Correlation between the severity of peptic ulcer bleeding and Helicobacter pylori type / 中华消化杂志

Objective@#To investigate the correlation between the severity of peptic ulcer bleeding (PUB) and the serum antibody typing of Helicobacter pylori (H.pylori).@*Methods@#From January 1, 2009 to December 31, 2018, at Guangzhou First People′s Hospital, 1 444 patients diagnosed with PUB and received H. pylori serum antibody test at the same time were enrolled and divided into high-risk group (324 cases) and low-risk group (1 120 cases) according to Forrest classification, and according to recurrent bleeding, the patients were divided into recurrent bleeding group (32 cases) and non-rebleeding group (1 412 cases). Serum H. pylori specific antibodies cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA) and urease were detected by protein array. The correlation between H. pylori positive rate, H. pylori type, PUB and rebleeding were analyzed. Chi-square test and logistic regression analysis were used for statistical analysis.@*Results@#Among 1 444 PUB patients, there were 709 patients with gastric ulcer bleeding (GUB) and 735 patients with duodenal ulcer bleeding (DUB). Previous history of peptic ulcer disease (odds ratio (OR)=1.501, P=0.006), the maximum diameter of ulcer over 2 cm (OR=2.484, P<0.01) and H. pylori infection (OR=1.508, P=0.005) were independent risk factors of the severity of PUB. The total H. pylori positive rate was 68.49% (989/1 444), H. pylori type Ⅰ was the main type. Of which, 61.34% (549/895) were CagA and VacA double positive strains, 31.73% (284/895) were VacA single positive bacteria and CagA single positive bacteria was only 6.93% (62/895). The positive rate of H. pylori of high-risk group was higher than that of low-risk group (75.31%, 244/324 vs. 66.52%, 745/1 120), and the difference was statistically significant (χ2=8.999, P=0.004). In addition, the more serious Forrest classification, the higher the detection rate of H. pylori, and the difference was statistically significant (χ2=11.840, P=0.037). There was no significant difference in the positive rate of H. pylori between recurrent bleeding group and non-rebleeding group (81.25%, 26/32 vs. 68.20%, 963/1 412; χ2=2.469, P>0.05). According to H. pylori antibody type, H. pylori type Ⅰ infection was mainly in both high-risk group and low-risk group. The positive rate of H. pylori type Ⅰ strain of high-risk group was higher than that of low-risk group (67.28%, 218/324 vs. 60.45%, 677/1 120), and the difference was statistically significant (χ2=4.986, P=0.026). There was no statistically significant difference in the positive rate of H. pylori between GUB group and DUB group (68.41%, 485/709 vs. 68.57%, 504/735; χ2=0.005, P>0.05).@*Conclusions@#The infection of H. pylori is positively correlated with the severity of PUB, but not correlated with early ulcer rebleeding. H. pylori type Ⅰ is the main pathogenic strain of GUB and DUB, and CagA and VacA double positive strain is the most common strain.

Correlation between the severity of peptic ulcer bleeding and Helicobacter pylori type / 中华消化杂志

Objective To investigate the correlation between the severity of peptic ulcer bleeding (PUB) and the serum antibody typing of Helicobacter pylori (H.pylori).Methods From January 1, 2009 to December 31, 2018, at Guangzhou First People's Hospital, 1 444 patients diagnosed with PUB and received H.pylori serum antibody test at the same time were enrolled and divided into high-risk group (324 cases) and low-risk group ( 1 120 cases ) according to Forrest classification , and according to recurrent bleeding , the patients were divided into recurrent bleeding group (32 cases) and non-rebleeding group (1 412 cases).Serum H.pylori specific antibodies cytotoxin-associated gene A (CagA), vacuolating cytotoxin A (VacA) and urease were detected by protein array .The correlation between H.pylori positive rate, H.pylori type, PUB and rebleeding were analyzed .Chi-square test and logistic regression analysis were used for statistical analysis . Results Among 1 444 PUB patients, there were 709 patients with gastric ulcer bleeding ( GUB) and 735 patients with duodenal ulcer bleeding ( DUB).Previous history of peptic ulcer disease ( odds ratio (OR)＝ 1.501, P＝0.006), the maximum diameter of ulcer over 2 cm (OR＝2.484, P?0.01) and H.pylori infection (OR＝1.508, P＝0.005) were independent risk factors of the severity of PUB .The total H.pylori positive rate was 68.49%(989/1 444), H.pylori type Ⅰwas the main type.Of which, 61.34%(549/895) were CagA and VacA double positive strains , 31.73%(284/895) were VacA single positive bacteria and CagA single positive bacteria was only 6.93%(62/895).The positive rate of H.pylori of high-risk group was higher than that of low-risk group (75.31%, 244/324 vs.66.52%, 745/1 120), and the difference was statistically significant (χ2 ＝8.999, P ＝0.004).In addition, the more serious Forrest classification , the higher the detection rate of H.pylori, and the difference was statistically significant (χ2 ＝11.840, P＝0.037).There was no significant difference in the positive rate of H.pylori between recurrent bleeding group and non-rebleeding group (81.25%, 26/32 vs.68.20%, 963/1 412; χ2 ＝2.469, P＞0.05).According to H.pylori antibody type, H.pylori typeⅠinfection was mainly in both high-risk group and low-risk group.The positive rate of H.pylori typeⅠstrain of high-risk group was higher than that of low-risk group (67.28%, 218/324 vs.60.45%, 677/1 120), and the difference was statistically significant ( χ2 ＝4.986, P ＝0.026).There was no statistically significant difference in the positive rate of H.pylori between GUB group and DUB group (68.41%, 485/709 vs. 68.57%, 504/735; χ2 ＝0.005, P＞0.05).Conclusions The infection of H.pylori is positively correlated with the severity of PUB, but not correlated with early ulcer rebleeding .H.pylori typeⅠis the main pathogenic strain of GUB and DUB, and CagA and VacA double positive strain is the most common strain .

Helicobacter pylori virulence factor vacuolating cytotoxin A ( VacA) promotes homologous recombi-nation repair in GES-1 cells / 中华微生物学和免疫学杂志

Objective To analyze the impact of Helicobacter pylori standard strain (Hp P12) and its virulence factor vacuolating cytotoxin A ( VacA) on DNA damage and homologous recombination ( HR) repair in a human gastric epithelial cell line (GES-1). Methods Strains of Hp P12 and vacA gene knock-out Hp P12 ( Hp P12 ΔvacA) were respectively used to infect GES-1 cells at a multiplicity of infection of 100. GES-1 cells treated with etoposide (50μmol/L) or mitomycin (0. 5μg/ml) for 2 h were used as posi-tive control. Western blot and immunofluorescence were performed to detect the expression of DNA damage marker protein γH2AX and key HR repair proteins (Rad51, pMRE11, CtIP and pCtIP) and the recruitment of them at DNA damage sites. Human embryonic kidney HEK-293 ( DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA strains to verify the impact of VacA on HR repair efficiency. Results The expres-sion and recruitment of γH2AX and key HR repair proteins ( Rad51, pMRE11, CtIP and pCtIP) were in-creased in Hp P12-infected cells as compared with that in uninfected and Hp P12 ΔvacA-infected cells ( all P<0. 05). To evaluate the HR repair efficiency, I-SceⅠ plasmid-transfected HEK-293 (DR-GFP) cells were infected with Hp P12 and Hp P12 ΔvacA and the results showed that green fluorescent protein ( GFP)-positive cells were decreased after infection, especially in Hp P12 ΔvacA-infected cells (both P<0. 05). Conclusions Hp P12 infection could cause DNA damage and promote HR repair in GES-1 cells, in which the virulence factor VacA played an important role.

Cytopathic effects of toxogenic strains of Helicobacter pylori on different cell lines.

Purpose: Many virulence factors are involved in the pathomechanism of infection caused by Helicobacter pylori. Toxins such as vacuolating cytotoxin, encoded by the vacA gene and the immunogenic protein cagA, encoded by the cagA gene (cytotoxin-associated gene) are major factors conferring the property of virulence. The current study is aimed at isolation of H. pylori and separation of its toxin from antral biopsies of patients. Materials and Methods: The following cell lines were used to demonstrate the cytopathic effect (CPE) of the separated toxin: African green monkey kidney (Vero), baby hamster kidney, human lung carcinoma (LLC-MK2), and human epithelial. Results: H. pylori was isolated from 27 out of 45 patients (60%) selected for the study. CPE of H. pylori toxin was highly signifi cant on Vero cells than other cell lines used as it reached a high dilution titer of toxin (1/16) in 13 isolated strains (48.15%). No signifi cant difference in CPE of toxin in different dilutions was detected among other cell lines used in different groups. H. pylori toxin could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis as a distinct band with a molecular weight ranging between 66 and 97 kDa and closely related to 87 kDa. Conclusion: H. pylori vacuolating cytotoxin plays a vital role in the pathogenesis of gastroduodenal diseases (gastritis, gastric ulcer, duodenal ulcer, and gastric cancer). The Vero cell lines were found to be the most suitable form of tissue culture when compared with other cell lines used in our study for demonstrating the activity of H. pylori toxin.

Isolation and purification of recombinant VacA and Helicobacter pylori-secreted VacA and VacA-induced cell vacuolar change and apoptosis / 军事医学

Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA , and to investigate the effect of VacA-induced cell vacuolar change and apoptosis .Methods VacA proteins were separated and pu-rified from the culture supernatant of H.pylori ( ATCC26695 ) or from the split products of genetically engineered bacteria (pQE30-VacA-E.coli M15) expressing recombinant VacA.The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng/ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay , respectively .Results H.pylori-secreted VacA and recombi-nant VacA were successfully separated and purified .The H.pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells (P<0.01) while the recombinant VacA did not.Conclusion H.pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis, but recombinant VacA can not, suggesting that the purified VacA protein secreted by H.pylori can be used to explore VacA-induced pathogenesis.

Differences in virulence markers between Helicobacter pylori strains from the Brazilian Amazon region

Introduction This study compares virulence markers of Helicobacter pylori isolated from patients in 2 cities in the Brazilian Amazon. Methods The study analyzed 168 patients with chronic gastritis from Belém and 151 from Bragança, State of Pará, Brazil. Levels of bacterial DNA associated with cagA and vacA alleles were checked by PCR, and hematoxylin-eosin staining was used for histologic diagnosis. Results In Bragança 87% of patients were genotype s1m1 cagA-positive (s1m1 cagA+), compared with 76% in Belém. In samples from patients in both cities, there was an association between s1m1 cagA+ strains and gastric mucosal damage. Conclusions Both cities have a high frequency of s1m1 cagA+ strains of H. pylori. .

Helicobacter pylori infection in relation to gastric cancer progression.

Gastric cancer is a major cause of cancer death worldwide, especially in developing countries. The incidence of gastric cancer varies from country to country, probably as a result of genetic, epigenetic, and environmental factors. H. pylori infection is considered as a major risk factor in the development of gastric cancer. However, the scenario varies in Asian countries, exhibiting a higher rate of H. pylori infection and low incidence of gastric cancer, which could be attributed to strain-specific virulence factors and host genetic makeup. In this review, we discuss the various virulence factors expressed by this bacterium and their interaction with the host factors, to influence pathogenesis.

The effects of H.pylori and its crude extracted proteins on isolated rabbit parietal cells acid secretion / 中华内科杂志

Objective To explore the effects of H.pylori and crude extracted proteins secreted by H.pylori(broth culture filtrate protein,BCF-P)on acid secretion from isolated rabbit parietal cells.Methods Parietal cells from rabbit gastric mucosa were isolated and enriched with digestion and elutriation.H.pylori(NCTC 11637,CagA+ VacA+)were grown in liquid broth culture and BCF-P was precipitated with ammonium sulfate.The vacuolation activity of BCF-P was evaluated with neutral red dye uptake test in HeLa cell.Isolated parietal cells were incubated with H.pylori(bacteria/cell=100∶1)for 2 h and 16 h,or BCF-P(100μg/ml)for 1 h and 12 h.Acid secretion from parietal cells was studied using 14C-aminopyrine(14C-AP)accumulation indirectly and H+-K+ ATPase α subunit mRNA expression was assessed using RT-PCR.Results (1)BCF-P containing vacuolating cytotoxin(VacA)with vacuolation activity on HeLa cells had positive result on neutral red uptake test.(2)The basal expression of H+-K+ ATPase α subunit mRNA could be detected in isolated parietal cells and 14C-AP accumulation was significantly increased in response to the stimulation of histamine with different concentrations for 30 min(P＜0.05).These results indicated that the isolated parietal cells retain relative intact acid secretion function.(3)The histamine(1.0×104 mol/L)stimulated acid secretion was inhibited sustainedly in response to H.pylori by 81% at 2 h and by 94% at 16 h(P＜0.05).However,H+-K+ ATPase α subunit mRNA expression was up-regulated in tlle acute period(2 h)and was down-regulated in the chronic period (16 h)by H.pylori(P＜0.05).(4)BCF-P significantly inhibited the histamine-stimulated acid secretion by 24% at 1 h and by 58% at 12 h(P＜0.05),and this inhibition was accompanied by the down-regulated expression of H+-K+ATPase α subunit mRNA.Conclusions Intact H.pylori and VacA secreted by H.pylori could directly inhibit histamine-stimulated acid secretion from parietal cells and this inhibition may be mediated by the down-regulated H+-K+ ATPase expression.

Characteristic typing of Helicobacter pylori phenotypes and its clinical significance in patients with duodenal bulb ulcers / 中国医师进修杂志

Objective to study the characteristic typing of Helicobacter pylor(Hp)phenotypes and their sub-phenotypes in the patients with duodenal bulb ulcers(DU),and its clinical significance. Methods One hundred thirty-five cases with DU and 140 casses with chronic superficial gastritis were enrolled in this study. Determinations of serum cytotoxin-associated gene protein A (CagA),vacuolating cyto-toxin A(VacA),urease (Ure)A,UreB antibodies and their sub-phenotypes by immunoblotting were carried ou. Results Positive rate of middle-phenotypes of Hp infection in DU was significantly lower than that in chronic superficial gastritis (21.5%vs 27.9%,P<0.05).VacA and CagA antibodies might express alone. There had no significant difference among the expression rate of phenotype CagA, VacA antibodies and their sub-phenotype. But expression rate of Ure antibodies in Du was higher than that in chronic superficial gasstritis (P<0.05).In infection of Hp type I, the expression rate of sub-phenotypes 30ku UreA in DU was hronic superficial gastritis (P<0.05). Conclusions The VacA is not for expressing higher than that inpression of Hp exists many sub-phenotypes ( 128 ku CagA 116 ku CagA, 95 ku VacA, 91 ku CagA 0 ku UreA),and it probably causes formation of DU by comprehensive effect. Hp type I with sub phenotype expressing 30 ku Urea may be more pathogenic in DU formation.

Expression of Eotaxin in Gastric Epithelial Cells Stimulated with Helicobacter pylori Vacuolating Cytotoxin

Helicobacter pylori-infected gastric mucosa is characterized by infiltration of various inflammatory cells such as neutrophils and eosinophils. Although several mechanisms for neutrophil infiltration are well known, there has been little known the role of eotaxin, which is a potent chemoattractant for eosinophils, on the inflammatory process of H. pylori infection. The present study was to investigate the mechanisms of eotaxin expression in gastric epithelial cells stimulated with H. pylori vacuolating cytotoxin (VacA). Stimulation with VacA purified from VacA+ H. pylori slightly increased eotaxin expression in MKN-45 gastric epithelial cells. In contrast, the combined stimulation with VacA and IL-4 synergistically increased the eotaxin expression as determined by quantitative RT-PCR and ELISA. In MKN-45 cells transfected with an eotaxin promoter-luciferase reporter plasmid, costimulation with VacA and IL-4 induced more luciferase activity than either VacA or IL-4 alone did. However, such up-regulation was significantly decreased in the cells transfected with luciferase reporter plasmid bearing an eotaxin promoter which has a mutation at STAT6 binding site. These results suggest that the up-regulation of eotaxin in VacA-stimulated gastric epithelial cells may be synergistically facilitated by IL-4 via a STAT6-dependent mechanism.

Expression of Eotaxin in Gastric Epithelial Cells Stimulated with Helicobacter pylori Vacuolating Cytotoxin

Helicobacter pylori-infected gastric mucosa is characterized by infiltration of various inflammatory cells such as neutrophils and eosinophils. Although several mechanisms for neutrophil infiltration are well known, there has been little known the role of eotaxin, which is a potent chemoattractant for eosinophils, on the inflammatory process of H. pylori infection. The present study was to investigate the mechanisms of eotaxin expression in gastric epithelial cells stimulated with H. pylori vacuolating cytotoxin (VacA). Stimulation with VacA purified from VacA+ H. pylori slightly increased eotaxin expression in MKN-45 gastric epithelial cells. In contrast, the combined stimulation with VacA and IL-4 synergistically increased the eotaxin expression as determined by quantitative RT-PCR and ELISA. In MKN-45 cells transfected with an eotaxin promoter-luciferase reporter plasmid, costimulation with VacA and IL-4 induced more luciferase activity than either VacA or IL-4 alone did. However, such up-regulation was significantly decreased in the cells transfected with luciferase reporter plasmid bearing an eotaxin promoter which has a mutation at STAT6 binding site. These results suggest that the up-regulation of eotaxin in VacA-stimulated gastric epithelial cells may be synergistically facilitated by IL-4 via a STAT6-dependent mechanism.

Cloning and expressing of vacA gene related segment with vacuolating activity of Helicobacter pylori / 重庆医科大学学报

Objective:To obtain the antigen of recombinant vacuolating segment of Helicobacter pylori vacuolating cytotoxing by gene-engineering.Methods:The objective gene was cloned into PQE-30,sequenced and expressed using PCR and recombinant techniques.After expression,the antigenicity of recombinant protein was analyzed by Western blotting.Results:DNA sequence analysis identified that the sequence was integrated.Compared with standard strain AF361700,1.5% of the cloned gene was mutated,one amino acid radicle was changed.SDS-PAGE revealed the molecular weight of objective recombinant protein was 27 000 as it was predicted;the level of expression product was over 30%.Western blotting results showed the objective protein could be recognized by anti-serum against HP.Conclusion:This recombinant protein has perfect antigenicity,so it may be used as a protective antigen in preventing HP infection.

Prevalence of Virulence Factors in Helicobacter pylori Isolated from Koreans and Proinflammatory Cytokine Gene Expression in Human Gastric Epithelial Cells Induced by H. pylori with Virulence Factors / 대한내과학회지

OBJECTIVES: CagA or cytotoxin-positive H. pylori may be associated with gastroduodenal diseases. However, controversies about this association also exist. Moreover, there could be geographic differences in the prevalence of virulence factors such as cagA or cytotoxin. In H. pylori infection, the gastric mucosa shows acute and chronic inflammation. However, the pathogenesis of such as an inflammation by H. pylori is not well elucidated. We performed this study 1) to determine prevalence of the genes of virulence factor such as cagA and cytotoxin in H. pylori, 2) to assess the correlation of their presence with clinical findings, and 3) to test whether the vacuolating cytotoxin of H. pylori could evoke proinflammatory cytokine gene expression in gastric epithelial cells. METHODS: 1) The prevalence of the cagA, vacA and adhesin genes in H. pylori strains isolated from Koreans was determined by PCR analysis. 2) H. pylori was cultured in Brucella broth containing 10% fetal bovine serum for 3 days using a shaker in a microaerophilic condition. Cytotoxin assay was performed by determining whether addition of the concentrated culture supernatants is able to cause vacuolization of HeLa cells. 3) After human gastric epithelial cells, Hs746T and AGS were incubated with the culture supernatants containing vacuolating cytotoxin, each RNAs were extracted from the gastric epithelial cells. And then various cytokine gene expression were assessed using RT-PCR. The expressed cytokine transcripts were quantified by RT-PCR and standard synthetic RNA. Among cytokines, IL-8 proteins were also measured by ELISA. RESULTS: 1) More than 95% of H. pylori isolates from Korean adults possessed cagA, vacA and adhesin genes. And 80.6% of H. pylori strains have expressed vacuolating cytotoxicity against HeLa cells within 24 hours. 2) There was no correlation between the virulence factors of H. pylori strains and clinical findings. 3) Cytotoxin-positive culture supernatants also caused vacuolization in gastric epithelial cells, both Hs746T and AGS. 4) Expression of mRNA for proinflammatory cytokines such as IL-1alpha: IL-8, MCP-1 and GM-CSF was much more upregulated by vacuolating cytotoxin-positive culture supernatants than cytotoxin-negative ones in both Hs746T and AGS cells. Number of molecules of the expressed IL-8 transcripts was parallel to the amounts of IL-8 protein secreted from gastric epithelial cells. CONCLUSION: These results suggest that virulence factors of H. pylori may not be factors determining disease entitiy in Korean patients infected with H. pylori. In addition, vacuolating cytotoxin secreted from H. pylori could give rise to vacuolization in gastric epithelial cells as well as induce proinflammatory cytokines from the cells.

Construction of vacA-ctxB prokaryotic expression vectors to Helicobacter pylori / 重庆医科大学学报

Objective:To lay a foundation of expressing VCTB fusion protein and preparation the oral vaccine for prophylaxis and therapy of H.pylori infection,the prokaryotic expression vectors contained the fusion gene vacA toxic subunit of H.pylori and cholera toxin subunit B(ctxB)was constructed.Methods:The high homogeneity gene fragment was found in comparing VacA toxic subunit gene of domestic typical H.pylori strains.The primers were designed according to vacA and ctxB sequences in the gene bank.VacA toxic subunit gene was amplified by PCR as the template of DNA genome of H.pylori and cloned into plasmid pQE30,which was plasmid pQE30-vacA.The ctxB gene was amplified by PCR as the template of pET32(?)+-ctxB plasmid.The purified ctxB gene was inserted into pQE30-vacA to construct expressing plasmid pQE-vctB of containing vacA and ctxB genes.pQE-vctB was transformed into prokaryotic E.coli Top10.The recombinant plasmid of bacteria cultivated was extracted and purified,and cutted with the incision enzyme to evaluated.Results:The sequence of vacA gene amplified was about 723 bp and the sequence of ctxB gene was about 372 bp.They were consistented with the anticipated length of the DNA.The expressing plasmid pQE-vctB was conformitied with objective gene inserted into pQE30.vctB fusion gene sequenced as 1 092 bp by the sequencing was conformitied with the genebank,and encodes polypeptides of 364 amino acid residues.Conclusion:The prokaryotic expression vectors contained vacA and ctxB fusion gene was constructed successfully and lay a foundation of expression VCTB fusion protein.

Relationship between VacA genotypes of Helicobacter pylori and gastric diseases / 重庆医科大学学报

Objective:To investigate the distribution of Helicobacter pylori (H.pylori) VacA genotypes in different gastrointestinal diseases and the relationship among each other.Methods:VacA genotypes of 108 H.pylori strains isolated from patients with chronic gastritis,peptic ulcer and gastric cancer were tested by polymerase chain reaction (PCR).Results:The frequencies of the signal allele sla,slb and the middle-region allele m1,m2 were 85 (79%),22 (20%),29 (27%),77 (71%) respectively.The numbers of H.pylori strains of VacA s1a/m2 positive were 66 (61.1%).The s2 form was not found.The positive rates of VacA s la of H.pylori strains isolated from patients with chronic gastritis,peptic ulcer and gastric cancer were 65.4% (34/52),90% (36/40) and 94% (15/16) respectively.Conclusion:The majority of H.pylori strains isolated from Chongqing patients express s1a/m2.The s1a/m2 genotype is dispersed in all of the gastrointestinal diseases associated with H.pylori.However the s2 form is not found.