusate. Results were independent of whether animals were fasted or fed, and only dependent on the imposed experimental conditions. Conclusions. Consistent with microelectrode measurements, PGE2-stimulated base secretion alkalinized pHs at very acid luminal pH. Results suggest that current understanding of gastric pHs is appropriate for conditions in fasted, but not fed, animals. Results have strong implications for understanding gastric mucosal defense and the coordinated regulation of acid and base secretion in the fed stomach. G0379 REGRESSION OF DUODENUM GASTRIC METAPLASIA IN H. P Y L O R I DUODENAL ULCER. G. Ciancio, B. Orsini, E. Surrenti, MR. Biagini, M. Nuti, A. Palomba*, A. Amorosi*, S. Milani, C. Surrenti. Department of Clinical Pathophysiology, Gastroenterology Unit, *Pathology Institute, University of Florence, Italy. H. pylori (Hp) has a pathogenic role in the development of duodenal ulcer (DU), neverthless the mechanisms are still debatable. Hp directly might cause DU by growing on areas of gastric metaplasia (GM) in duodenum. AIM: We investigated that Hp gastric infection and its eradication had a direct role on GM duodenum regression in DU patients. METHODS: Duodenal (anterior, inferior, superior walls of the first part of duodenum) and gastric antrum biopsies were obtained from 10 Hp-positive patients. Hp gastric infection was diagnosed by rapid urease test, histology and PCR for urease (6 M and 4 F, median age 55.50, range 28-69 years). The patients were treated with 20 mg omeprazole 2 times daily associated with 250 mg clarithromicin and 500 mg amoxicillin 4 times daily for a period of 10 days. Then all patients were submitted to 20 mg omeprazole daily for the duration of the study. Control endoscopy was carried out at 6 and 18 weeks from enrollment. RESULTS: GM was observed in 8/10 (80%) patients in at least one duodenal biopsy specimen and Hp colonization on areas of GM was 100%. At the end of eradicant treatment 8/10 (80%) patients resulted Hp-negative while 2/10 (20%) were Hp-positive. At the last endoscopy we found a GM regression only in 6/8 (75%) patients, vice versa in 2/8 (25%) patients GM was unchanged. Moreover, we observed that in 6 patients with GM regression, 5 (83.3%) eradicated while 1 (16.7%) remained Hp-positive. Vice versa 2 (100%) patients without GM regression resulted eradicated (P=NS). CONCLUSIONS: Our data showed that Hp itself has not a direct role on GM regression. Since the total percentage of GM regression was 80% we might suppose that gastric acid secretion plays an important role in this phenomenon. In our opinion, probably, Hp indirectly contributes by modifying acid secretion to GM development. G0380 IS GRAM STAIN USEFUL FOR CLINICIANS TO ASSESS HELICOB A C T E R P Y L O R I INFECTION? ANTIBIOTICS RESISTANCE IN OUR AREA. C. Ciriza, R. L6pez-Medrano, C. Fuster, S. Dajil. Gastroenterology and Microbiology departments. Hospital del Bierzo. Ponferrada. Le6n. Spain. Culture and histology are classically considered the gold standard to confirm Helicobacter pylori (Hp) infection. However, there are simple and cheaper tests to achieve similar results for clinicians while histology or culture are available. An ongoing study is being carried out in Hospital del Bierzo to determine Gram stain sensitivity and specificity vs histology. Methods. To date, upper endoscopy was performed in 191 symptomatic patients. 6 gastric biopsies were taken from antrum in all patients and in 42 of them corpus biopsies were also obtained. 3 of 6 biopsies were for histological examination (Giemsa stain) and the remaining 3 for microbiological analysis (1 for Gram stain, 2 for culture). Each Hp isolate was tested for metronidazole, clarithromycin, ampicillin and amoxicillin-clavulanic by the epsilometer method E-test. 1994 NCCLS MIC (minimum inhibitory concentration) breakpoints were applied to assess Hp resistance. Results. Sensitivity and specificity for Gram stain and culture were 59% and 84% vs 47% and 86% respectively. If we consider antrum and corpus separately sensitivity was 47% vs 68% for Gram stain and 46% vs 52% for culture. Specificity was lower for both, Gram stain and culture, in corpus compared to antrum (64% vs 88% for Gram stain and 76% vs 88% for culture). Metronidazole resistance was 29.41% and 13.72% for clarithromycin. We did not find resistance to the other antibiotics tested. Conclusions. Sensitivity and specificity for Gram stain and culture were similar, indicating that Gram stain is a simple test that might be useful to obtain faster Hp results for clinicians. We found higher sensitivity and lower specificity for both, Gram stain and culture, in corpus vs antrum suggesting a more homogeneous Hp colonization in corpus than in antrum. Therefore, corpus and antrum biopsies should be taken to improve accuracy in Hp diagnosis.
April 1998 and HM-CAP, but the FLEX PACK assay failed to detect two (33%). Conclusions: (1) The Chiron lysate and HM-CAP are equally sensitive in detecting HP antibody but the lysate assay is twice as specific; (2) the FLEX PACK assay is significantly less sensitive than the other two assays; (3) 29% of pediatric patients with abdominal pain have antibodies to CagA or VacA. These patients are accurately detected by Chiron lysate or HM-CAP assays, but not by FLEX PACK. This study was funded in part by a grant from EPI. • G0377 CORRELATION OF SURFACE pH VERSUS MUCUS GEL THICKNESS BY I N VIVO CONFOCAL MICROSCOPY OF RAT GASTRIC MUCOSA. S. Chu, S. Tanaka, J.D. Kannitz and M.H. Montrose. Depts. Of Medicine, Johns Hopkins Univ., West LA VAMC & UCLA, Los Angeles CA and Baltimore MD The mucus gel layer on the gastric surface is believed to protect the epithelium from secreted gastric acid by sequestering bicarbonate secreted from the surface epithelium. The aim of our study was to measure mucus gel thickness in our preparation and to use the spatial resolution of confocal microscopy to test if surface pH (pHs) was protected by gastric mucus. Methods. Anesthetized rats were supine on a Zeiss LSM410 confocal microscope stage, and the exposed gastric mucosal surface (Peptides 17:155, 1996) was imaged in living animals while superfused continuously with 37°C Kreb's solution containing 10pM CI-NERF (pH-sensitive dye) and 0.5mM Lucifer yellow (reference dye). pHs was calculated from emission ratios of dye 0-25pm from the surface. Mucus gel was visualized by endogenous suspended particles in the gel, and the thickness of mucus reported by this method correlated precisely with the thickness detected by placing latex microspheres on the surface. Results. Mucus gel thickness was calculated from 24 rats under nonstimulated conditions. Gel thickness was highly variable (range 0-270 pm) in local regions of the gastric surface ( < lmm2), so a maximum and a minimum thickness were recorded for each image. All values are plotted in the frequency histogram. Average gel thickness was 70 +- 39 mm (SD, n=78), pHs was independent of overlying gel thickness when the superfusate was pH 3 or pH 5 (pHs~4 in all conditions). However, after stimulation of bicarbonate secretion (10pg/ml dimethyl-PGE2), surface pH was 4.5 under thin (0-25 pm) gel layers but was pH 5.5 under thicker gels ( > 50 pm). Conclusions. In unstimulated tissue, mucus gel thickness is similar to that observed by others, but does not have significant effect on pHs. Only thicker mucus gels enhance the gastric barrier after stimulation of bicarbonate secretion. 3O 25 20 15 10 5 0 ~
0 ~
~ ~
0 o
~ ~
0 ~
0 o
0 o
• G0378 SURFACE pH OF RAT STOMACH IS REGULATED BY LUMINAL pH AND HORMONAL AGONISTS. S. Chu, S. Tanaka, LD. Kaunitz and M.H. Montrose. Depts. Of Medicine, Johns Hopkins Univ., West LA VAMC & UCLA, Los Angeles, CA and Baltimore, MD It is believed that secreted mucus and bicarbonate form an alkaline juxtamucosal mucus gel layer which protects the gastric surface from secreted acid. However, in response to luminal pH 5, a value which approximates the luminal pH of the fed rat stomach (pH 4.5 -+0.2, mean -+SEM n=10), in vivo confocal microscopy reported a gastric surface pH (pHs) of ~pH 3.7. The aim of this study was to measure pHs when the gastric mucosa was exposed to pH values which approximate the luminal pH of the rat stomach after an overnight fast (pH 2.7 + 0.2, n=4), and to examine whether a stimulator of bicarbonate secretion (PGE2) affected pHs. Methods. Gastric mucosa was exposed in anesthetized rats as described (Peptides 17:155, 1996). Rats were supine on a Zeiss LSM 410 confocal microscope stage, and the gastric mucosal surface was imaged in living animals while superfused continuously with 37°C Kreb's solution. Superfusates contained 10 laM CI-NERF (pH-sensitive dye) and 0.5 mM Lucifer yellow (non pH-sensitive reference dye). Extracelfular pH was calculated from emission ratios calibrated for pH 2-6, and pHs was defined as pH measured 0-25 lam adjacent to the surface. Results. Under pH 5 superfusion, pHs was 3.76 _ 0.1 (n=5) and 50 pg/kg i.v. pentagastrin acidified pHs by 0.5 pH unit. When superfusates were switched between pH 5 and 3, it took ~30 min to re-establish a stable pHs. With pH3 superfusion, steady state pHs was relatively alkaline; pH 4.5 _+0.2 (n=4). In
some areas pHs was higher than pH 6.5, beyond calibration range. If pH 3 superfusion was stopped transiently ( < 2 rain) a wave of alkaline secretion emanated from the tissue: implying continuous secretion of base equivalents. Under pH 3 superfusion, topical dimethyl-PGE 2 (10 pg/ml) further alkalinized pHs by 1 pH unit, and accelerated base secretion. Dimethyl-PGE 2 was not able to reverse the acidic surface observed in the presence of gastrin with pH 5 superfusate. Results were independent of whether animals were fasted or fed, and only dependent on the imposed experimental conditions. Conclusions. Consistent with microelectrode measurements, PGE2-stimulated base secretion alkalinized pHs at very acid luminal pH. Results suggest that current understanding of gastric pHs is appropriate for conditions in fasted, but not fed, animals. Results have strong implications for understanding gastric mucosal defense and the coordinated regulation of acid and base secretion in the fed stomach. G0379 REGRESSION OF DUODENUM GASTRIC METAPLASIA IN H. P Y L O R I DUODENAL ULCER. G. Ciancio, B. Orsini, E. Surrenti, MR. Biagini, M. Nuti, A. Palomba*, A. Amorosi*, S. Milani, C. Surrenti. Department of Clinical Pathophysiology, Gastroenterology Unit, *Pathology Institute, University of Florence, Italy. H. pylori (Hp) has a pathogenic role in the development of duodenal ulcer (DU), neverthless the mechanisms are still debatable. Hp directly might cause DU by growing on areas of gastric metaplasia (GM) in duodenum. AIM: We investigated that Hp gastric infection and its eradication had a direct role on GM duodenum regression in DU patients. METHODS: Duodenal (anterior, inferior, superior walls of the first part of duodenum) and gastric antrum biopsies were obtained from 10 Hp-positive patients. Hp gastric infection was diagnosed by rapid urease test, histology and PCR for urease (6 M and 4 F, median age 55.50, range 28-69 years). The patients were treated with 20 mg omeprazole 2 times daily associated with 250 mg clarithromicin and 500 mg amoxicillin 4 times daily for a period of 10 days. Then all patients were submitted to 20 mg omeprazole daily for the duration of the study. Control endoscopy was carried out at 6 and 18 weeks from enrollment. RESULTS: GM was observed in 8/10 (80%) patients in at least one duodenal biopsy specimen and Hp colonization on areas of GM was 100%. At the end of eradicant treatment 8/10 (80%) patients resulted Hp-negative while 2/10 (20%) were Hp-positive. At the last endoscopy we found a GM regression only in 6/8 (75%) patients, vice versa in 2/8 (25%) patients GM was unchanged. Moreover, we observed that in 6 patients with GM regression, 5 (83.3%) eradicated while 1 (16.7%) remained Hp-positive. Vice versa 2 (100%) patients without GM regression resulted eradicated (P=NS). CONCLUSIONS: Our data showed that Hp itself has not a direct role on GM regression. Since the total percentage of GM regression was 80% we might suppose that gastric acid secretion plays an important role in this phenomenon. In our opinion, probably, Hp indirectly contributes by modifying acid secretion to GM development. G0380 IS GRAM STAIN USEFUL FOR CLINICIANS TO ASSESS HELICOB A C T E R P Y L O R I INFECTION? ANTIBIOTICS RESISTANCE IN OUR AREA. C. Ciriza, R. L6pez-Medrano, C. Fuster, S. Dajil. Gastroenterology and Microbiology departments. Hospital del Bierzo. Ponferrada. Le6n. Spain. Culture and histology are classically considered the gold standard to confirm Helicobacter pylori (Hp) infection. However, there are simple and cheaper tests to achieve similar results for clinicians while histology or culture are available. An ongoing study is being carried out in Hospital del Bierzo to determine Gram stain sensitivity and specificity vs histology. Methods. To date, upper endoscopy was performed in 191 symptomatic patients. 6 gastric biopsies were taken from antrum in all patients and in 42 of them corpus biopsies were also obtained. 3 of 6 biopsies were for histological examination (Giemsa stain) and the remaining 3 for microbiological analysis (1 for Gram stain, 2 for culture). Each Hp isolate was tested for metronidazole, clarithromycin, ampicillin and amoxicillin-clavulanic by the epsilometer method E-test. 1994 NCCLS MIC (minimum inhibitory concentration) breakpoints were applied to assess Hp resistance. Results. Sensitivity and specificity for Gram stain and culture were 59% and 84% vs 47% and 86% respectively. If we consider antrum and corpus separately sensitivity was 47% vs 68% for Gram stain and 46% vs 52% for culture. Specificity was lower for both, Gram stain and culture, in corpus compared to antrum (64% vs 88% for Gram stain and 76% vs 88% for culture). Metronidazole resistance was 29.41% and 13.72% for clarithromycin. We did not find resistance to the other antibiotics tested. Conclusions. Sensitivity and specificity for Gram stain and culture were similar, indicating that Gram stain is a simple test that might be useful to obtain faster Hp results for clinicians. We found higher sensitivity and lower specificity for both, Gram stain and culture, in corpus vs antrum suggesting a more homogeneous Hp colonization in corpus than in antrum. Therefore, corpus and antrum biopsies should be taken to improve accuracy in Hp diagnosis.