Increased oxidative DNA damage, inducible nitric oxide synthase, nuclear factor кB expression and enhanced antiapoptosis-related proteins in Helicobacter pylori-infected non-cardiac gastric adenocarcinoma

AIM: Several epidemiological studies have demonstrated a dose association between Helicobacter pylori (H Pylon) infection and non-cardiac carcinoma of the stomach. H pylori infection induces active inflammation with neutrophilic infiltrations as well as production of oxygen free radicals that can cause DNA damage. The DNA damage induced by oxygen free radicals could have very harmful consequences,leading to gene modifications that are potentially mutagenic and/or carcinogenic. The aims of the present study were to assess the effect of H pyloriinfection on the expression of inducible nitric oxidative synthase (iNOS) and the production of 8-hydroxy-deoxyguanosine (8-OHdG), a sensitive marker of oxidative DNA injury in human gastric mucosa with and without tumor lesions, and to assess the possible factors affecting cell death signaling due tooxidative DNA damage.METHODS: In this study, 40 gastric carcinoma specimens and adjacent specimens were obtained from surgical resection. We determined the level of 8-OHdG formation by HPLC-ECD, and the expression of iNOS and mechanism of cell death signaling [including nuclear factor-κB(NFκB),MEKK-1, Caspase 3, B Cell lymphomal leukemia-2 (Bcl-2),inhibitor of apoptosis protein (IAP) and myeloid cellleukemia-1 (Mcl-1)] by Western-blot assay.RFSULTS: The concentrations of 8-OHdG, iNOS, NFx3, Mcl-1 and IAP were significantly higher in cancer tissues than in adjacent non-cancer tissues. In addition, significantly higher concentrations of 8-OHdG, iNOS, NFxB, Mcl-1 and lAP were detected in patients infected with H pyloricompared with patients who were not infected with HpylorL Furthermore,8-OHdG, iNOS, NFκB, Mcl-1 and IAP concentrations were significantly higher in stage 3 and 4 patients than in stage1 and 2 patients.CONCLUSION: Chronic Hpylori infection induces iNOS expression and subsequent DNA damage as well as enhances anti-apoptosis signal transduction. This sequence of events supports the rihypothesis that oxygen-free radical-mediated damage due to Hpyloriplays a pivotal role in the development of gastric carcinoma in patients with chronic gastritis.

Maastricht Ⅱ treatment scheme and efficacy of different proton pump inhibitors in eradicating Helicobacter pylori

AIM: The Maastricht Ⅱ criteria suggest the use of amoxicillin and clarithromycin in addition to a proton pump inhibitor over 7-10 d as a first line therapy in the eradication of Helicobacter pylori (Hpylori). For each proton pump inhibitor, various rates of eradication have been reported. The present study was to compare the efficacy of different proton pump inhibitors like omeprazole, lansoprazole and pantoprazole in combination with amoxicillin and clarithromycin in the first line eradication of Hpylonand to investigate the success of H pylonrieradication in our district. METHODS: A total of 139 patients were included having a Helicobacter pylori (+) gastroduodenal disorders diagnosed by means of histology and urease test. Besides amoxicillin (1000 mg twice a day) and darithromycin (500 mg twice a day), they were randomized to take omeprazole (20 mg twice a day), or lansoprazole (30 mg twice a day), or pantoprozole (40 mg twice a day) for 14 d. Four weeks after the therapy, the eradication was assessed by means of histology and urease test. It was evaluated as eradicated if the Hpyloriwas found negative in both. The complaints (pain in epigastrium, nocturnal pain, pyrosis and bloating) were graded in accordance with the Licert scale. The compliance of the patients was recorded. RESULTS: The eradication was found to be 40.8% in the omeprazole group, 43.5% in the lansoprazole group and 47.4% in the pantoprazole group. Sixty-three out of 139 patients (45%) had eradication. No statistically significant difference was observed between the groups. Significant improvements were seen in terms of the impact on the symptom scores in each group. CONCLUSION: There was no difference between omeprazole, lansoprazole and pantoprazole in H pylori eradication, and the rate of eradication was as low as 45%. Symptoms were improved independent of the eradication in each treatment group. The low eradication rates suggest that the antibiotic resistance or the genetic differences of the microorganism might be in effect. Further studies are required to verify these suggestions.

Helicobacter pylori cagA,iceA and vacA genotypes in patients with gastric cancer in Taiwan

AIM: Helicobacter pylori( H pylori) has been linked to chronic gastritis, peptic ulcer, gastric cancer and MALT-lymphoma.The link of genotypes of Hpylorito gastric cancer remains controversial. The aim of this study was to investigate the Hpylori vacA alleles, cagA and iceA in patients with gastric cancer in Taiwan.METHODS: Patients with gastric cancer, peptic ulcer and chronic gastritis were enrolled in this study. We obtained biopsy specimens from the stomach at least 2 cm away from the tumor margin in patients with gastric cancer, and from the antrum of stomach in patients with peptic ulcer or chronic gastritis. DNA extraction and polymerase chain reaction were used to detect the presence or absence of cagA and to assess the polymorphism of vacA and iceA.RESULTS: A total of 168 patients (gastric ulcer: 77, duodenal ulcer: 66, and chronic gastritis: 25) were found to have positive PCR results of the biopsy specimens from patients with peptic ulcer and chronic gastritis. We found positive cagA (139/168, 83%), m2 (84/168, 50%) and iceA1 (125/168,74%) strains in the majority of patients. In patients with gastric cancer, the vacA sla and slc subtypes were less commonly found than those in non-cancer patients (35/66 vs 127/168, P= 0.0001 for sla and 13/66 vs93/168, P<0.0001 for slc). In the middle region, the mlT strain in patients with gastric cancer was more than that of non-cancer patients(23/66 vs 33/168, P = 0.02).CONCLUSION: In Taiwan, H pylori with positive vacA sla,cagA and iceA1 strains are found in the majority of patients with gastric cancer or non-cancer patients. In patients with gastric cancer, the vacA s1a and slc subtypes are less and m1T is more than in patients with peptic ulcer and chronic gastritis.

Coordinate increase of telomerase activity and c-Myc expression in Helicobacterpylori-associated gastric diseases

AIM:To detect the telomerase activity and c-Myc expression in gastric diseases and to examine the relation between these values and Helicobacter pylori(H pylori)as a risk factor for gastric cancer. METHODS:One hundred and seventy-one gastric samples were studied to detect telomerase activity using a telomerase polymerase chain reaction enzyme linked immunosorbent assay(PCR-ELISA),and c-Myc expression using immunohistochemistry. RESULTS:The telomerase activity and c-Myc expression were higher in cancers(87.69% and 61.54%)than in noncancerous tissues.They were higher in chronic atrophic gastritis with severe intestinal metaplasia(52.38% and 47.62%)than in chronic atrophic gastritis with mild intestinal metaplasia (13.33% and 16.67%).In chronic atrophic gastritis with severe intestinal metaplasia,the telomerase activity and c-Myc expression were higher in cases with Hpylori infection (67.86% and 67.86%)than in those without infection(21.43% and 7.14%).c-Myc expression was higher in gastric cancer with H pylor/infection(77.27%)than in that without infection(28.57%).The telomerase activity and c-Myc expression were coordinately up-regulated in H pylori infected gastric cancer and chronic atrophic gastritis with severe intestinal metaplasia. CONCLUSION:H pylori infection may influence both telomerase activity and c-Myc expression in gastric diseases, especially in chronic atrophic gastritis.

Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells

AIM: To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylon) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in Hpylori-infected gastric METHODS: Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from Hpyiori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry.RESULTS: The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pyiorialone. Interestingly,the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori(Control vs TRAIL and Hpylori. 0.51±0.06 vs 2.29±0.27,P = 0.018). A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis. Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pyloriwas dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori.CONCLUSION: H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis. Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection.

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pyloriconservative region of adhesin antigen and its immunogenicity

AIM: To construct a non-resistant and attenuated Salmonella typhimurium ( S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori(Hpylon) and evaluate its immunogenicity.METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya1 delta Crp1 delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supematant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments.RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed.Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supematant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, bhe entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0×l0^10 cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response.CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro,which providing a new live oral vaccine candidate for protection and care of H pylori infection.

Antigen epitope of Helicobacter pylorivacuolating cytotoxin A

AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori ( H pylori ) infection.METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E.coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA, cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro.RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20% and 30.41% respectively.CONCLUSION: Two of the three candidates, LZ-VacAland LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

Helicobacter pylori lipopolysaccharide:Biological activities in vitro and in vivo,pathological correlation to human chronic gastritis and peptic ulcer

AIM: To determine the biological activity of Helicobacter pylori(H pylori) lipopolysaccharide (H-LPS) and understand pathological correlation between H-LPS and human chronic gastritis and peptic ulcer.METHODS: H-LPS of a clinical Hpylori strain and LPS of Escherichia coli strain O55:B5(E-LPS) were extracted by phenol-water method. Biological activities of H-LPS and E-LPS were detected by limulus lysate assay, pyrogen assay,blood pressure test and PBMC induction test in rabbits, cytotoxicity test in NIH 3T3 fibroblast cells and lethality test in NIH mice. By using self-prepared rabbit anti-H-LPS serum as the first antibody and commercial HRP-labeled sheep anti-rabbit sera as the second antibody, H-LPS in biopsy specimens from 126 patients with chronic gastritis (68 cases) or gastric ulcer (58 cases) were examined by immunohistochemistry.RESULTS: Fibroblast cytotoxicity and mouse lethality of H-LPS were weaker than those of E-LPS. But the ability of coagulating limulus lysate of the two LPSs was similar (+/0.5 ng/mL).At 0.5 h after H-LPS injection, the blood pressures of the 3 rabbits rapidly declined. At 1.0 h after H-LPS injection, the blood pressures in 2 of the 3 rabbits fell to zero causing death of the 2 animals. For the other one rabbit in the same group, its blood pressure gradually elevated. At 0.5 h after E-LPS injection, the blood pressures of the three rabbits also quickly declined and then maintained at low level for approximately 1.0 h. At 0.5 hafter injection with H-LPS or E-LPS, PBMC numbers of the rabbits showed a remarkable increase. The total positivity rate of H-LPS from 126 biopsy specimens was 60.3%(76/126). H-LPS positivity rate in the biopsy specimens from chronic gastritis (50/68, 73.5%) was significantly higher than that from gastric ulcer (26/58, 44.8%) (χ^2=10.77,P＜0.01). H-LPS positivity rates in biopsy specimens from chronic superficial gastritis (38/48, 79.2%) and chronicactive gastritis (9/10, 90.0%) were significantly higher than that of the patients with atrophic gastritis (3/10, 30.0%)(χ^2=7.50-9.66,P＜0.01). CONCLUSION: The biological activities of H-LPS were weaker than those of E-LPS, the activities of H-LPS of lowering rabbit blood pressure and inducing rabbit PBMC were relatively stronger. H-LPS may play a critical role in inducing inflammatory reaction in human gastritis.

Cloning and expression and immunogenicity of Helicobacter pylori BabA_2 gene

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori(H pylori) and to study the immunogenicity of BabA.METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore,BabA immunogenicity was studied by animal test.RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank.The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA.CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of Hpyloriinfection.

A new subtype of 3' region of cagA gene in Helicobacter pyloristrains isolated from Zhejiang Province in China

AIM: To isolate the subtypes of 3′ region of cagA gene in Helicobacter pylori (H pylon) strains from Zhejiang Province in China and to investigate their relations to H pylori-associated gastroduodenal diseases. METHODS: One hundred and thirty-seven H pylori clinical strains were isolated from the gastric mucosa specimens of 74 patients with chronic gastritis, 61 with peptic ulceration, and 2 with gastric cancer. Bacterial genomic DNA was extracted and 3′ region of cagA gene was amplified by polymerase chain reaction (PCR). Subtypes of 3′ region of cagA gene were determined by the size of PCR amplified segments. The sequences of the subtypes were analyzed by PCR-based sequencing. RESULTS: Of the 137 H pyloriisolates from Zhejiang Province, 132 (96.4%) yielded PCR products that could be classified into three groups of subtypes, named as subtypes Ⅰ, Ⅱ, and Ⅲ according to their sizes. The sizes of subtypes Ⅰ, Ⅱ, and Ⅲ were 648-650bp, 705-707bp, and 815bp, respectively. Among the 132 cagA-positive H pylori strains, 123(93.2%) belonged to the group of subtype Ⅰ, 6 (4.5%) presented subtype Ⅱ, 1(0.8%) was subtype Ⅲ, and 2(1.5%) presented subtypes Ⅰ and Ⅲ both. The primary structure of subtype Ⅰ was composed of 3 repeats of R1, 1 repeat of R2 and 1 repeat of R3. Subtype Ⅱ possessing 4 repeats of R1, 2 repeats of R2 and 1 repeat of R3 was a newly found type of 3′ region of cagA gene which had not been reported before. The primary structure of subtype Ⅲ consisted of 4 repeats of R1, Ⅰ repeat of R2 and 2 repeats of R3. Comparison of the sequences of subtype Ⅰ strains with the corresponding sequences deposited in GenBank, showed a similarity of 95.0% (94.0-96.1%) for nucleotide sequences and 95.9% (94.9-97.4%) for deduced amino acid sequences. Comparison of the sequences of subtype Ⅲ strains with the corresponding sequences deposited in GenBank, showed a similarity of 93.9% (90.8-96.9%) for nucleotide sequences and 93.2% (90.2-96.2%) for deduced amino acid sequences. Among subtype Ⅱ strains, the nucleotide and deduced amino acid sequences showed a similarity of 95.2% (94.1-96.5%) and 96.4% (93.8-97.9%),respectively. There were no statistical differences in the distribution of subtypes of 3′ region of cagA gene among different H pylori-associated gastroduodenal diseases (x^2=11.544, P>0.05). CONCLUSION: There are three subtypes (Ⅰ,Ⅱ, and Ⅲ) of 3′ region of cagA gene in Hpylori strains isolated from Zhejiang Province, and subtype Ⅰ is predominant. Subtype Ⅱ is a newly found subtype of 3′ region of cagA gene. The result of this study does not support the view that the subtypes of 3′ region of cagA gene in H pylori isolated from Zhejiang Province are correlated with the clinical outcomes of H pylori infection.

Effects of fucosylated milk of goat and mouse on Helicobacter pylori binding to Lewis b antigen

AIM:To evaluate the effects of animal milk containing fucosylated antigens on Helicobacter pylori (H pylon) binding to Lewis b antigen.METHODS:A mammary gland expression vector containing human α1-3/4-fucosyltransferase cDNA sequences was constructed. Transient expression of human(α1-3/4-fucosyltransferase cDNA in goat mammary cell and establishment of transgenic mice were performed. The adhesion inhibitory properties of milk samples were analyzed by using Hpylori.RESULTS: Goat milk samples were found to inhibit bacterial binding to Lewis b antigen. The highest inhibition was observed 42 h after injection of the plasmid. The binding activity of Hpylori to Lewis b antigen reduced mostly, by 83%, however milk samples from transgenic mice did not inhibit Hpylori binding to Lewis b antigen.CONCLUSION: The use of 'humanized' animal milk produced by the transgenic introduction of fucosylated antigen can perhaps provide an alternative therapy and preventive measure for Hpylori infection.

Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori

AIM: To construct a recombinant E. col/strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori ( H pylon).METHODS: The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E. col/strain. This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis. Finally this sequence AF479754 was compared with HP0638 and JHP0581.RESULTS: The sequence of the aim gene was obtained. It had 924 base pairs. The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581.CONCLUSION: Although the aim gene was obtained, but it was different from the published sequence of GenBank. It is not clear what makes this difference. Maybe it is because different strain was used or because there were some variations. So more researches are required to prove it.

Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia colirLTKA63 and rLTB

AIM: To demonstrate the effect of lactose as an inducer on expression of the recombinant proteins encoded by Helicobacter pylori ureB and hpaA, and Escherichia coli LTB and LTKA63 genes and to determine the optimal expression parameters. METHODS: By using SDS-PAGE and BIO-RAD gel image analysis system, the outputs of the target recombinant proteins expressed by pET32a-ureB-E.coliBL21, pET32a-hpaA-E, coliBL21, pET32a-L TKA63-E. coliBL21 and pET32a-LTB-E.coliBL21 were measured when using lactose as inducer at different dosages, original bacterial concentrations, various inducing temperatures and times. The results of the target protein expression induced by lactose were compared to those by isopropyl-β-D-thiogalactoside (IPTG). The proteins were expressed in E.coli. RESULTS: Lactose showed higher efficiency of inducing the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG. The expression outputs of the target recombinant proteins induced at 37℃ were remarkably higher than those at 28℃. Other optimal expression parameters for the original bacterial concentrations, dosages of lactose and inducing time were 0.8, 50 g/L and 4 h for rHpaA; 0.8, 100 g/L and 4 h for rLTKA63; 1.2, 100 g/L and 5 h for both rUreB and rLTB, respectively. CONCLUSION: Lactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG. The results in this study establish a beneficial foundation for industrial production of Hpylorigenetic engineering vaccine.

Diagnosis of Helicobacterpyloriinfection and diseases associated with Helicobacter pylori by Helicobacter pylori outer membrane proteins

AIM:TO examine the serological response of patients with upper gastrointestinal diseases and Helicobocter pylori(H pylon) infection to two Hpyloriouter membrane proteins(OMPs) (M,18 000 and M,26 000)acquired by gene recombinant technique,and to determine the diagnostic significance of serological tests derived from these OMPs. METHODS:Recombinant vectors encoding the two Hpylori OMPs were used to transform and express in BL21(DE3) E.COli.After purification with Ni^(2+)-NTA agarose resin,colloid gold kits were prepared with purified recombinant proteins to detect H pylori infectJon and H pylori-associated diseases by the immunity-marker technology.We selected 150 patients with Hpyloriinfection and digestive symptoms without previous treatment,induding chronic gastritis(n=60),duodenal ulcer (n=30),gastric ulcer(n=30),and gastric cancer(n=30). As controls,33 Hpylori-negative healthy volunteers were also recruited.Serum samples were collected from all subjects,and the antibodies to specific proteins of H pylori were tested with the colloid gold test kits.The sensitivity, specificity and accuracy of the colloid gold tests were evaluated,by using the combination of standard diagnostic methods(^(13)C urea breath test and bacteria culture)and classic enzyme-linked immunosorbent assay(ELISA)as reference. RESULTS:After purification with Ni^(2+)-NTA agarose resin, the purity of recombinant fusion proteins was about 95%. The recombinant fusion proteins were recognized by the specific monodonal antibodies against the two Hpylori OMPs, as demonstrated by the ELISA.Of the 150 serum samples from patients infected with Hpylori 141(94.0%)responded positively to the recombinant protein with M_126 000,while the seropositive rates were 95.0%,96.7%,96.7% and 90.0% for patients with H pylori-associated chronic gastritis, duodenal ulcer,gastric ulcer,and gastric cancer respectively. The sensitivity,specificity,and accuracy of the colloid gold kit with M_1 26 000 protein were 94.0%,97.0%,and 94.5%, respectively.Compared with the classic ELISA,bacteria culture and ^(13)C urea breath test results in detecting Hpylori- infection,there was no significant difference(P>0.05).For the colloid gold kit with M,18 000,the seropositive rates were 52.0%,40.0%,40.0%,53.3% and 86.7%,respectively, in Hpylori-infected patients,and those with Hpylori-assodated chronic gastritis,duodenal ulcer,gastric ulcer,and gastric cancer.There was a significant difference(P<0.05)in seropositivity between patient with gastdc cancer(86.7%) and those with other diseases(43.3%). CONCLUSION:The two colloid gold kits derived from the recombinant OMPs are useful tools either for detecting Hpyloriinfection,or for,predicting Hpylori-assodated gastric malignancy.

Gene distribution of cagII in Helicobacter pylori-infected patients of Zhejiang Province

AIM: To determine the prevalence of genotypes of cagⅡ in Helicobacter pylori( H pylon)-infected patients in Zhejiang Province and investigate the relationship between these genotypes and the types of gastroduodenal diseases.METHODS: One hundred and seventy one clinical isolates were collected from 70 chronic superficial gastritis, 31 chronic atrophic gastritis, 41 gastric ulcer, 21 duodenal ulcer, 3 gastric and duodenal ulcer, and 5 gastric adenocarcinoma patients. Polymerase chain reaction assays were performed for analysis of cagT, ORF13 and ORF10 genes in the cagⅡ region.RESULTS: Of 171 Hpyloriisolates from Zhejiang patients,159(93.0%) were positive for all the three loci. One isolate (0.6%) was negative for all the three loci, and 11(6.4%) were partially deleted in cagⅡ. The positive rates of cagT,ORF13 and ORF10 genes were 97.1%, 94.7% and 99.4%,respectively. In the strains isolated from the patients with diseases including chronic superficial gastritis, chronic atrophic gastritis, gastric ulcer and duodenal ulcer, the sitive rates of cagT were 95.7%, 100.0%, 95.1% and 100.0%, respectively. The positive rates of ORF13 were 94.3%, 93.5%, 95.1% and 100.0%, respectively. The sitive rates of ORF10 were 98.6%,100.0%,100.0% and 100.0%, respectively. The three genes were all positive in the three H pylori strains isolated from the patients with both gastric and duodenal ulcer. In the five strains isolated from the patients with gastric adenocarcinoma,only one isolate was negative for ORF13. There were no significant differences of the cagT, ORF13 and ORF10 genes among the different gastroduodenal diseases including chronic superficial gastritis, chronic atrophic gastritis,gastric ulcer, duodenal ulcer, both gastric and duodenal ulcer and gastric adenocarcinoma (χ^2=3.098, P＞0.05 for cagT;χ^2=3.935, P＞0.05 for ORF13 and χ^2=6.328,P＞0.05 for ORF10).CONCLUSION: The cagⅡ is not a uniform and conserved entity. Although the genes in cagⅡ are highly associated with the gastroduodenal diseases, the clinical outcome of Hpyloriinfection is not reliably predicted by the three genes in cagⅡ in patients from Zhejiang Province.

Prominent role of y-glutamyl-transpeptidase on the growth of Helicobacter pylori

AIM: γ-glutamyl transpeptidase (GGT) has been reported as a virulence and colonizing factor of Helicobacter pylori (Hpylori). This study examined the effect of GGT on thegrowth of H pylori. METHODS: Standard H pylori strain NCTC 11637 and 4 dinical isolates with different levels of GGT activity as measured by an enzymatic assay were used in this study. Growth inhibilJon and stimulation studies were carried out by culturing H pyloriin brain heart infusion broth supplemented with specific GGT inhibitor (L-serine sodium borate complex, SBC) or enhancer (glutathione together with glycyl-glycine), respectively. The growth profiles of Hpyloriwere determined based on viable bacterial count at time interval. RESULTS: Growth was more profuse for Hpyloriisolates with higher GGT activity than those present with lower GGT activity. However, in the presence of SBC, growth of Hpylori was retarded in a dose dependent manner (P = 0.034). In contrast, higher growth rate was observed when GGT activity was enhanced in the presence of glutathione and glycyl-glycine. CONCLUSION: Higher GGT activity provides an advantage to the growth of Hpy/oriin vitro. Inhibition of GGT activity by SBC resulted in growth retardation. The study shows that GGT plays an important role on the growth of Hpy/ori.

Effects of Helicobacterpyloriinfection on gastric emptying rate in patients with non-ulcer dyspepsia

AIM:The pathogenesis of delayed gastric emptying in patients with non-ulcer dyspepsia(NUD)remains unclear. We aimed to examine whether gastric emptying rate in NUD patients was associated with Helicobacter pylori(Hpylori) infection and whether it was affected by eradication of the infection. METHODS:Gastric emptying rate of a mixed solid-liquid meal was assessed by the paracetamol absorption method in NUD patients and asymptomatic controls(n=17).Hpylori status was assessed by serology and biopsy urease test. H pylori-positive NUD patients(n=23)received 10-day triple eradication therapy.Hpyloristatus was re-assessed by biopsy urease test four weeks later,and if eradication was confirmed,gastric emptying rate was re-evaluated. RESULTS:Thirty-three NUD patients and 17 controls were evaluated.NUD patients had significantly delayed gastric emptying compared with controls.The mean maximum plasma paracetamol concentration divided by body mass (Cmax/BM)was 0.173 and 0.224 mg/L.kg respectively (P=0.02),the mean area under plasma paracetamol concentration-time curve divided by body mass(AUC/BM) was 18.42 and 24.39 mg.min/L.kg respectively(P=0.01). Gastric emptying rate did not differ significantly between H pylori-positive and H pylori-negative NUD patients.The mean Cmax/BM was 0.172 and 0.177 mg/L·kg respectively (P=0.58),the mean AUC/BM was 18.43 and 18.38 mg·min/ L·kg respectively(P=0.91).Among 14 NUD patients who were initially H pylori-positive,confirmed eradication of the infection did not significantly alter gastric emptying rate. The mean Cmax/BM was 0.171 and 0.160 mg/L.kg before and after Hp eradication,respectively(P=0.64),the mean AUC/BM was 17.41 and 18.02 mg.min/L.kg before and after eradication,respectively(P=0.93). CONCLUSION:Although gastric emptying is delayed in NUD patients compared with controls,gastric emptying rate is not associated with H pylori status nor it is affected by eradication of the infection.

Loss of FHIT expression in gastric mucosa of patients with family histories of gastric cancer and Helicobacter pylori infection

AIM: To answer the question whether FHIT gene expression is affected by the family history of gastric carcinoma and the presence of Helicobacter pylori (Hpylori) in the gastric mucosa of patients with dyspepsia.METHODS: FHIT gene expression in two different topographic sites of the gastric mucosa of twenty-one patients with dyspepsia and with or without familial gastric carcinoma, infected or not infected with H pylori, was evaluated by reverse transcription-PCR (RT-PCR) and IMAGE QUANT methods. A rapid urease test and histopathological examination were used to determine H pylori colonization.RESULTS: In the gastric mucosa of patients with family histories of gastric carcinoma, the amount of FHIT protein mRNA was reduced down to 32%, and for patients with H pylori colonization, to 24% in comparison to controls with dyspepsia and without cancer in the family. FHIT expression was independent of the topography of specimens (corpus vsantrum), and for the control patients it was less sensitive to infection with H pylori. A considerable statistical difference in FHIT levels was observed in the gastric mucosa from the corpus of patients with family histories of gastric carcinoma in respect to H pylori colonization (P = 0.06). Macroscopic evaluation of the gastric mucosa demonstrated that pathologic changes classified according to the Sydney system had no significant influence on FHIT expression within each tested group of patients.CONCLUSION: Loss of FHIT expression was observed in patients with dyspepsia and family histories of gastric carcinoma, especially those infected with H pylori. Such results may constitute an early indication of the development of gastric carcinoma, which is associated with family factors including heredity and H pylori infection. The loss of the FHIT gene may serve as a marker for early diagnosis and prevention of gastric carcinoma, especially in context of early monitoring of H pylori infection in individuals with a record of familial stomach cancer.

Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens

AIM:To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and/or culture) in order to determine which of the three PCR methods (ureA,glmM and 26-kDa,SSA gene) was most appropriate in the diagnosis of Helicobacterpylori(Hpylori) infection and also to evaluate the detection of a putative virulence marker of H pylori,the cage,gene,by PCR in biopsy specimens. METHODS:One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms.The PCR methods used to detect H pylori DNA directly from biopsies were the glmM,26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS:Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods,while 68% of these were positive for the cagA gene.Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened.The remaining 41% were either positive for ureA gene only,glmM only,26-kDa only,or ureA+glmM, ureA+26-kDa,glmM+26-kDa.Out of the 35% positive biopsies,41% and 82% were positive by culture and CLO respectively,while all negative biopsies were also negative by culture and cagA.Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION:This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.

Neither gastric topological distribution nor principle virulence genes of Helicobacter pylori contributes to clinical outcomes

AIM: Studies on Helicobacter pylori (H pylon) and gastroduodenal diseases have focused mainly on the distal sites of the stomach, but relationship with the gastric cardia is lacking. The aim of this study is to determine if the gastric topology and genotypic distribution of Hpyloriwere associated with different upper gastrointestinal pathologies in a multiethnic Asian population. METHODS: Gastric biopsies from the cardia, body/corpus and antrum were endoscoped from a total of 155 patients with dyspepsia and/or reflux symptoms, with informed consent. H pylori isolates obtained were tested for the presence of 26kDa, ureC, cagA, vacA, iceA1, iceA2 and babA2 genes using PCR while DNA fingerprints were generated using random amplification polymorphic DNA (RAPD). RESULTS: Hpyloriwas present in 51/155 (33%) of patients studied. Of these, 16, 15 and 20 were isolated from patients with peptic ulcer diseases, gastroesophageal reflux diseases and non-ulcer dyspepsia, respectively. Of the Hpyloripositive patients, 75% (38/51) had Hpyloriin all three gastric sites. The prevalence of various genes in the H pylori isolates was shown to be similar irrespective of their colonization sites as well as among the same site of different patients. The RAPD profiles of H pylori isolates from different gastric sites were highly similar among intra-patients but varied greatly between different patients. CONCLUSION: Topographic colonization of H pylori and the virulence genes harboured by these isolates have no direct bearing to the clinical state of the patients. In multiethnic Singapore, the stomach of each patient is colonized by a predominant strain of H pylori，irrespective of the clinical diagnosis.