Inhibitory Effect of Ponciretin on Helicobacter pylori VacA Toxin-induced Vacuolation in HeLa Cells

( Jong Mi Kim ),( Ji Eun Shin ),( Eun Ah Bae ),( Myung Joo Han ),( Dong Hyun Kim ) 한국미생물생명공학회 2006 Journal of microbiology and biotechnology Vol.16 No.1

위 질환 환자들에서 분리된 Helicobacter pylori 의 공포화 독소 ( Vacuolating Toxin ) 생성

김충기(Chung Kee Kim),정승수(Sung Soo Jeong),이겸철(Gyum Cheol Lee),서대홍(Dae Hong Suh),송갑영(Gap Young Song),최경현(Kyung Hun Choi),장명웅(Myung Woong Chang) 대한내과학회 1998 대한내과학회지 Vol.54 No.5

N/A Objectives: This study was carried out to survey the prevalence of Helicobacter pylori infection and the incidence of vacuolating toxin producing H pylori. A further aim of this study was to evaluate the quantitative assay for cell vacuolation on the basis of the rapid uptake of neutral red dye by vaculoes of the cells. Methods: We studied the gastric biopsy specimens of patients with 154 cases of gastritis, 74 cases of gastric ulcer, and 167 cases of gastric cancer and in 44 cases of healthy persons, One of the biopsy specimen was placed into a CLOtest plate for rapid urease test and the other one of the biopsy spcimen was inoculated on Brain Heart Infusion blood agar for culture. The culture supernatant of isolated H. pylari was serially diluted with BHI broth. After 24 hour incubation of cultured RK-13 cells treated with the culture supernatgnt of H. pylori, cytoplasmic vacuolation of the cells were observed microscopically. Results: The positivity of urease test and the rate of isolation of K pylori from urease positive gastric biopsy materials were 34.1% and 93.3% in healthy person, 55.8% and 70.9% in gastritis, 60.8% and 71.1% in gastric ulcer, and 56.3% and 96.8% in gastric cancer. The isolation rate of H. pylari from patients between 20 and 39 years old was 16.8%, far patients between 40 and 59 years old it was 51.9%, and for patients above 60 years old it was 31.2%. The isolation rate of the vacuolating toxin producing H pylori from gastric biopsy specimens was 66.7% in a healthy person, 76.6% in gastritis, 79.4% in gastric ulcer, and 80% in gastric cancer. Conclusions: The isolation rate of H. pylori from the patients with gastric diseases is higher than the rate of H. pylori from healthy persons, but the isolation rate of the vacuolating toxin producing H. pylori is not different between the patients with gastric diseases and healthy persons. The titers of vacuolating toxin produced by some H, pylori isolated from the patients with gastric diseases are higher than those from healthy persons.

소화효소가 Helicobacter pylori의 증식과 공포화독소 생성에 미치는 영향

홍대식,강경희,장명웅 고신대학교(의대) 고신대학교 의과대학 학술지 2001 고신대학교 의과대학 학술지 Vol.16 No.1

Background : Global high prevalence rate of Helicobacter pylori infection is well-known fact. However, a large discrepancies exist between the diagnostic data obtained by the PCR and culture method, because the coccoid form of H.pylori which represent the majority form in the biological samples are not viable in the standard BHI broth with 5% horse serum. The component parts of saliva and gastric juice acted as neither stimulator nor inhibitor to the growth of H.pylori remain unclear. Objective : The purpose of this study was to evaluate the influence of amylase, pepsin, mucin, and lysozyme on the growth and vacuolating toxin production and titer of H.pylori in vitro. Materials and Methods : H.pylori 13,39,46,59,72,94,98 and 125 strains were isolated from patients with gastric disease. The digestive enzymes were inoculated in the 5% horse serum-BHI broth following final concentration of amylase (A; 0.20ug/ml), pepsin (P; 0.25ug/ml), mucin (M; 2,5ug/ml), and lysozyme (L; 1.5ug/ml). The vacuolating toxin titers of the culture supernatant were evaluated in the monolayer culture of RK-13 cells. Results : The morphologic conversion from bacillary to horse-shoe to coccoid forms of H.pylori was observed in the BHI broth containing 5% horse serum on day 5 culture. Half of H.pylori was changed from bacillary to coccoidal form on day 7 culture, most population of H.pylori was changed to coccoidal form on day 10 culture, and almost all H.pylori was changed to coccoidal form on day 14 culture. The growth of H.pylori 72, and 94 strains was increased in BHI broth containing each enzyme such as amylase, pepsin, mucin and mixture of two or three enzymes on day 5 and 7 culture. The growth of H.pylori 94 strains was increased in BHI broth containing each enzyme such as amylase, pepsin, mucin, and mixture of two or three enzymes on day 10 and 14 culture. The vacuolating toxin production of H.pylori strains was not influenced by the addition of amylase, pepsin, mucin, and lysozye or mixture of two or three enzymes. The neutralization of vacuolating toxin of H.pylori was not affected by the addition of amylase, pepsin, mucin, and lysozyme or mixture of two or three enzymes. Conclusion : We found that coccoid forms of H.pylori were viable in the standard 5% horse serum BHI media in the presence of amylase, pepsin, mucin, and lysozyme. The present study demonstrates that gastric juice of the stomach supports the viability of H.pylori and that oral route of infection is a plausible hypothesis. This work also demonstrates that gastric environment may not provide a selective advantage for the toxigenic strains of H.pylori.

Lactobacillus helveticus CU 631 에 의한 Helicobacter pylon 의 Urease 및 공포 생성 독소 억제활성

송의한,원병렬,윤영호,강경희,장명웅 한국동물자원과학회 2001 한국축산학회지 Vol.43 No.6

표준균주 혹은 유제품으로부터 분리된 Lactobacillus spp.와 Bifidobacterium spp. 32균주를 사용하여 H. pylori 생장을 현저하게 억제하는 L. helveticus CU631을 선발하고, urease와 공포생성 독소의 활성을 억제하는 효과를 측정하여 다음의 결과를 얻었다. L. helveticus CU631의 억제대의 직경이 10.0±1.5㎜ 나타내어 가장 강력한 생장 억제 능력을 보였으며 L. plantarum과 L. fermentum은 직경 4.0㎜ 내외의 억제대를 나타내어 비교적 약한 억제 활성을 보였으며 Bifidobacterium spp.에서 억제 활성을 보이지 않았다. L. helveticus CU631의 배양액과 배양 상층액 모두, H. pylori NCTC11637의 urese 억제 활성을 나타내었다. L. helveticus CU631를 H. pylori G88016를 같이 배양했을시 공포생성 독소의 역가가 50%로 감소하였으며 L. helvesticus CU631의 배양 상층액과 H. pylori G88016의 배양 상층액을 5:5와 6:4 비율로 혼합하였을 때 억제 활성이 나타났다. The inhibitory effects of 32 strains of lactobacilli against Helicobacter. pylori were determined and Lactobacillus. helveticus CU631 has been selected as the strain which possessed the strongest inhibitory effect against H. pylori NCTC11637 in inhibition zone test showing inhibition zone with the average diameter of 10±1.5㎜, whereas Lactobacillus. plantarum and L. fermentum made inhibition zone with the average diameter of 4.0㎜, H. pylori G88016 revealed the highest vacuolating toxin activity among the 8 strains of H. pylori, which showed positive reaction of vacuolating toxin gene in PCR amplification test. Both L. helveticus CU631 and cell free culture supernatant had a strong inhibitory activity on the urease activity of H. pylori NCTC11637. The inhibitory activity of L. helveticus CU631 on the vacuolating toxin activity of H. pylori manifested in the co-culture of two strains and in the 5:5 mixture of supernatant of the two strains.

소화효소가 Helicobater pylori의 증식과 공포화독소 생성에 미치는 영향

홍대식,강경희,장명웅 고신대학교 의학부 2001 高神大學校 醫學部 論文集 Vol.16 No.1

Background Global high prevalence rate of Helicobacter pylori infection is well-known fact. However, a large discrepancies exist between the diagnostic data obtained by the PCR and culture method, because the coccoid form of H. pylori which represent the majority form in the biological samples are not viable in the standard BHI broth with 5% horse serum. The component parts of saliva and gastric juice acted as neither stimulator nor inhibitor to the growth of H. pylori remain unclear. Objective The purpose of this study was to evaluate the influence of amylase. pepsin, mucin, and lysozyme on the growth and vacuolating toxin production and titer of H. pylori in vitro. Materials and Methods H pylori 13, 39, 46, 59, 72, 94, 98, and 125 strains were isolated from patients with gastric diseases. The digestive enzymes were inoculated in the 5% horse serum-BHI broth following final concentration of amylase (A: 0.25㎍/㎖), pepsin(P: 0.25㎍/㎖), mucin(M: 2.5㎍/㎖), and lysozyme(L: 1.5㎍/㎖). The vacuo-lating toxin titers of the culture supernatant were evaluated in the monolayer culture of RK-13 cells. Results The morphologic conversion from bacillary to horse-shoe to coccoid forms of H pylori was observed in the BHI broth containing 5% horse serum on day 5 culture. Half of H. pylori was evaluated from bacillary to coccoidal form on day 7 culture, most population of H. pylori was changed to coccoidal form on day 10 culture, and almost all H. pylori was changed to coccoidal form on day 14 culture. The growth of H. pylori 72, and 94 strains was increased in BHI broth containing each enzyme such as amylase, pepsin, mucin and mixture of two or three enzymes on day 5 and 7 culture. The growth of H. pylori 94 stains was increased in BHI broth containing each enzyme such as amylase, pepsin, mucin, and mixture of two or three enzymes on day 10 and 14 culture. The vacuolating toxin production of H. pylori strains was not influenced by the addition of amylase, pepsin, mucin and lysozyme or mixture of two or three enzymes. The neutralization of vacuolating toxin of H. pylori was not affected by the addition of amylase, pepsin mucin and lysozyme or mixture of two or three enzymes. Conclusion We found that coccoid forms of H. pylori were viable in the standard 5% horse serum BHI media in the presence of amylase, pepsin, mucin, and lysozyme. The present study demonstrates that gastric juice of the stomach supports the viability of H. pylori and that oral route of infection is a plausible hypothesis. This work also demonstrates that gastric environment may not provide a selective advantage for the toxigenic strains of H pylori.