Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

Amino acid deletions at positions 893 and 894 of cytotoxinassociated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

BACKGROUND Helicobacter pylori(H.pylori)persistently colonizes the human gastric mucosa in more than 50%of the global population,leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma.Cytotoxin-associated gene A(CagA)protein,an important oncoprotein,has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus,which play crucial roles in pathogenesis.Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer.AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function.METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894.The cagA gene was amplified and mutated into cagA-NT and cagA-NE(sequence characteristics of strains from nongastric cancer patients),cloned and inserted into pAdtrack-CMV,and then transfected into AGS cells.The expression of cagA and its mutants was examined using realtime polymerase chain reaction and Western blotting,cell elongation via cell counting,F-actin cytoskeleton visualization using fluorescence staining,and interleukin-8(IL-8)secretion via enzyme-linked immunosorbent assay.RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE(40.88±3.10 vs 32.50±3.17,P<0.001 and 40.88±3.10 vs 32.17±3.00,P<0.001)at 12 hours after transfection.At 24 hours,pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT(46.02±2.12 vs 53.90±2.10,P<0.001 and 46.02±2.12 vs 51.15±3.74,P<0.001).The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE(27.54±17.37 vs 41.51±11.90,P<0.001 and 27.54±17.37 vs 41.39±14.22,P<0.001)at 12 hours after transfection.Additionally,pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection.CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity,which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H.pylori.

Ability of Helicobacter pylori to internalize into Candida

The following are our views regarding the“letter to the editor”(Helicobacter is preserved in yeast vacuoles!Does Koch’s postulates confirm it?)by Alipour and Gaeini,and the response“letter to the editor”(Candida accommodates nonculturable Helicobacter pylori in its vacuole-Koch’s postulates aren’t applicable)by Siavoshi and Saniee.Alipour and Gaeini rejected the methods,results,discussion,and conclusions summarized in a review article by Siavoshi and Saniee.The present article reviews and discusses evidence on the evolutionary adaptation of Helicobacter pylori(H.pylori)to thrive in Candida cell vacuoles and concludes that Candida could act as a Trojan horse,transporting potentially infectious H.pylori into the stomach of humans.

Role of Helicobacter pylori virulence factor cytotoxin-associated gene A in gastric mucosa-associated lymphoid tissue lymphoma

Helicobacter pylori(H.pylori)infection might initiate and contribute to the progression of lymphoma from gastric mucosa-associated lymphoid tissue(MALT).Increasing evidence shows that eradication of H.pylori with antibiotic therapy can lead to regression of gastric MALT lymphoma and can result in a 10-year sustained remission.The eradication of H.pylori is the standard care for patients with gastric MALT lymphoma.Cytotoxin-associated gene A(CagA)protein,one of the most extensively studied H.pylori virulence factors,is strongly associated with the gastric MALT lymphoma.CagA possesses polymorphisms according to its C-terminal structure and displays different functions among areas and races.After being translocated into B lymphocytes via typeⅣsecretion system,CagA deregulates intracellular signaling pathways in both tyrosine phosphorylation-dependent and-independent manners and/or some other pathways,and thereby promotes lymphomagenesis.A variety of proteins including p53and protein tyrosine phosphatases-2 are involved in the malignant transformation induced by CagA.Mucosal inflammation is the foundational mechanism underlying the occurrence and development of gastric MALT lymphoma.

Helicobacter pylori virulence genes

Helicobacter pylori(H.pylori)is one of the most important human pathogens,infecting approximately half of the global population.Despite its high prevalence,only a subset of H.pylori infected individuals develop serious gastroduodenal pathology.The pathogenesis of H.pylori infection and disease outcome is thus thought to be mediated by an intricate interplay between host,environmental and bacterial virulence factors.H.pylori has adapted to the harsh milieu of the human stomach through possession of various virulence genes that enable survival of the bacteria in the acidic environment,movement towards the gastric epithelium,and attachment to gastric epithelial cells.These virulence factors enable successful colonization of the gastric mucosa and sustain persistent H.pylori infection,causing chronic inflammation and tissue damage,which may eventually lead to the development of peptic ulcers and gastric cancer.Numerous studies have focused on the prevalence and role of putative H.pylori virulence genes in disease pathogenesis.While several virulence factors with various functions have been identified,disease associations appear to be less evident,especially among different study populations.This review presents key findings on the most important H.pylori virulence genes,including several bacterial adhesins and toxins,in children and adults,and focuses on their prevalence,clinical significance and potential relationships.

Role of polymorphisms in genes that encode cytokines and Helicobacter pylori virulence factors in gastric carcinogenesis

The Helicobacter pylori(H. pylori) infection is a determinant factor in gastric cancer(GC) development. However, the infection outcomes are variable and depend on both host and bacterial characteristics. Some host cytokines such as interleukin(IL)-1β, IL-1 Ra, IL-8, IL-10 and tumor necrosis factor-α play important roles in the host immune system response to the pathogen, in the development of gastric mucosal lesions and in cell malignant transformation. Therefore, these host factors are crucial in neoplastic processes. Certain polymorphisms in genes that encode these cytokines have been associated with an increased risk of GC. On the other hand, various virulence factors found in distinct H. pylori bacterial strains, including cytotoxinassociated antigen A, vacuolating cytotoxin, duodenal ulcer promoting gene A protein, outer inflammatory protein and blood group antigen binding adhesin, have been associated with the pathogenesis of different gastric diseases. The virulent factors mentioned above allow the successful infection by the bacterium and play crucial roles in gastric mucosa lesions, including malignant transformation. Moreover, the role of host polymorphisms and bacterial virulence factors in gastric carcinogenesis seems to vary among different countries and populations. The identification of host and bacterium factors that are associated with an increased risk of GC development may be useful in determining the prognosis of infection in patients, what could help in clinical decision-making and in providing of an optimized clinical approach.

What exists beyond cag A and vacA? Helicobacter pylori genes in gastric diseases

Helicobacter pylori(H. pylori) infection is present in more than half the world's population and has been associated with several gastric disorders, such as gastritis, peptic ulceration, and gastric adenocarcinoma.The clinical outcome of this infection depends on host and bacterial factors where H. pylori virulence genes seem to play a relevant role. Studies of cag A and vac A genes established that they were determining factors in gastric pathogenesis. However, there are gastric cancer cases that are cag A-negative. Several other virulence genes have been searched for, but these genes remain less well known that cag A and vac A. Thus, this review aimed to establish which genes have been suggested as potentially relevant virulence factors for H. pylori-associated gastrointestinal diseases. We focused on the cag-pathogenicity island, genes with adherence and motility functions, and ice A based on the relevance shown in several studies in the literature.

Helicobacter pylori infection: Host immune response, implications on gene expression and microRNAs

Helicobacter pylori(H. pylori) infection is the most common bacterial infection worldwide. Persistent infection of the gastric mucosa leads to inflammatory processes and may remain silent for decades or progress causing more severe diseases, such as gastric adenocarcinoma. The clinical consequences of H. pylori infection are determined by multiple factors, including host genetic predisposition, gene regulation, environmental factors and heterogeneity of H. pylori virulence factors. After decades of studies of this successful relationship between pathogen and human host, various mechanisms have been elucidated. In this review, we have made an introduction on H. pylori infection and its virulence factors, and focused mainly on modulation of host immune response triggered by bacteria, changes in the pattern of gene expression in H. pylori-infected gastric mucosa, with activation of gene transcription involved in defense mechanisms, inflammatory and immunological response, cell proliferation and apoptosis. We also highlighted the role of bacteria eradication on gene expression levels. In addition, we addressed the recent involvement of different microRNAs in precancerous lesions, gastric cancer, and inflammatory processes induced by bacteria. New discoveries in this field may allow a better understanding of the role of major factors involved in the pathogenic mechanisms of H. pylori.

Gastric precancerous lesions are associated with gene variants in Helicobacter pylori -susceptible ethnic Malays

AIM: To identify genes associated with gastric pre-cancerous lesions in Helicobacter pylori (H. pylori )susceptible ethnic Malays. METHODS: Twenty-three Malay subjects with H. pylori infection and gastric precancerous lesions identified during endoscopy were included as "cases". Thirtyseven Malay subjects who were H. pylori negative and had no precancerous lesions were included as "controls". Venous blood was collected for genotyping with Affymetrix 50K Xba1 kit. Genotypes with call rates < 90% for autosomal single nucleotide polymorphisms (SNPs) were excluded. For each precancerous lesion, associated SNPs were identified from Manhattan plots, and only SNPs with a χ2 P value < 0.05 and Hardy Weinberg Equilibrium P value > 0.5 was considered as significant markers. RESULTS: Of the 23 H. pylori -positive subjects recruited, one sample was excluded from further analysis due to a low genotyping call rate. Of the 22 H. pylori positive samples, atrophic gastritis only was present in 50.0%, complete intestinal metaplasia was present in 18.25%, both incomplete intestinal metaplasia and dysplasia was present in 22.7%, and dysplasia only was present in 9.1%. SNPs rs9315542 (UFM1 gene), rs6878265 (THBS4 gene), rs1042194 (CYP2C19 gene) and rs10505799 (MGST1 gene) were significantly associated with atrophic gastritis, complete intestinal metaplasia, incomplete metaplasia with foci of dysplasia and dysplasia, respectively. Allele frequencies in "cases" vs "controls" for rs9315542, rs6878265, rs1042194 and rs10505799 were 0.4 vs 0.06, 0.6 vs 0.01, 0.6 vs 0.01 and 0.5 vs 0.02, respectively. CONCLUSION: Genetic variants possibly related to gastric precancerous lesions in ethnic Malays susceptible to H. pylori infection were identified for testing in subsequent trials.

Helicobacter pylori and cytokine gene variants as predictors of premalignant gastric lesions

Gastric cancer remains the third leading cause of mortality from cancer worldwide and carries a poor prognosis,due largely to late diagnosis.The importance of the interaction between Helicobacter pylori(H.pylori)infection,the main risk factor,and host-related genetic factors has been studied intensively in recent years.The genetic predisposition for non-hereditary gastric cancer is difficult to assess,as neither the real prevalence of premalignant gastric lesions in various populations nor the environmental risk factors for cancer progression are clearly defined.For non-cardiac intestinal-type cancer,identifying the factors that modulate the progression from inflammation toward cancer is crucial in order to develop preventive strategies.The role of cytokines and their gene variants has been questioned in regard to non-self-limiting H.pylori gastritis and its evolution to gastric atrophy and intestinal metaplasia;the literature now includes various and non-conclusive results on this topic.The influence of the majority of cytokine single nucleotide polymorphisms has been investigated for gastric cancer but not for preneoplastic gastric lesions.Among the investigated gene variants onlyIL10T-819C,IL-8-251,IL-18RAP917997,IL-22 rs1179251,IL1-B-511,IL1-B-3954,IL4R-398 and IL1RN were identified as predictors for premalignant gastric lesions risk.One of the most important limiting factors is the inhomogeneity of the studies(e.g.,the lack of data on concomitant H.pylori infection,methods used to assess preneoplastic lesions,and source population).Testing the modifying effect of H.pylori infection upon the relationship between cytokine gene variants and premalignant gastric lesions,or even testing the interaction between H.pylori and cytokine gene variants in multivariable models adjusted for potential covariates,could increase generalizability of results.

Effect of Helicobacter pylori VacA on gene expression of gastric cancer cells

AIM: To determine the effect of Helicobacter pylori VacA on gene expression of gastric cancer cells. METHODS: Gene expression profile of a gastric cancer cell line, SGC7901, after challenged by VacA^+ and VacA^- H pylori broth culture supernatants (BCS), was detected by the cDNA microarray technique. Cytoskeleton changes of SGC7901 and HeLa cells were observed through high-resolution laser scanning confocal microscopy. RESULTS: A total of 16 000 cDNA clones were detected. The percentage of genes with heterogeneous expression in SGC7901 cells challenged by VacA^+ BCS reached 5%, compared with that challenged by VacA^- BCS. There were 865 genes/EST with 2-fold differential expression levels and 198 genes/EST with 3-fold differential expression levels. Most of these genes were involved in vital cell events including signal transduction, regulation of gene expression, cytoskeleton, apoptosis, stress response and inflammation, cell cycle and tumor development. Cells co-cultured with VacA^+ BCS showed collapsed and disrupted microtubular cytoarchitecture. CONCLUSION: VacA^+ BCS can disrupt cytoskeletal architecture, likely through affecting the expression of cytoskeleton-associated genes, directly induce the expression of tumor promoter-related genes and inhibit the expression of tumor suppressor genes, thus favoring the development of tumors. VacA^+ BCS can also alter the expression of inflammation and stress response genes. This suggests that VacA may play an important role in the pathogenicity of H pylori.

Gene polymorphisms of pathogenic Helicobacter pylori in patients with different types of gastrointestinal diseases

Helicobacter pylori(H. pylori) is a kind of chronic infectious pathogen which can cause chronic gastritis, peptic ulcer, gastric cancer and other diseases. The genetic structure of the pathogenic genes of H. pylori varies largely, which contributes to the differences in virulence among various strains, and in clinical symptoms. Virulence genes of H. pylori can be categorized into three main classes: those related to adhesion and colonization, those related to gastric mucosal injury, and others. This review focuses on the relationship between genetic polymorphisms of the three classes of virulence genes of H. pylori and diseases. Most of the genetic polymorphisms of the main virulence factors of H. pylori are summarized in this paper.

Toll-like receptor 9 polymorphisms and Helicobacter pylori influence gene expression and risk of gastric carcinogenesis in the Brazilian population

BACKGROUND Toll-like receptors(TLRs)are the first line of host defense,and are involved in Helicobacter pylori(H.pylori)recognition and activation of both inflammatory and carcinogenic processes.The presence of single nucleotide polymorphisms(SNPs)in genes that activate the immune response may modulate the risk of precancerous lesions and gastric cancer(GC).Among them,Toll-like receptor 9(TLR9)polymorphisms have emerged with a risk factor of infectious diseases and cancer,however the studies are still inconclusive.AIM To evaluate whether TLR9 rs5743836 and rs187084 SNPs contribute to the risk of gastric carcinogenesis,and its influence on mRNA expression.METHODS A case-control study was conducted to evaluate two TLR9 SNPs(TLR9-1237 TCrs5743836 and TLR9-1486 CT-rs187084)in chronic gastritis(CG)and GC patients.A total of 609 DNA samples of peripheral blood[248 CG,161 GC,and 200 samples from healthy individuals(C)]were genotyped by polymerase chain reaction-restriction fragment length polymorphism.All samples were tested for the H.pylori infection using Hpx1 and Hpx2 primers.Quantitative polymerase chain reaction by TaqMan?assay was used to quantify TLR9 mRNA from fresh gastric tissues(48 GC,26 CG,and 14 C).RESULTS For TLR9-1237,the TC+CC or CC genotypes were associated with a higher risk of GC than C[recessive model odds ratio(OR)=5.01,95%confidence interval(CI):2.52-9.94,P<0.0001],and the CG(recessive model OR=4.63;95%CI:2.44-8.79,P<0.0001)groups.For TLR9-1486,an association between the CT+TT genotypes and increased risk of both GC(dominant model OR=2.72,95%CI:1.57-4.72,P<0.0001)and CG(dominant model OR=1.79,95%CI:1.15-2.79,P=0.0094)was observed when compared to the C group.Moreover,the presence of TLR9-1237 TC/CC+TLR9-1486 CC genotypes potentiate the risk for this neoplasm(OR=18.57;95%CI:5.06-68.15,P<0.0001).The TLR9 mRNA level was significantly higher in the GC group(RQ=9.24,P<0.0001)in relation to the CG group(RQ=1.55,P=0.0010)and normal mucosa(RQ=1.0).When the samples were grouped according to the polymorphic genotypes and the presence of H.pylori infection,an influence of TLR9-1237 TC+CC polymorphic genotypes(P=0.0083)and H.pylori infection(P<0.0001)was observed on the upregulation of mRNA expression.CONCLUSION Our findings show that TLR9 rs5743836 and rs187084 polymorphisms are associated with a higher risk of carcinogenesis gastric,and that TLR9 mRNA levels can be modulated by TLR9-1237 TC+CC variant genotypes and H.pylori infection.

Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori

BACKGROUND The drug resistance rate of clinical Helicobacter pylori(H.pylori)isolates has increased.However,the mechanism of drug resistance remains unclear.In this study,drug-resistant H.pylori strains were isolated from different areas and different populations of Chinese for genomic analysis.AIM To investigate drug-resistant genes in H.pylori and find the genes for the early diagnosis of clarithromycin resistance.METHODS Three drug-resistant H.pylori strains were isolated from patients with gastritis in Bama County,China.Minimal inhibitory concentrations of clarithromycin,metronidazole,and levofloxacin were determined and complete genome sequencing was performed with annotation.Hp1181 and hp1184 genes were found in these strains and then detected by reverse transcription polymerase chain reaction.The relationships between hp1181 or hp1184 and clarithromycin resistance were ascertained with gene mutant and drug-resistant strains.The homology of the strains with hp26695 was assessed through complete genome detection and identification.Differences in genome sequences,gene quantity,and gene characteristics were detected amongst the three strains.Prediction and analysis of the function of drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains,which was the same in the artificially induced clarithromycin-resistant bacteria.After gene knockout,the drug sensitivity of the strains was assessed.RESULTS The strains showing a high degree of homology with hp26695,hp1181,and hp1184 genes were found in these strains;the expression of the genes hp1184 and hp1181 was associated with clarithromycin resistance.CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance,and they may be the potential target genes for the diagnosis,prevention,and treatment of clarithromycin resistance.CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance,and they may be the potential target genes for the diagnosis,prevention,and treatment of clarithromycin resistance.

Time-series gene expression prof iles in AGS cells stimulated with Helicobacter pylori

AIM: To extend the knowledge of the dynamic interaction between Helicobacter pylori (H. pylori) and host mucosa. METHODS: A time-series cDNA microarray was performed in order to detect the temporal gene expression prof iles of human gastric epithelial adenocarcinoma cells infected with H. pylori. Six time points were selected to observe the changes in the model. A differential expression prof ile at each time point was obtained by comparing the microarray signal value with that of 0 h. Real-time polymerase chain reaction was subsequently performed to evaluate the data quality. RESULTS: We found a diversity of gene expression patterns at different time points and identifi ed a group of genes whose expression levels were significantly correlated with several important immune response and tumor related pathways. CONCLUSION: Early infection may trigger some important pathways and may impact the outcome of the infection.

Association of NOD1 and NOD2 genes polymorphisms with Helicobacter pylori related gastric cancer in a Chinese population

AIM:To investigate the association between the tag single nucleotide polymorphisms(TagSNPs) of NOD1 and NOD2 and the risk of developing gastric cancer.METHODS:We conducted a hospital-based case-control study including 296 incident gastric cancer patients and 160 gastritis controls.Eight TagSNPs in the NOD1 and NOD2 genes were selected from the Hapmap database using the haploview software and genotyped by the Sequenom MassArray system.The serum levels of anti-Helicobacter pylori(H.pylori) IgG were measured by enzyme-linked immunosorbent assay to indicate H.pylori infection.The odds ratios(OR) and 95% confidence intervals(CI) were calculated by unconditional logistic regression,including sex and age as confounding factors.RESULTS:The NOD1 rs2907749 GG genotype showed a decreased risk for gastric cancer(OR 0.50,95% CI:0.26-0.95,P = 0.04) while the rs7789045 TT genotype showed an increased risk(OR 2.14,95% CI:1.20-3.82,P = 0.01).An elevated susceptibility to gastric cancer was observed in the subjects with H.pylori infection and the NaOD1 rs7789045 TT genotype(OR 2.05,95% CI:1.07-3.94,P = 0.03) or the NOD2 rs7205423 GC genotype(OR 2.52,95% CI:1.05-6.04,P = 0.04).Haplotype analysis suggested that the distribution of AGT(rs2907749,rs2075820 and rs7789045) in NOD1 between the cases and control groups was significantly different(P corrected:0.04),and the diplotype AGT/AGT was associated with an elevated gastric cancer risk(OR 1.98,95% CI:1.04-3.79,P = 0.04).The association of the NOD1 rs7789045 TT genotype and the diplotype AGT/AGT was significant with H.pylori-related diffuse-type gastric cancer(OR 3.00,95% CI:1.38-6.53,P = 0.01;OR 4.02,95% CI:1.61-10.05,P < 0.01,respectively).CONCLUSION:Genetic polymorphisms in NOD1 and NOD2 may interact with H.pylori infection and may play important roles in promoting the development of gastric cancer in the Chinese population.

Screening Helicobacter pylori genes induced during infection of mouse stomachs

AIM:To investigate the effect of in vivo environment on gene expression in Helicobacter pylori(H.pylori) as it relates to its survival in the host.METHODS:In vivo expression technology(IVET) systems are used to identify microbial virulence genes.We modified the IVET-transcriptional fusion vector,pIVET8,which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors,pIVET11 and pIVET12.Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H.pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase.Additionally,each vector contains a kanamycin resistance gene.We used a mouse macrophage cell line,RAW 264.7 and mice,as selective media to identify specific genes that H.pylori expresses in vivo.Gene expression studies were conducted by infecting RAW 264.7 cells with H.pylori.This was followed by real time polymerase chain reaction(PCR) analysis to determine the relative expression levels of in vivo induced genes.RESULTS:In this study,we have identified 31 in vivo induced(ivi) genes in the initial screens.These 31 genes belong to several functional gene families,including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs.Virulence factors,vacA and cagA,were found in this screen and are known to play important roles in H.pylori infection,colonization and pathogenesis.Their detection validates the efficacy of these screening systems.Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H.pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae.Transcription profiles of allivi genes were confirmed by real time PCR analysis of H.pylori RNA isolated from H.pylori infected RAW 264.7 macrophages.We compared the expression profile of H.pylori and RAW 264.7 coculture with that of H.pylori only.Some genes such as cag A,vac A,lpx C,mur I,tlp C,trx B,sod B,tnp B,pgi,rbf A and inf B showed a 2-20 fold upregulation.Statistically significant upregulation was obtained for all the above mentioned genes(P < 0.05).tlp C,cag A,vac A,sod B,rbf A,inf B,tnp B,lpx C and mur I were also significantly upregulated(P < 0.01).These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal.CONCLUSION:The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H.pylori gene expression in the host environment.

Detection and amplification of Helicobacter pylori urease gene A in gastric biopsies by using nested polymerase (?)hain reaction

A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.

Cloning of the Gene Encoding Urease Subunit A in Helicobacter Pylori

The gene encoding urease subunit A (ureA) of Helicobacter pylori (H.pylori) was cloned from H.pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined. Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet. 0.8 kb PCR product was amplified from all H.pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44 % G+C content. The DNA sequence was 98 % homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino acid sequences of the ureA were 99 % homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H.pylori in the NCBI Entrez database.

Diagnostic Performance of glmM Gene and Histological Stains for Detection of Helicobacter pylori in Gastric Biopsy from Patients Admitted to Wad Madani Teaching Hospital, Sudan

Helicobacter pylori is the microbial agent most responsible for gastro-duodenal ulcer and chronic gastritis, which can develop into carcinoma of the stomach. This study was performed in Wad Medani Teaching Hospital, Sudan to detect Helicobacter pylori in stomach samples, and evaluate the performance of the tests used, which were histological stains and PCR. Gastric biopsies were obtained from 105 referred patients during endoscopy, and fixed specimens examined by haematoxylin-eosin and Warthin-Starry silver stains, while DNA was extracted for glmM gene amplification. Epigastric pain was the most common symptom at 78% (82/105) and chronic gastritis recorded with 71% (68/105) of endoscopy results. Warthin-Starry silver stain gave 31% (33/105) as positive for Helicobacter pylori followed by glmM gene 27% (28/105) and haematoxylin-eosin 24% (25/105). The study indicated good performance of histological staining and high specificity of glmM gene in detection of Helicobacter pylori from gastric biopsies.