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Stability of the cag pathogenicity island in U.S. and asian Helicobacter pylori isolates
A1256 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118. No, 4
5747 RELATIONSHIP BETWEEN HELICOBACTER PYLORI ICEA GENOTYPES, VACA ALLELES AND CLINICALLY SIGNIFICANT DISEASE IN SOUTH AFRICA. Mark Kidd, Richard M. Peek, Albert 1. Lastovica, Alex F. Kummer, Japie A. Louw, Univ of Cape Town, Cape Town, South Africa; Vanderbilt Med Ctr. Nashville, TN. Introduction: South African Helicobacter pylori isolates are characterized by an almost universal presence of cagA, but have differences in vacuolating cytotoxin gene (vacA) alleles which correlate with clinically significant disease. The candidate virulence marker gene, iceA, has not been investigated. The aims of this study were to characterize the iceA genotype of South African H. pylori isolates and to investigate whether variants of this locus were associated with differences in vacA status and clinical outcome. Methods: We studied 109 H. pylori strains isolated from dyspeptic patients (36 with peptic ulceration [PUD], 26 with gastric adenocarcinoma [GCl and 47 with no pathology other than gastritis). DNA was isolated from cultured isolates and PCR-amplified for the presence of iceAI or iceA2 genes. and for differences in the genetic organization of iceA2. Results: In this study population, iceAI was detected in 74 (68%) of isolates and iceA2 in 85 (80%). Fifty-four isolates (50%) were positive for both iceAl and iceA2. One isolate did not have a detectable iceA genotype. A statistically higher prevalence (p < 0.01) of iceAI was noted in isolates from patients with GC than isolates from patients with gastritis alone while a significantly higher prevalence (p < 0.003) of iceA2 was found in the latter group. No difference was noted for isolates from patients with PUD. Analysis of the organization of iceA2 revealed that the iceA2-1 variant was found more commonly in isolates from patients with gastritis alone (61%, p < 0.008 vs. PUD) while the iceA2-2 was more common in PUD isolates (48%, p < 0.03 vs. gastritis alone). Analysis of the vacA gene demonstrated a strong relationship between PUD or GC and vacA sl/iceAI (p < 0.(05) and between gastritis alone and vacA s2/iceA2 (p < 0.001). Conclusion: In this study, the iceAI genotype was significantly associated with gastric adenocarcinoma but not peptic ulcer disease. In addition, no isolate from patients with clinically significant disease exhibited either the vacA s2/iceAI or the vacA s2/iceA2 genotypes. Analysis of iceA is useful in South Africa, and combination analysis of virulence factors may provide excellent negative markers for disease associated organisms.
5748 ALTERATIONS IN THE CAG PATHOGENICITY ISLAND ARE ASSOCIATED WITH VACA ALLELES AND DISEASE IN SOUTH AFRICAN HELICOBACTER PYLORI ISOLATES. Mark Kidd, Albert J. Lastovica, John C. Atherton, Japie A. Louw, Univ of Cape Town, Cape Town, South Africa; Univ of Nottingham. Nottingham, United Kingdom. Introduction: The development of clinically significant gastroduodenal disease in South Africa is associated with the vacuolating cytotoxin gene (vocA) s I genotype of H. pylori but not with ilie presence of the cytotoxin associated gene (cagA). cagA occurs in >95% of South African isolates and is a variable marker for the entire cagpathogenicity island (PAl). The aims of this study were to characterize the cagPAI in South African H. pylori isolates and to investigate whether structural variants of this multigene locus were associated with variations in vacAstatus and clinical outcome. Methods: We studied 109 H. pylori strains isolated from dyspeptic patients (36 with peptic ulceration, 26 with gastric adenocarcinoma and 47 with no pathology other than gastritis). DNA was isolated from cultured isolates and PCR amplified for differences in selected marker genes (cagA,E,M,Q~S.T.6-7) of the cagPAI and alleles of vacA. Results: The distribution of cag marker genes are tabulated. *p < 0.00 I vs. gastritis (Fisher's test). Analysis of the vacA signal sequence and mid-region demonstrated a strong relationship between the virulence associated alleles vacA sl (p < 0.005) and vacA ml (p = 0.05) and an intact cagPAI. Conclusion: In this study, the presence of a contiguous cagPAI was significantly associated with peptic ulcer disease and gastric adenocarcinoma, suggesting the importance of genes in this island to pathogenesis of disease in South Africa. This hypothesis is supported by the strong relationship between virulence alleles vacA s Im I and the PAL The findings of similar virulence markers in isolates from dissimilar diseases, however, indicates that further work is required to differentiate the relationship between specific genes, host factors and disease processes. Patient
5'·cag6·7
cagT
cagQ-S
cagM
cagE
cagA·3'
Gastritis PUD GC
45% 81%' 73%'
53% 97%' 88%'
43% 86%' 77%'
91% 94% 96%
72% 94%' 92%'
100% 100% 100%
5749 DOES THE DEVELOPMENT OF BARRETT'S ESOPHAGUS DEPEND ON DURATION OF HELICOBACTER PYLORI INFECTION? Uta Kiltz, Juergen Baier, Dirk Hagemann, Wolfgang E. Schmidt, Romuald J. Adamek, Dept of Medicine, St Josef Hosp, Bochum, Germany; St Vinzenz-Krankenhaus. Bochum, Germany. Background: It has been estimated that the colonization of gastric epithelium with Helicobacter pylori (Hp) might have an influence on develop-
ment of Barrett's mucosa. Prevalence of Hp infection reflects longstanding infection principal acquired in childhood. Little data are available about the relationship between patients age & Hp status on the development of Barrett's esophagus (BE). Aim: To assess patients age & Hp status as a risk factor for BE. Material&Method: We studied 464 consecutive patients with a diagnosis of GERD and 116 consecutive patients with no abnormalities in the upper GI tract (controls). During an upper GI endoscopy antral and corpus biopsies were taken in order to determine Hp status. In all patients 4 radial biopsies from just below the Z-line were obtained to identify globelet cells. Results: 400 patients presented with reflux esophagitis (group A), 64 patients with histopathologic prooven Barrett's mucosa (group B). 116 patients fulfilled the inclusions criteria of the control group (group C). The median age of all patients was 60 yrs. (range: 19-89); 47% female. The overall Hp positivity was 39.8%. In patients with reflux esophagitis the Hp prevalence was 39.5%, in BE 33% and 45% in controls (p<0.05). Patients younger than 60 yrs. with BE had a significantly lower prevalence of Hp compared to patients older than 60 yrs. (14% vs 52%, p<0.05, Table I). The Hp prevalence did not differ in young and old patients of group A and C (37%/41 % vs 44%/45%). Conclusion: Our data imply that the presence of Hp might delay the development of BE. Due to the fact that first infection with Hp took place in childhood, duration of Hp infection is believed to be a negative risk factor for development of Barrett's mucosa. Table 1:Median values oftheanalyzed parameters HP pos/neg A B C
all patients
<60 a
>60 a
p-value
158/400(39.5%) 21/64(33%) 521116(45%)
721194(37%) 4/28(14%) 25/59(44%)
86/206(41 %) 19/36(52%) 26/57(45%)
p=n.s. p<0.05 p=n.s.
5750 HELICOBACTER PYLORI INFECTION OF HUMAN GASTRIC EPITHELIAL CELLS UPREGULATES INOS EXPRESSION AND INDUCES THE POLARIZED SECRETION OF NITRIC OXIDE. Jung Mogg Kim, Joo Sung Kim, Hyun Chae Jung, In Sung Song, Chung Yong Kim, Dept of Microbiology, Hanyang Univ Coli of Medicine, Seoul, South Korea; Seoul National Univ, Soeul, South Korea; Seoul National Univ, Seoul, South Korea. Background: Gastric epithelial cells can provide signals for the initiation of an acute mucosal inflammatory response following H. pylori infection. Nitric oxide (NO), generated through the conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS), is known to be an important modulator of the mucosal inflammatory response. In this study, we asked whether H. pylori infection of human gastric epithelial cells could upregulate epithelial cell inducible NOS (iNOS) gene expression and NO production could show polarity. Methods: Human gastric epithelial cell lines (AGS, Hs746T, SNU-5) were infected with H. pylori, or stimulated with proinflammatory agonists. Expression of iNOS mRNA and protein was assessed by quantitative RT-PCR using an internal standard and Western blot analysis. respectively. NO production was assessed by determining nitrite levels in culture supernatants. To determine the polarity of NO secretion by H. pylori-infected epithelial cells, Caco-2 cells were cultured as polarized monolayers in transwell chambers and NO production was measured. Results: iNOS mRNA levels were significantly upregulated in the cells infected with H. pylori (26-fold increase in H. pylori-infected Hs746T cells 8 h post-infection compared with non-infected controls). Expression of iNOS protein of H. pylori-infected Hs746T cells was confirmed by Western blot analysis 12 and 24 h after infection. Increased NO production in gastric epithelial cells was seen as early as 4-h post-infection, reached maximal levels by 24 h post-infection, and was abrogated by inhibitors of NO synthesis. After H. pylori infection of polarized epithelial cell monolayers, nitrite was released predominantly into the apical compartment, but IL-8 was released predominantly into basolateral compartment. Moreover, addition of IFN-y to H. pylori-infected polarized epithelial cells showed synergistic increased apical and basolateral NO release. Conclusion: These results suggest that upregulation of iNOS expression and NO production by gastric epithelial cells infected with H. pylori play a role in the modulation of gastric mucosal inflammation.
5751 STABILITY OF THE CAG PATHOGENICITY ISLAND IN U.S, AND ASIAN HELICOBACTER PYLORI ISOLATES. Sung-Kook Kim, Michele Sozzi, David M. Aronoff, Martin J. Blaser, Kyungpook National Univ Hosp. Taegu, Korea; Cro, Aviano, Italy; Vanderbilt Univ, Nashville, TN. Background: The cag island (CI), marked by cagA, is present in 50-70% of Western and 80-95% of Asian H. pylori (Hp) strains. Occasionally, Hp isolates from the same patient with identical RAPD-PCR profiles differ in cagA status. To investigate this phenomenon, we performed PCR using primers to the genes (HP0519 and HP0549) flanking the CI. In cagG strains, the site of the CI is empty; thus the empty site (ES) PCR yields a product of 360bp. In a cag+ strain. due to the 35-40 kb cag island, the ES-PCR should be negative. Methods: We performed the ES-PCR on 87 Hp isolates from 4 countries (US, n=22; China, n=30; India, n=24; Japan, n = 11). For selected strains with positive products > 360bp, the products
AGAA1257
April 2000
were cloned and the sequences analyzed. For strain 84- 183, serial dilution PCR compared the frequency of the ES-product with that from glmM, a urease-related gene, and with picB, a CI gene. 8 single colonies (SC) of 84-183 were picked for further analysis. Results: Among 7 cagG US isolates, all had the expected 360bp ES-PCR product. Among 15 cag+ strains, 12 had no product, as expected, but 3 (20%) had products of 1.0 or 1.6kb. For strain 84-183, sequence analysis of the 1.0kb product revealed a complete deletion of the CI except for a fragment from IS606. Seria l dilution PCR indicated that the deletion frequency was 10--4 compared to both glmM and picB. Examination of 8 single colonies showed no deletions, indicating that it was not occurring at high frequency in vitro. Of the 65 Asian cag + isolates, a 0.9 to 1.2kb ES-PCR product was observed in 10 (43%) from India. 15 (50%) from China, and 6 (55%) from Japan. However, sequence analysis of 3 ES-PCR products did not show any sequences for HP0519, HP0549. or the CI, indicating non-specific amplification by the primers which were based on the Western sequences for these two genes. Conclusions: Deletion of the CI appears to not be a rare event in H. pylori isolates from US patients and in vitro occurs at a frequency of approximately 10--4. This phenomenon may be facilitated by the direct repeats flanking the CI, as well as by IS606 (and IS605). The failure of the ES-PCR primers to detect this phenomenon in Asian strains may reflect essential differences in the biology of their CI, or sequence diversity in the flanking HP05 I9 and HP0549 genes.
5752 TRANSEPITHELIAL MIGRATION OF NEUTROPHILS INDUCED BY HEUCOBACTER PYLORI IN INVERTED CACO·2 CELL MONOLAYER. Jae Gyu Kim, Hyun Chae Jung, Byung Chul Yoo, In Sung Song, Sill Moo Park, Chung Yong Kim, Chung Ang Univ Coli of Medicine, Seoul, South Korea; Seoul National Univ, Seoul, South Korea. One of the characteristic histologic features of Helicobacter pylo ri (HP) infection is neutrophil infiltration of the gastric mucosa. It is thought to be a direct response to the presence of HP. But little is known concerning the mechanisms by which neutrophils migrate across epithelia. Aims: To induce transepithelial migration (TEM) of neutrophils by HP in inverted Caco-2 cell monolayer and analyze the mechanism of TEM of neutroph ils in HP infection. Method s: HP were inoculated to confluent monolayers of Caco-2 cells (ATCC HTB-37) till 24 hours. Total cellular RNA was extracted from monolayers of Caco-2 cells. Reverse transcription (RT)PCR was performed to quantify expression of IL-8 mRNA. Inverted Caco-2 cell monolayers were grown on polycarbonate filters (area, 0.33 crrr'; pore diameter. 3/Lm; Transwell; Costar). Confluence and integrity of monolayers were assessed by measuring the transepithelial electrical resistance. TEM of neutrophils was induced by HP (VacA+, CagA + jin inverted Caco-2 cell monolayers. And we observed TEM of neutrophils with light microscope and electron microscope till 8 hours. Isolated neutrophils were labeled with chromium-5 1 e lCr) and applied to the basal compartment of inverted Caco-2 cell monolayers. And also HP were inoculated to apical surface of inverted Caco-2 cell monolayers (HP:cell ratio, 200: 1). We quantify TEM of neutrophils induced by HP till 8 hours. Expression of IL-8 mRNA after HP inoculation and TEM of neutrophil s induced by HP were compared with those of condition without HP. Results: Inoculation of HP to monolayers of Caco-2 cells resulted in increased level of IL-8 mRNA till 8 hours. Migrating and migrated neutrophils across inverted Caco-2 monolayer were found after HP inoculation till 8 hours. TEM of neutroph ils in inverted Caco-2 cell monolayers increased till 8 hours after HP inoculation. Conclusions: HP might induce TEM of neutrophils in inverted Caco-2 cell monolayer through increase of IL-8 mRNA expression.
5753 HEUCOBACTER PYWRI ERADIC ATION AND INCIDENCE OF REFLUX ESOPHAGITIS IN PATIENTS WITH DUODENAL AND BENIGN GASTRIC ULCERS IN KORE A. Nayoung Kim, Seon Hee Lim, Kye Heui Lee, Wook Ryul Choi, Kangnam Gen Hosp, Seoul. South Korea. Little is known about the relationship between Helicobacter pylori (Hp) infection and reflux esophagitis (RE). This study was conducted to investigate whether or not Hp plays the protective role in the pathogenesis of RE in patients with duodenal (DU) and benign gastric ulcers (BGU). The prevalence rates of RE depending on Hp status in the consecutively diagnosed DU or BGU patients were evaluated. In addition, the incidence rates of RE depending on Hp status were evaluated for those patients who received follow-up endoscopy at least 6 months after eradication treatment. The prevalence rates of RE were 5.6% (2 patients) in the 36 Hp-negative DU group and 7.1% (45 patients) in the 63 1 Hp-positive DU group, and there was no statistical difference. Similarly, those of BGU patient s was 9.4% (4 1 patients) in the 436 Hp-positive group. slightly higher than that of 86 Hp-negative group, 3.5% (3 patients), but without statistical significance. After eradication treatment the RE incidence rates were 2.3% (2 patients) in the 87 Hp-eradicated DU group and 7.7% (3 patients) in the 39 non-eradicated DU group, and there was no statistical difference. Similarly , those of BGU patients were 6.8% (3 patients) in the 44 Hp-eradicated and 8.7% (2 patients) in the 23 non-eradicated BGU group, again without statistical difference. These results suggest that Hp might not play a protective role in the pathogenesis of RE in patients with DU or BGU in Korea.
5754 " ENDOSCOPIC" REFLUX ESOPHAGITIS IS ATTRIBUTED TO " HISTOLOGICAL" ESOPHAGITIS, BUT INFLAMMATORY CHANGES DO NOT ALWAYS CONTRIBUTE TO REFLUX SYMP· TOMS. Arata Kimura, Shingo Tuji, Yoshimi Kakiuchi, Masakazu Yasumaru, Naoki Kawai, Masahiko Tujii, Yutaka Sasaki, Masatsugu Hori, Sunao Kawano, Osaka Univ Grad Sch of Medicine, Suita, Japan; Osaka Univ Faculty of Medicine. Suita, Japan. Background and Aim: Gastroesophageal reflux disease(GERD) is a complex and multifactorial disease. But the precise mechanism of reflux symptoms is not clearly elucidated. And it is not clear that whether these reflux symptoms and endoscopic findings are associated with histological inflammation. The aim of this study is to investigate the relationship between endoscopic, histological esophagitis and reflux symptoms. Subjec ts and Methods: 19 patients (12 males, 7 females) with or without reflux symptom (heartburn or acid regurgitation) underwent endoscopy after informed consent had been obtained. If reflux esophagitis is present endoscopicaly, the mucosal break is classified according to the classification of Los Angeles. From each patient, at least two biopsy specimens were obtained from esophageal mucosa adjacent to squamocolumnar jun ction. The specimens were fixed in 10% formalin, embedded in paraffin, stained with Hematoxyline and eosin. Histological esophagitis was defined as the presence of the following findings; intraepitheIial neutrophilic or eosinophilic infiltration, capillary congestion or hemorrhage in papillae , basal cell hyperplasia, erosions or ulcers in the epithelium. Results: Of 9 patients with endoscopy-positive esophagitis, all 9 (100%) patients had histologically-positive esophagitis , whereas 3 out of 10 (30%) endoscopy-negative patients also had esophagitis histologically. There was a significant correlation between endoscopic findings and histological findings (p = 0.003 by Fisher' s exact test). However, 3 of the 4 (75%) patients with reflux symptom had histologically-positive esophagitis, 9 out of 15 (60%) patients who showed no symptom had histologically-positive esophagitis . There was no correlation between reflux symptom and histological findings. Nor no correlation was found between reflux symptom and endoscopic findings. Conclusion: These data suggest that "endoscopic" reflux esophagitis is significantly correlated with "histological" esophagitis, but inflammatory changes do not always contribute to the occurrence of reflux symptoms.
5755 DOES HEUCOBACTER PYLORI INFECTION INDUCE DIFFUSE· TYPE GASTRIC CANCER ? Fumiaki Kitahara, Tadashi Sato, Yuichiro Kojima, Toshiya Nakamura, Atsuro Morozumi, Masayuki A. Fujino, Yamanashi Med Univ, Yamanashi, Japan. Introduction: Several reports have revealed that there is a significant correlation between atrophic gastritis and intestinal-type gastric cancer. But the effect of HP infection on diffuse-type gastric cancer is unsure. We conducted a case control study to evaluate whether the cases with severe atrophy really have a high risk of diffuse-type gastric cancer. Subjects and Methods: The sera at the time of diagnosis from 37 signet ring cell carcinoma cases (male: 23, female:14, mean age: 62 years old), 37 sex- and age-matched poorly differentiat ed adenocarcinoma cases, 74 sex- and age-matched intestinal-type gastric cancer cases, and 148 sex- and agematched cancer-free controls were tested for the presence of anti-HP IgG antibody (HM-CAP ELISA kit) and serum pepsinogen(PG ) levels (PG I and II Riabead). The cut-off point were serum PG I level less than 70 nglmL and IIII ratio less than 3.0. Four catego ries were made, according to serum PG levels and anti-HP IgG antibody. We compared characteristics of three-type ca ncer and control groups, and conducted case-control studies on three cancer types. Results: In signet-type gastric carcinomas, serum PG I and II levels were higher than those in controls (87.8 vs 54.3, 23.1 vs 17.8).In poorly differentiated-type and intestinal-type gastric cancers, 1/11 ratios were lower than controls (2.6, 2.5 vs 3.7), and positive rates of PG levels were higher than controls (56.8%, 54.6% vs 37.8%). The odds ratios were 0.97(95%CI 0.48-1.9), 1.03(0.62-1.63) and 0.87(0.50-1.53) for positive in anti-HP IgG antibody and 1.0. 1.98(1.12-3.42) and 2. 18(1.23-3.78) for positive in serum PG levels of signet-type, poorly differentiated-type and intestinal-type respectively. The odds ratios in the category with severe atrophy (positive PG levels and negative anti-HP IgG antibody) were 1.95 (0.64-5.89),5.42(2.80- 10.52) and 5.89(2.89- 12.97) of signet-type, poorly differentiated-type and intestinal-type respectively. Conclusion: These results suggest the severe atrophic state of low serum PG levels and negative IgG antibody to HP, really has a high risk of poorly differentiated-type and intestinal-type gastric cancer. However, carcinogenesis of the signet-type is affected by HP infection and atrophic gastritis.
GASTROENTEROLOGY Vol. 118. No, 4
5747 RELATIONSHIP BETWEEN HELICOBACTER PYLORI ICEA GENOTYPES, VACA ALLELES AND CLINICALLY SIGNIFICANT DISEASE IN SOUTH AFRICA. Mark Kidd, Richard M. Peek, Albert 1. Lastovica, Alex F. Kummer, Japie A. Louw, Univ of Cape Town, Cape Town, South Africa; Vanderbilt Med Ctr. Nashville, TN. Introduction: South African Helicobacter pylori isolates are characterized by an almost universal presence of cagA, but have differences in vacuolating cytotoxin gene (vacA) alleles which correlate with clinically significant disease. The candidate virulence marker gene, iceA, has not been investigated. The aims of this study were to characterize the iceA genotype of South African H. pylori isolates and to investigate whether variants of this locus were associated with differences in vacA status and clinical outcome. Methods: We studied 109 H. pylori strains isolated from dyspeptic patients (36 with peptic ulceration [PUD], 26 with gastric adenocarcinoma [GCl and 47 with no pathology other than gastritis). DNA was isolated from cultured isolates and PCR-amplified for the presence of iceAI or iceA2 genes. and for differences in the genetic organization of iceA2. Results: In this study population, iceAI was detected in 74 (68%) of isolates and iceA2 in 85 (80%). Fifty-four isolates (50%) were positive for both iceAl and iceA2. One isolate did not have a detectable iceA genotype. A statistically higher prevalence (p < 0.01) of iceAI was noted in isolates from patients with GC than isolates from patients with gastritis alone while a significantly higher prevalence (p < 0.003) of iceA2 was found in the latter group. No difference was noted for isolates from patients with PUD. Analysis of the organization of iceA2 revealed that the iceA2-1 variant was found more commonly in isolates from patients with gastritis alone (61%, p < 0.008 vs. PUD) while the iceA2-2 was more common in PUD isolates (48%, p < 0.03 vs. gastritis alone). Analysis of the vacA gene demonstrated a strong relationship between PUD or GC and vacA sl/iceAI (p < 0.(05) and between gastritis alone and vacA s2/iceA2 (p < 0.001). Conclusion: In this study, the iceAI genotype was significantly associated with gastric adenocarcinoma but not peptic ulcer disease. In addition, no isolate from patients with clinically significant disease exhibited either the vacA s2/iceAI or the vacA s2/iceA2 genotypes. Analysis of iceA is useful in South Africa, and combination analysis of virulence factors may provide excellent negative markers for disease associated organisms.
5748 ALTERATIONS IN THE CAG PATHOGENICITY ISLAND ARE ASSOCIATED WITH VACA ALLELES AND DISEASE IN SOUTH AFRICAN HELICOBACTER PYLORI ISOLATES. Mark Kidd, Albert J. Lastovica, John C. Atherton, Japie A. Louw, Univ of Cape Town, Cape Town, South Africa; Univ of Nottingham. Nottingham, United Kingdom. Introduction: The development of clinically significant gastroduodenal disease in South Africa is associated with the vacuolating cytotoxin gene (vocA) s I genotype of H. pylori but not with ilie presence of the cytotoxin associated gene (cagA). cagA occurs in >95% of South African isolates and is a variable marker for the entire cagpathogenicity island (PAl). The aims of this study were to characterize the cagPAI in South African H. pylori isolates and to investigate whether structural variants of this multigene locus were associated with variations in vacAstatus and clinical outcome. Methods: We studied 109 H. pylori strains isolated from dyspeptic patients (36 with peptic ulceration, 26 with gastric adenocarcinoma and 47 with no pathology other than gastritis). DNA was isolated from cultured isolates and PCR amplified for differences in selected marker genes (cagA,E,M,Q~S.T.6-7) of the cagPAI and alleles of vacA. Results: The distribution of cag marker genes are tabulated. *p < 0.00 I vs. gastritis (Fisher's test). Analysis of the vacA signal sequence and mid-region demonstrated a strong relationship between the virulence associated alleles vacA sl (p < 0.005) and vacA ml (p = 0.05) and an intact cagPAI. Conclusion: In this study, the presence of a contiguous cagPAI was significantly associated with peptic ulcer disease and gastric adenocarcinoma, suggesting the importance of genes in this island to pathogenesis of disease in South Africa. This hypothesis is supported by the strong relationship between virulence alleles vacA s Im I and the PAL The findings of similar virulence markers in isolates from dissimilar diseases, however, indicates that further work is required to differentiate the relationship between specific genes, host factors and disease processes. Patient
5'·cag6·7
cagT
cagQ-S
cagM
cagE
cagA·3'
Gastritis PUD GC
45% 81%' 73%'
53% 97%' 88%'
43% 86%' 77%'
91% 94% 96%
72% 94%' 92%'
100% 100% 100%
5749 DOES THE DEVELOPMENT OF BARRETT'S ESOPHAGUS DEPEND ON DURATION OF HELICOBACTER PYLORI INFECTION? Uta Kiltz, Juergen Baier, Dirk Hagemann, Wolfgang E. Schmidt, Romuald J. Adamek, Dept of Medicine, St Josef Hosp, Bochum, Germany; St Vinzenz-Krankenhaus. Bochum, Germany. Background: It has been estimated that the colonization of gastric epithelium with Helicobacter pylori (Hp) might have an influence on develop-
ment of Barrett's mucosa. Prevalence of Hp infection reflects longstanding infection principal acquired in childhood. Little data are available about the relationship between patients age & Hp status on the development of Barrett's esophagus (BE). Aim: To assess patients age & Hp status as a risk factor for BE. Material&Method: We studied 464 consecutive patients with a diagnosis of GERD and 116 consecutive patients with no abnormalities in the upper GI tract (controls). During an upper GI endoscopy antral and corpus biopsies were taken in order to determine Hp status. In all patients 4 radial biopsies from just below the Z-line were obtained to identify globelet cells. Results: 400 patients presented with reflux esophagitis (group A), 64 patients with histopathologic prooven Barrett's mucosa (group B). 116 patients fulfilled the inclusions criteria of the control group (group C). The median age of all patients was 60 yrs. (range: 19-89); 47% female. The overall Hp positivity was 39.8%. In patients with reflux esophagitis the Hp prevalence was 39.5%, in BE 33% and 45% in controls (p<0.05). Patients younger than 60 yrs. with BE had a significantly lower prevalence of Hp compared to patients older than 60 yrs. (14% vs 52%, p<0.05, Table I). The Hp prevalence did not differ in young and old patients of group A and C (37%/41 % vs 44%/45%). Conclusion: Our data imply that the presence of Hp might delay the development of BE. Due to the fact that first infection with Hp took place in childhood, duration of Hp infection is believed to be a negative risk factor for development of Barrett's mucosa. Table 1:Median values oftheanalyzed parameters HP pos/neg A B C
all patients
<60 a
>60 a
p-value
158/400(39.5%) 21/64(33%) 521116(45%)
721194(37%) 4/28(14%) 25/59(44%)
86/206(41 %) 19/36(52%) 26/57(45%)
p=n.s. p<0.05 p=n.s.
5750 HELICOBACTER PYLORI INFECTION OF HUMAN GASTRIC EPITHELIAL CELLS UPREGULATES INOS EXPRESSION AND INDUCES THE POLARIZED SECRETION OF NITRIC OXIDE. Jung Mogg Kim, Joo Sung Kim, Hyun Chae Jung, In Sung Song, Chung Yong Kim, Dept of Microbiology, Hanyang Univ Coli of Medicine, Seoul, South Korea; Seoul National Univ, Soeul, South Korea; Seoul National Univ, Seoul, South Korea. Background: Gastric epithelial cells can provide signals for the initiation of an acute mucosal inflammatory response following H. pylori infection. Nitric oxide (NO), generated through the conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS), is known to be an important modulator of the mucosal inflammatory response. In this study, we asked whether H. pylori infection of human gastric epithelial cells could upregulate epithelial cell inducible NOS (iNOS) gene expression and NO production could show polarity. Methods: Human gastric epithelial cell lines (AGS, Hs746T, SNU-5) were infected with H. pylori, or stimulated with proinflammatory agonists. Expression of iNOS mRNA and protein was assessed by quantitative RT-PCR using an internal standard and Western blot analysis. respectively. NO production was assessed by determining nitrite levels in culture supernatants. To determine the polarity of NO secretion by H. pylori-infected epithelial cells, Caco-2 cells were cultured as polarized monolayers in transwell chambers and NO production was measured. Results: iNOS mRNA levels were significantly upregulated in the cells infected with H. pylori (26-fold increase in H. pylori-infected Hs746T cells 8 h post-infection compared with non-infected controls). Expression of iNOS protein of H. pylori-infected Hs746T cells was confirmed by Western blot analysis 12 and 24 h after infection. Increased NO production in gastric epithelial cells was seen as early as 4-h post-infection, reached maximal levels by 24 h post-infection, and was abrogated by inhibitors of NO synthesis. After H. pylori infection of polarized epithelial cell monolayers, nitrite was released predominantly into the apical compartment, but IL-8 was released predominantly into basolateral compartment. Moreover, addition of IFN-y to H. pylori-infected polarized epithelial cells showed synergistic increased apical and basolateral NO release. Conclusion: These results suggest that upregulation of iNOS expression and NO production by gastric epithelial cells infected with H. pylori play a role in the modulation of gastric mucosal inflammation.
5751 STABILITY OF THE CAG PATHOGENICITY ISLAND IN U.S, AND ASIAN HELICOBACTER PYLORI ISOLATES. Sung-Kook Kim, Michele Sozzi, David M. Aronoff, Martin J. Blaser, Kyungpook National Univ Hosp. Taegu, Korea; Cro, Aviano, Italy; Vanderbilt Univ, Nashville, TN. Background: The cag island (CI), marked by cagA, is present in 50-70% of Western and 80-95% of Asian H. pylori (Hp) strains. Occasionally, Hp isolates from the same patient with identical RAPD-PCR profiles differ in cagA status. To investigate this phenomenon, we performed PCR using primers to the genes (HP0519 and HP0549) flanking the CI. In cagG strains, the site of the CI is empty; thus the empty site (ES) PCR yields a product of 360bp. In a cag+ strain. due to the 35-40 kb cag island, the ES-PCR should be negative. Methods: We performed the ES-PCR on 87 Hp isolates from 4 countries (US, n=22; China, n=30; India, n=24; Japan, n = 11). For selected strains with positive products > 360bp, the products
AGAA1257
April 2000
were cloned and the sequences analyzed. For strain 84- 183, serial dilution PCR compared the frequency of the ES-product with that from glmM, a urease-related gene, and with picB, a CI gene. 8 single colonies (SC) of 84-183 were picked for further analysis. Results: Among 7 cagG US isolates, all had the expected 360bp ES-PCR product. Among 15 cag+ strains, 12 had no product, as expected, but 3 (20%) had products of 1.0 or 1.6kb. For strain 84-183, sequence analysis of the 1.0kb product revealed a complete deletion of the CI except for a fragment from IS606. Seria l dilution PCR indicated that the deletion frequency was 10--4 compared to both glmM and picB. Examination of 8 single colonies showed no deletions, indicating that it was not occurring at high frequency in vitro. Of the 65 Asian cag + isolates, a 0.9 to 1.2kb ES-PCR product was observed in 10 (43%) from India. 15 (50%) from China, and 6 (55%) from Japan. However, sequence analysis of 3 ES-PCR products did not show any sequences for HP0519, HP0549. or the CI, indicating non-specific amplification by the primers which were based on the Western sequences for these two genes. Conclusions: Deletion of the CI appears to not be a rare event in H. pylori isolates from US patients and in vitro occurs at a frequency of approximately 10--4. This phenomenon may be facilitated by the direct repeats flanking the CI, as well as by IS606 (and IS605). The failure of the ES-PCR primers to detect this phenomenon in Asian strains may reflect essential differences in the biology of their CI, or sequence diversity in the flanking HP05 I9 and HP0549 genes.
5752 TRANSEPITHELIAL MIGRATION OF NEUTROPHILS INDUCED BY HEUCOBACTER PYLORI IN INVERTED CACO·2 CELL MONOLAYER. Jae Gyu Kim, Hyun Chae Jung, Byung Chul Yoo, In Sung Song, Sill Moo Park, Chung Yong Kim, Chung Ang Univ Coli of Medicine, Seoul, South Korea; Seoul National Univ, Seoul, South Korea. One of the characteristic histologic features of Helicobacter pylo ri (HP) infection is neutrophil infiltration of the gastric mucosa. It is thought to be a direct response to the presence of HP. But little is known concerning the mechanisms by which neutrophils migrate across epithelia. Aims: To induce transepithelial migration (TEM) of neutrophils by HP in inverted Caco-2 cell monolayer and analyze the mechanism of TEM of neutroph ils in HP infection. Method s: HP were inoculated to confluent monolayers of Caco-2 cells (ATCC HTB-37) till 24 hours. Total cellular RNA was extracted from monolayers of Caco-2 cells. Reverse transcription (RT)PCR was performed to quantify expression of IL-8 mRNA. Inverted Caco-2 cell monolayers were grown on polycarbonate filters (area, 0.33 crrr'; pore diameter. 3/Lm; Transwell; Costar). Confluence and integrity of monolayers were assessed by measuring the transepithelial electrical resistance. TEM of neutrophils was induced by HP (VacA+, CagA + jin inverted Caco-2 cell monolayers. And we observed TEM of neutrophils with light microscope and electron microscope till 8 hours. Isolated neutrophils were labeled with chromium-5 1 e lCr) and applied to the basal compartment of inverted Caco-2 cell monolayers. And also HP were inoculated to apical surface of inverted Caco-2 cell monolayers (HP:cell ratio, 200: 1). We quantify TEM of neutrophils induced by HP till 8 hours. Expression of IL-8 mRNA after HP inoculation and TEM of neutrophil s induced by HP were compared with those of condition without HP. Results: Inoculation of HP to monolayers of Caco-2 cells resulted in increased level of IL-8 mRNA till 8 hours. Migrating and migrated neutrophils across inverted Caco-2 monolayer were found after HP inoculation till 8 hours. TEM of neutroph ils in inverted Caco-2 cell monolayers increased till 8 hours after HP inoculation. Conclusions: HP might induce TEM of neutrophils in inverted Caco-2 cell monolayer through increase of IL-8 mRNA expression.
5753 HEUCOBACTER PYWRI ERADIC ATION AND INCIDENCE OF REFLUX ESOPHAGITIS IN PATIENTS WITH DUODENAL AND BENIGN GASTRIC ULCERS IN KORE A. Nayoung Kim, Seon Hee Lim, Kye Heui Lee, Wook Ryul Choi, Kangnam Gen Hosp, Seoul. South Korea. Little is known about the relationship between Helicobacter pylori (Hp) infection and reflux esophagitis (RE). This study was conducted to investigate whether or not Hp plays the protective role in the pathogenesis of RE in patients with duodenal (DU) and benign gastric ulcers (BGU). The prevalence rates of RE depending on Hp status in the consecutively diagnosed DU or BGU patients were evaluated. In addition, the incidence rates of RE depending on Hp status were evaluated for those patients who received follow-up endoscopy at least 6 months after eradication treatment. The prevalence rates of RE were 5.6% (2 patients) in the 36 Hp-negative DU group and 7.1% (45 patients) in the 63 1 Hp-positive DU group, and there was no statistical difference. Similarly, those of BGU patient s was 9.4% (4 1 patients) in the 436 Hp-positive group. slightly higher than that of 86 Hp-negative group, 3.5% (3 patients), but without statistical significance. After eradication treatment the RE incidence rates were 2.3% (2 patients) in the 87 Hp-eradicated DU group and 7.7% (3 patients) in the 39 non-eradicated DU group, and there was no statistical difference. Similarly , those of BGU patients were 6.8% (3 patients) in the 44 Hp-eradicated and 8.7% (2 patients) in the 23 non-eradicated BGU group, again without statistical difference. These results suggest that Hp might not play a protective role in the pathogenesis of RE in patients with DU or BGU in Korea.
5754 " ENDOSCOPIC" REFLUX ESOPHAGITIS IS ATTRIBUTED TO " HISTOLOGICAL" ESOPHAGITIS, BUT INFLAMMATORY CHANGES DO NOT ALWAYS CONTRIBUTE TO REFLUX SYMP· TOMS. Arata Kimura, Shingo Tuji, Yoshimi Kakiuchi, Masakazu Yasumaru, Naoki Kawai, Masahiko Tujii, Yutaka Sasaki, Masatsugu Hori, Sunao Kawano, Osaka Univ Grad Sch of Medicine, Suita, Japan; Osaka Univ Faculty of Medicine. Suita, Japan. Background and Aim: Gastroesophageal reflux disease(GERD) is a complex and multifactorial disease. But the precise mechanism of reflux symptoms is not clearly elucidated. And it is not clear that whether these reflux symptoms and endoscopic findings are associated with histological inflammation. The aim of this study is to investigate the relationship between endoscopic, histological esophagitis and reflux symptoms. Subjec ts and Methods: 19 patients (12 males, 7 females) with or without reflux symptom (heartburn or acid regurgitation) underwent endoscopy after informed consent had been obtained. If reflux esophagitis is present endoscopicaly, the mucosal break is classified according to the classification of Los Angeles. From each patient, at least two biopsy specimens were obtained from esophageal mucosa adjacent to squamocolumnar jun ction. The specimens were fixed in 10% formalin, embedded in paraffin, stained with Hematoxyline and eosin. Histological esophagitis was defined as the presence of the following findings; intraepitheIial neutrophilic or eosinophilic infiltration, capillary congestion or hemorrhage in papillae , basal cell hyperplasia, erosions or ulcers in the epithelium. Results: Of 9 patients with endoscopy-positive esophagitis, all 9 (100%) patients had histologically-positive esophagitis , whereas 3 out of 10 (30%) endoscopy-negative patients also had esophagitis histologically. There was a significant correlation between endoscopic findings and histological findings (p = 0.003 by Fisher' s exact test). However, 3 of the 4 (75%) patients with reflux symptom had histologically-positive esophagitis, 9 out of 15 (60%) patients who showed no symptom had histologically-positive esophagitis . There was no correlation between reflux symptom and histological findings. Nor no correlation was found between reflux symptom and endoscopic findings. Conclusion: These data suggest that "endoscopic" reflux esophagitis is significantly correlated with "histological" esophagitis, but inflammatory changes do not always contribute to the occurrence of reflux symptoms.
5755 DOES HEUCOBACTER PYLORI INFECTION INDUCE DIFFUSE· TYPE GASTRIC CANCER ? Fumiaki Kitahara, Tadashi Sato, Yuichiro Kojima, Toshiya Nakamura, Atsuro Morozumi, Masayuki A. Fujino, Yamanashi Med Univ, Yamanashi, Japan. Introduction: Several reports have revealed that there is a significant correlation between atrophic gastritis and intestinal-type gastric cancer. But the effect of HP infection on diffuse-type gastric cancer is unsure. We conducted a case control study to evaluate whether the cases with severe atrophy really have a high risk of diffuse-type gastric cancer. Subjects and Methods: The sera at the time of diagnosis from 37 signet ring cell carcinoma cases (male: 23, female:14, mean age: 62 years old), 37 sex- and age-matched poorly differentiat ed adenocarcinoma cases, 74 sex- and age-matched intestinal-type gastric cancer cases, and 148 sex- and agematched cancer-free controls were tested for the presence of anti-HP IgG antibody (HM-CAP ELISA kit) and serum pepsinogen(PG ) levels (PG I and II Riabead). The cut-off point were serum PG I level less than 70 nglmL and IIII ratio less than 3.0. Four catego ries were made, according to serum PG levels and anti-HP IgG antibody. We compared characteristics of three-type ca ncer and control groups, and conducted case-control studies on three cancer types. Results: In signet-type gastric carcinomas, serum PG I and II levels were higher than those in controls (87.8 vs 54.3, 23.1 vs 17.8).In poorly differentiated-type and intestinal-type gastric cancers, 1/11 ratios were lower than controls (2.6, 2.5 vs 3.7), and positive rates of PG levels were higher than controls (56.8%, 54.6% vs 37.8%). The odds ratios were 0.97(95%CI 0.48-1.9), 1.03(0.62-1.63) and 0.87(0.50-1.53) for positive in anti-HP IgG antibody and 1.0. 1.98(1.12-3.42) and 2. 18(1.23-3.78) for positive in serum PG levels of signet-type, poorly differentiated-type and intestinal-type respectively. The odds ratios in the category with severe atrophy (positive PG levels and negative anti-HP IgG antibody) were 1.95 (0.64-5.89),5.42(2.80- 10.52) and 5.89(2.89- 12.97) of signet-type, poorly differentiated-type and intestinal-type respectively. Conclusion: These results suggest the severe atrophic state of low serum PG levels and negative IgG antibody to HP, really has a high risk of poorly differentiated-type and intestinal-type gastric cancer. However, carcinogenesis of the signet-type is affected by HP infection and atrophic gastritis.