The development of “virtual endoscopy” by transabdominal ultrasonography

GASTROENTEROLOGYVoL 114, No. 4

A338 AGA ABSTRACTS

• G1378 FREQUENT ALLELIC IMBALANCE AT CHROMOSOME 13q IN GASTRIC CANCER IN KOREA. Y. Yamaoka, D. Y. Graham, A. R. Sepulveda VA Medical Center and Baylor College of Medicine, Houston, TX; J. G. Kim. Guru Hospital, Korea University College of Medicine, Seoul, Korea. Background: Gastric cancer is one of the most frequent cancers in the world. The genetic mechanisms that result in the development of gastric neoplasia are poorly understood. Several studies have suggested that intestinal type and diffuse carcinomas may have different genetic lesions. H. pylori has been implicated as a co-factor in gastric carcinoma, but only a small number of individuals infected by H. pylori develops this cancer. Specific types of host genetic makeup may in part explain this outcome. The availability of highly polymorphic microsatellite markers throughout the genome can accelerate the search for new genes involved in cancer development. A high percentage of gastric cancers display allelic imbalance (AI) at various loci, characterized both by replicator errors (RER) and loss of heterozignsity (LOH). Aims: To identify genetic loci frequently altered in gastric carcinoma, by using allelic imbalance analysis in intestinal and diffuse gastric carcinomas. Methods: Biopsies from 15 gastric carcinomas from Korean patients were stained with the Genta stain, evaluated and graded for the presence of H. pylori and carcinoma. Genomic DNA was extracted from paraffin embedded tissues. End-labeled microsatellite markers D1S191, D2S123, D13S170 and TP53 were used to amplify tumor and non-neoplastic DNA. PeR products were separated in 6% denaturing polyacrylamide gels. Microsatellite instability (MI) was defined as a band shift in either of the two alleles in two or more markers. LOH was determined by a loss or decrease in intensity of a band present in the tumor, relative to the same band in the normal allele. Results: Eleven tumors were of intestinal type and four were diffuse carcinomas. Microsatellite instability was found less frequently in non-signet ring cell diffuse carcinomas. Six out of fifteen tumors (40%) displayed MI at 2 or more loci. D13S170 was the marker with more frequent MI 9/15 tumors (60%). In addition, there was frequent loss of heterozigosity (LOH) at the D 13S 170 locus in carcinoma tissues. Conclusions: The microsatellite marker D13S170 is very sensitive to detect allelic instability in gastric carcinomas in a population in Korea. This marker revealed a high level of LOH in this region of the genome (13q22-q31) suggesting that a tumor suppressor gene located in this region may be involved in gastric carcinogenesis. • G1379 HOW GOOD ARE THE SEROLOGICAL TESTS THAT HAVE BEEN USED TO MAKE H. PYLORI-DISEASE ASSOCIATIONS WITH CagA OR VacA? Y. Yamaoka, T. Kodama, K. Kashima, Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan; D.Y. Graham, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX. Background: There has been considerable interest in the putative virulence factors VacA and CagA. One approach to investigate whether they have any association with a particular H. pylori-related disease is to do a seroepidemiologic study for the presence of anti-CagA or anti-VacA antibodies. Interpretation of the results is critically dependent on the accuracy of the tests used. A number of immunoblotting tests have been introduced and have been used for making associations. Aim: To compare 4 such tests for antibodies to CagA or VacA. Methods: Sera from patients with H. pylori infection with H. pylori isolates known to be CagA-positive (by PeR and immunoblotting for 1-1.pylori) or VacA-positive (by vacuolating cytotoxin activities using Vero cells) were tested by immunoblotting with the Helicoblot 2.0 (Genelabs Diagnostics, Singapore), RIDA-Blot Helieobacter (R-Biopharm GmbH, Germany) and RIBA 11. pylori SIA (Chiron), and by ELISA with recombinant CagA antigen (OraVax). Results: False negative tests for anti-CagA were found (78 positive isolates) with the OraVax test (13%), the RIBA test (15%) and the HB 2.0 tests (10%). The RIDA-BH had no false negative tests. Only 2 cases were CagA negative and although no false positive tests were found, the sample is too small to exclude a high percentage of false positive tests (95% CI for 0/2 = 0-84%). Immunoblotting for VacA showed both false negatives and false positives (13 neg. and 67 pos. cases). The HB 2.0 had 49% false negatives and no false positives, RIDA-BH had no false negatives but 92% false positives, RIBA had 12% false negatives and 15% false positives. Conclusion: None of the available serological (immunoblots) tests for antibody to the putative virulence factors CagA and VacA can be recommended. Associations made using these tests should be viewed with caution. • G1380 TEST OF

THE

HELICOBACTER

HYPOTHESIS THAT vacA GENOTYPES OF PYLORI CORRELATE WITH CagA STATUS,

CYTOTOXIN PRODUCTION, OR CLINICAL OUTCOME. Y. Yamaoka. T. Kodama, M. Kita, J. Imanishi, K. Kashima, Third Department of Internal Medicine and Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, Japan; D.Y. Graham, Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX. Background: Recently, mosaicism in vacA alleles with three distinct families of vacA signal sequences (s I a, s lb and s2) and two distinct families of middle region alleles (ml and m2) were reported and, based on a small number of isolates, proposed to be associated with cytotoxin activity, histological, and clinical findings. Aim: To test whether this hypothesis could be confirmed. In addition, we hypothesized that if the vacA or cagA genotypes were to be associated with a

specific H. pylori-related disease, the proportion of the population with the genotype would fall in parallel with the decline in incidence of peptic ulcer and gastric cancer in this population. Methods: Three hundred and forty-three Japanese isolates of [t. pylori from individuals with gastritis alone, duodenal ulcer, gastric ulcer, or gastric cancer were characterized by vacA typing by polymerase chain reaction (PER) and DNA sequencing. Vacuolating cytotoxin activity was measured by in vitro assay using the Vero cell. Results: Three hundred and eighteen strains (92.7%) isolated were classified as sla/ml, 2% were sla/m2, 0.9% were slb/ml, and 0.3% was slb/m2; no type s2 strains were found. Cytotoxin activity was found with both ml and m2 vacA genotypes. There was no association between the vacA genotype or clinical outcome (e.g., gastritis, ulcer, or cancer). In the gastritis only individuals there was no fall in predominant vacA or cagA genotype in younger age individuals compared to the elderly populations. Conclusion: cagA gene-positive, sl/ml strains are the dominant type of H. pylori in Japan. The hypotheses that ml and not m2 vacA genotype was associated with cytotoxin activity, that cagA positivity was associated with genotype sla, or that cagA or vacA genotypes could be used to identify H. pylori of increased virulence related to clinical outcome were not supported. G1381 THE POSITIVE RATES OF 1t. PYLORI DECLINED MARKEDLY DURING TIlE FIRST TWO YEARS OF HEMODIALYSIS. Y. Yamaoka, T. Kodama, K. Kashima, Third Departments of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan. Background: Since patients with end-stage renal failure have an increased gastric urea concentration they may be more susceptible to colonization with H. pylori, however, the data to support that hypothesis is controversial. Aim: To identify the roles of/-/, pylori in hemodialysis patients. Methods: Four hundred and seventy-five hemodialysis patients were enrolled in this study. H. pylori status was determined by serological tests. Furthermore, 168 hemodialysis patients had undergone an endoscopy. As a control, 486 patients without chronic renal failure who had undergone serological test and endoscopy were also enrolled in this study. Results: Only 48% of hemodialysis patients were positive for H. pylori, significantly lower rates compared with control (71%). There were no relationships between the infection rates and clinical outcome (e.g., chronic gastritis: 46%, duodenal ulcer 50%). Diabetic derived patients were infected with H. pylori significantly higher than glomerulonephritis derived patients (n=89, 59.6% vs. n=339, 44.5% p < 0.01). In both hemodiaiysis patients and control, the positive rates of H. pylori were increased with age. Positive rates of H. pylori in patients having undergone hemodialysis within 2 years were significantly higher than those more than 3 years [duration < 1 years; 74.1%(n=27), 2 years; 75.0% (n=32), 3 years; 51.2%(n=43), > 3 years; 43.4% (n=373)]. Conclusion: Hemodialysis patients have similar positive rates of H. pylori infection to control in the first 1 to 2 years of hemodialysis. Certain factors prevent the infection of 11. pylori during the first 2 years of hemodialysis. • G1382

THE DEVELOPMENT OF "VIRTUAL ENDOSCOPY" BY TRANSABDOMINAL ULTRASONOGRAPHY. N. Yamashita. J. Hata, K. Haruma, S. Aoki, S. Yoshida, H. Kusunoki, M. Hananoki, E. Okamoto, S. Tanaka, M. Yoshihara, K. Sumii, G. Kajiyama. First Dept. of Internal Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. Three-dimensional ultrasonography (3D-US) has been developed recently. However, 3D-US by transabdominal ultrasonography (US) for the evaluation of gastrointestinal diseases has not yet been reported. The AIM of this study was to evaluate the usefulness of this method. We advocate the term "virtual endoscopy" for the demonstration of virtual endoscopic figures by 3D-US. METHODS: Sixteen patients (9 men, 7 women, mean age 52.0 years) with 2 early and 4 advanced gastric cancer, 1 advanced colon cancer, 4 gastric submucosal tumors, and 5 Crohn's disease (2 colonic type, 3 small intestinal type) entered the study. The gastric disease patients were scanned immediately after the ingestion of distilled water (300 mL), and the small intestinal / colonic disease patients were scanned an hour after the ingestion of Golytely solution (1~2 L). The US equipment employed was SSA-380A (Toshiba, Japan) with the 3.0~5.0 MHz transducer, and 3D-US equipment was COMPACT 3D (Tomtec, Germany) with freehand acquisition device using an electromagnetic sensor system. Virtual endoscopic images were obtained by the surface mode, which is the accumulation of the echogenic lines in each sonographic cross-sections with the site recognition. When virtual endoscopic figures (e.g. size, shape, location) were similar to those of the real endoscopy, we classified these figures as being well, otherwise virtual endoscopic figures were classified as being poor. RESULTS: Results are shown in Table. V'rtu Disease

early

colon Ca

~mhn's tltseas¢ Small intestinal donic type I type

GC

adv~d GC

gastric SM'T

WELL

1

4

3

1

2

2

POOR

1

0

1

0

0

1

Good images were obtained in 13 of 16 patients (81.2%). Poor images of 2 cases with gastric disease were due to floating mucous in the stomach, and poor image of one case with small intestinal disease was due to bowel residua and highly bended intestine. CONCLUSION: Virtual endoscopy can be an alternative modality to endoscopy to obtain the non-invasive images of gastrointestinal diseases. Also, this method is useful in the evaluation of

April

Esophageal, Gastric, and Duodenal Disorders A339

1998

lesions where the endoscopic approach is difficult (e.g. small bowel, the lesions beyond the severe stenosis). G1383 SEQUENTIAL CHANGE OF ESOPHAGEAL NITRIC OXIDE LEVEL IN EXPERIMENTAL ESOPHAGITIS IN RATS. S. Yamato, K. Matsueda, M. Uchida*, R. Shoda, A. Muraoka, M. Matsukawa, K. Sekigawa, J. Akiyama, S. Fukushima, H. Ohara, A, Niihata, N. Masaki, S. Hayashi, E. Shimojo, and N. Umeda. Division of Gastroenterology, International Medical Center of Japan, Tokyo, Japan, and *Laboratory of Pharmacology, Meiji Institute of Health Science, Kanagawa, Japan. Background & Aims: Increased nitric oxide (NO) level in colonic mucosa has been reported in patients with inflammatory bowel disease. However, the level of NO in the esophageal mucosa in reflux esophagitis has not been clarified. It is possible that mueosal NO could decrease the lower esophageal sphincter pressure, and this would aggravate esophagitis further. No adequate experimental model for reflux esophagitis has been established. Aims of this study were 1) to develop an acute experimenral model of reflux esophagitis in rats and 2) to measure NO in the esophagus. Methods: 1) Male Wister rats were used. Acute experimental esophagitis was produced by ligation of the duodenum and the forestomach for 4 hours. After ligation was released, 0(immediately after), 1, and 5 days of the esophagus were examined histologically and compared with control (N=5 in each groups). NO level of the esophageal tissue in each groups were also measured. The measurement of NO was due to conversion of L-arginine to L-citrulline. Results: 1) After 4 hours of ligation of the duodenum and the forestamach (0 day), edema, bleeding and ulceration of the esophagus developed. After 1 and 5 days from induction of the esophagitis, esophageal ulceration further deteriorated and some of them showed perforation. Histologically, massive reduction of the esophageal mucosa was observed on 0 day and remarkable inflammatory cell infiltration in the submucosa were noted on 1 and 3 days. 2) The esophageal NO level decreased on 0 day compared with that of control (246 vs 181 dpm/mg, p<0.05). On 1 and 5 days after producing acute esophagitis, the level of NO increased above that of control level (1 and 5 days: 2.3 and 3.1 times of the control, p < 0.05). Coudnsion: Acute experimental esophagitis in rats could be induced by 4 hours-ligation of the duodenum and the forestomach. The esophageal NO in these experimental esophagitis decreased immediately after the production of esophagitis, then increased thereafter. • G1384 A RAT NORMAL GASTRIC SURFACE MUCOUS CELL LINE (RGM-1) MONOLAYER: AS AN EXCELLENT IN VITRO MODEL FOR STUDYING GASTRIC MUCOSAL BARRIER FUNCTIONS. A. Yanaka, H. Suzuki, H. Matsui, M. Muto, T. Shibahara, Y Murata, H. Matsui, A. Nakahara, and H. Muto. Department of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan. RGM-1 is a cell line established from a normal rat gastric mucosa. Recent studies have shown that RGM-1 cells secrete mucus in response to prostaglandins, as do normal surface mucous ceils (Digestion 57:196, '96). It seems, therefore, reasonable to assume that monolayers of the RGM-1 cells show epithelial cell polarity as normal gastric surface mucous ceils. In the present study, we examined epithelial cell polarity of the RGM-1 cell monolayers, with special reference to the difference in permeability of H÷ across the apical and the basolateral membranes. Methods_: RGM-1 cells, seeded on permeable membranes (Falcon culture inserts) were incubated with the culture medium containing DMEM, Ham's F12, and FCS for 5 to 7 days in CO: incubator. After confirming that the cells are confluent, the monolayers were mounted in Lucite-Ussing chambers. The serosal and the luminal sides were bathed with a Ringer's solution buffered with HEPES (pH 7.4), and with an unbuffered 150 mM NaCI solution (pH 7.4), respectively. Transmucosal potential difference (PD) and electrical resistance (R) were monitored. Some cells, loaded with a pH-sensitive fluorescein, BCECF, were mounted in a special cuvette, which was designed to allow perfusion of both the luminal and the serosal sides separately. Intracellular pH (pHi) in the RGM-1 cell monolayers was fluorometrically measured, as described previously (Am J Physiol 258:G815, '90). The effects of graded doses of luminal acid (luminal pH: pilL 7.4~1.0), or serosal acid (serosal pH: pHs 7.4~3.0) on PD, R and pHi were examined. Some cells were assessed morphologically. Results: 1) Morphology of the RGM-1 cell monolayers showed numerous microvilli on the apical membranes, and some mucus granules within the cytoplasm. A number of tight junctions are observed between the cells. 2) During serosal acidification, R and pHi decreased at pHs 7.0-6.5, effects augmented by serosal amiloride, an inhibitor of Na÷/H+ exchanger, and prevented by monensin or EGF, agents known to stimulate Na÷/H+ exchange. 3) During luminal acidification, R and phi did not change at pilL 7.4-5.0, At pilL 5.0-2.5, R increased significantly, while phi did not change. Further acidification to pilL 2.0-1.0 caused marked reduction in both R and phi, effects enhanced by amiloride in the serosal but not in the luminal bath. Conclusions: These results suggest that 1) the apical membranes of RGM-1 cell monolayers are remarkably impermeable to H÷, while the basolateral membranes are relatively permeable, 2) the amiloride-sensitive Na÷/H+ exchangers are present only on the basolateral membranes, 3) RGM-1 cell monolayers establish excellent polarity as normal gastric epithelial cells.

• G1385 ACID-ACTIVATED VACUOLATING CYTOTOXINS OF HELICOBACTER PYLORI (HP) INHIBIT BOTH THE PROSTAGLANDIN-DEPENDENT AND THE GUANYLIN-DEPENDENT BICARBONATE SECRETIONS IN ISOLATED RAT DUODENAL MUCOSA IN VITRO. A. Yanaka, H. Suzuki, H. Matsui, A. Nakahara, S. Takahashi*, T Ito**, and H. Muto. Dept. of Gastroanterol, Univ of Tsukuba, Ibaraki, Japan. *3rd Dept. of Int. Med, Kyorin Univ, Tokyo, Japan. **Tokyo Metropolitan Research Laboratory of Public Health, Tokyo, Japan. It has been reported that acid-stimulated duodenal mucosal HCO~- secretion is impaired in HP-infected duodenal ulcer patients, and that eradication of HP restores the HCO~- secretion (Gastro 110:705, '96). The mechanisms by which HP inhibits HCO~- secretion, however, have not been well clarified. Recent studies in rat duodenal mucosa have shown that acidstimulated HCO~secretion is mediated by both the prostaglandin-cyclic AMP (Am J Physiol 243: G348, '82), and the guanylin-cyclic GMP pathways (Gastro 111: 1558, '97). We have previously shown that supematants of HP inhibit basolateral Na+/HCO3- cotransport in duodenal enterocytes (Gastro 110: A300, '96). The present study was designed to determine if vacuolating cytotoxins of HP affect duodenal HCO~- secretion. Methods: Intact sheets of rat proximal duodenal mucosae were incubated in Ussing chambers in vitro. The luminal and the serosal sides were bathed with 150 mM NaCI/100%O 2 (pH 7.4), and with HCO~-Ringer/95%O2-5%CO2 (pH 7A), respectively. Transmucosal potential difference (PD), electrical resistance (R), and short circuit current (Isc) were monitored. HCO~- secretion was measured by titration of the luminal solution at pH 7.4 with lmM HC1. Vacuolating cytotoxins (Vac A proteins) were prepared from Cag A (+), Vac A (+)-HIP strains, as described elsewhere (J Biol Chem 287: 10570, '92). Vac A was pre-activated by decreasing pH to 2.0 for 10 rain by addition of HCL Mucosal contents of cAMP and cGMP were measured by a radioimmunoassay. The effects of Vac A on electrophysiologyand HCO3- secretion were examined in the presence or absence of PGE2 and guanylin. Results: I) Exposure of the luminal surface to 10 mM HCI for 10 rain induced increases in PD, lsc and mucosal contents of cAMP and cGMP, followed by a marked elevation of HCO~- secretion. 2) Vac A prevented the HCl-stimulated increases in PD, Isc, and HCO~- secretion, effects restored by dbcAMP and/or by dbcGMP, but not affected by PGEz or guanylin. 3) Vac A inhibited PGEz-stimulated cAMP production and HCO3-secretion, effects reversed by dbcAMP. 4) Vac A also inhibited guanylin-stimulated cGMP production and HCO3- secretion, effects reversed by dbcGMP. Conclusions: These results suggest that acid-activated Vac A proteins of HP inhibit HCl-stimulated duodenal mucosal HCO3- secretion by blocking both the prostaglandin-cAMP and the guanylin-cGMP pathways. • G1386 EFFICACY OF SUCRALFATE SUSPENSIONS IN "tHE ERADICATION OF HELICOBACTER PYLORI. A. Yanaka, H. Suzuki, M. Muto, T. Shibahara, Y Murata, H. Matsui, H. Yokota, A. Nakahara, H. Muto. Department of Gastroenterology, University of Tsukuba, Ibaraki, Japan. It has been generally accepted that inhibition of acid secretion by high doses of proton pump inhibitors (PPIs), in combination with antibiotics, are essential tools for eradication of Helicobacter pylori (HP). Such high doses of PPIs, however, have been shown to cause prolonged inhibition of acid secretion and subsequent hypergastrinemia. On the other hand, sucralfate, which has no effect on acid secretion, strongly inhibits activity of HP urease. Based on this background, we hypothesized that suspensions of sucralfate, which diffuse over the gastric luminal surfaces, may render the HP more susceptible to antibiotics, thereby enhancing eradication of HP. The present study was designed first to determine if the sucralfate suspensions enhance eradication of HP during antibiotics therapy, and second to determine if the effect of the sucralfate suspensionson HP eradication is equivalent with that of PPIs. Methods: 150 patients with gastric and/or duodenal ulcer scars were endoscopically confirmed HP-positive by rapid urease test and by culture of gastric mucosal biopsies. 126 patients agreed to take part in the study by submitting written informed consents. The patients were randomized to receive one of the following three regimens for 2 weeks. A; amoxicillin (Amx) 2000 mg/day+clarithromycin (Clm) 800 mg/day. B; Amx 2000+Clm 800+sucralfate suspension (Sue) 30 ml/day. C; Amx 2000+Clm 800+omeprazole (Omp) 40 mg/day (Omeprazole 40 mg/day is double the ordinary daily dose in Japan). In 119 patients, who had completed the 2 weeks therapy, eradication of HP was assessed by urea breath test, which was performed at 8 weeks after the end of the treatment. Results: Protocol

# of eradicated/total

eradication rate (%)

..............................................................................................................................

A; Amx + Clm B; Amx + Clm + Suc C; Amx + Clm + Omp ...................

18/38 35/40 35/41

i'?'76".iSi";;7~roui;'X'"'"~'noi'si~/;ii:/~it'v2~oui;'i~

47.3 87.5* 85.4*# ..................

Conclusions: These results suggest that 1) sucralfate suspensions enhance eradication of HP during treatment with amoxicillin and clarithromycin, and that 2) efficacy of the sucralfate suspensions in HP eradication is almost equivalent with that of the high dose of omeprazole. This research was supported in part by a grant from Chugai Pharmaceutical Co. Ltd., Japan.