iuuq ey epj psh - downloads.hindawi.comdownloads.hindawi.com/archive/2014/418063.pdf · research...

Research ArticleEvaluation of the Pattern of EPIYA Motifsin the Helicobacter pylori cagA Gene of Patientswith Gastritis and Gastric Adenocarcinomafrom the Brazilian Amazon Region

Adenielson Vilar e Silva1 Mario Ribeiro da Silva Junior12

Ruth Maria Dias Ferreira Vinagre3 Kemper Nunes Santos12

Renata Aparecida Andrade da Costa12 Amanda Alves Fecury12

Juarez Antocircnio Simotildees Quaresma12 and Luisa Caricio Martins12

1 Laboratorio de Patologia Clınica Nucleo de Medicina Tropical Universidade Federal do ParaAvenida Generalissimo Deodoro No 92 Umarizal 66095-360 Belem PA Brazil

2 Nucleo de Medicina Tropical Laboratorio de Patologia Clınica das Doencas Tropicais Universidade Federal do ParaBelem PA Brazil

3 Departamento de Gastroenterologia Hospital Ophir Loyola Belem PA Brazil

Correspondence should be addressed to Luisa Caricio Martins caricioufpabr

Received 30 December 2013 Accepted 7 April 2014 Published 24 April 2014

Academic Editor Rodrigo E Mendes

Copyright copy 2014 Adenielson Vilar e Silva et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

TheHelicobacter pylori is associated with the development of different diseasesThe clinical outcome of infection may be associatedwith the cagA bacterial genotype The aim of this study was to determine the EPIYA patterns of strains isolated from patients withgastritis and gastric adenocarcinoma and correlate these patterns with the histopathological features Gastric biopsy samples wereselected from 384 patients infected with H pylori including 194 with chronic gastritis and 190 with gastric adenocarcinoma Thepresence of the cagA gene and the EPIYAmotif was determined by PCRThe cagA gene was more prevalent in patients with gastriccancer and was associated with a higher degree of inflammation neutrophil activity and development of intestinal metaplasiaThenumber of EPIYA-C repeats showed a significant association with an increased risk of gastric carcinoma (OR = 379 95 CI =192ndash746 and 119875 = 0002) A larger number of EPIYA-Cmotifs were also associated with intestinal metaplasia In the present studyinfection with H pylori strains harboring more than one EPIYA-C motif in the cagA gene was associated with the development ofintestinal metaplasia and gastric adenocarcinoma but not with neutrophil activity or degree of inflammation

1 Introduction

Helicobacter pylori is a spiral Gram-negative bacterium thatinfects the stomach and causes chronic gastritis In additionthe bacterium plays an important role in the pathogenesisof gastroduodenal ulcer and gastric carcinoma [1] Thediverse clinical outcomes of H pylori infection depend onfactors such as bacterial virulence host susceptibility andenvironmental factors [2]

Protein-associated gene A (CagA) is an important vir-ulence factor of H pylori that is found in 70 to 80 of

strains isolated in Brazilian cities and is associated with thedevelopment of peptic ulcers and gastric carcinoma [3 4]This protein is encoded by the cagA gene which is locatedon the cag pathogenicity island (cag-PAI) After adhesion ofcagA-positive H pylori strains to the gastric epithelium theCagA protein is injected directly into the host cell througha type IV secretion system encoded by the cag-PAI Insidethe epithelial cell CagA is phosphorylated in its carboxy-terminal region This region is highly variable and containsa polymorphic pattern of Glu-Pro-Ile-Tyr-Ala amino acidrepeats (EPIYA motif) [5 6] Four types of EPIYA segments

Hindawi Publishing CorporationInternational Journal of BacteriologyVolume 2014 Article ID 418063 6 pageshttpdxdoiorg1011552014418063

2 International Journal of Bacteriology

have been described (EPIYA-A -B -C and -D) [7 8] CagAproteins always possess the EPIYA-A and EPIYA-B sites butsome proteins also contain one ormore repeats of the EPIYA-C site This pattern is found normally in strains circulatingin Western countries such as Europe North America andAustralia (western CagA) whereas the presence of EPIYA-Aand EPIYA-B sites followed by the EPIYA-D site has beendescribed for H pylori strains isolated in Asian countries[6 8 9]

The EPIYA-C and -D motifs are the main sites forphosphorylation of CagA Phosphorylated CagA forms aphysical complex with SHP-2 phosphatase and triggersabnormal cellular signals that lead to the deregulation of cellgrowth cell-to-cell contact and cell migration elongationof epithelial cells and increased epithelial cell turnoverincreasing the risk of acquiring precancerous genetic changesThe presence of the EPIYA-D motif or of multiple EPIYA-C repeats is associated with increased SHP-2 phosphataseactivity induced by CagA [10 11] In Western countriesinfection with strains carrying EPIYA-C repeats has beenshown to predispose to the development of precancerouslesions and gastric cancer [8 11]

Studies conducted in Para state have demonstrated ahigh frequency of the cagA gene among circulating strainsIn addition CagA was found to be associated with pepticulcers and gastric cancer [3 12] However there are no studiesof polymorphism of CagA in bacterial strains isolated in theAmazon region And even in Brazil such studies are scarce[13 14]

The objective of the present study was to determine theprevalence of variants of the 31015840-region of the cagA gene amongstrains isolated from patients with chronic gastritis andgastric carcinoma and to investigate the association betweenthese variants and histopathological features of the diseases

2 Materials and Methods

21 Patients Participated in the study were a total of 384patients infected with H pylori (222 men and 162 women)seen between May 2010 and June 2011 at Hospital OphirLoyola Belem Para Brazil All subjects included in thestudy were of the same socioeconomic level had similarcultural habits were born in the State of Para and had thesame ethnic background (approximately 50 Portuguese40 Amerindian and 10 African) [15]

The study was approved by the Ethics Committee of theTropical Medicine Center Belem Para Brazil All patientsgave their informed consent to participate in the study

During endoscopy two biopsies of the area adjacent to thelesion (perilesion) were obtained from patients with a sus-picion of carcinoma for histological analysis and two antralbiopsy specimens were obtained for analysis by molecularmethods Four antral biopsies (two for histological analysisand two for molecular analysis) were obtained from patientswith gastritis

22 Histological Analysis The two antral biopsy specimens(one from the greater curvature and one from the incisura

angularis) were fixed in 10 buffered formalin embeddedin paraffin cut into sequential 04 120583m sections and stainedwith hematoxylin and eosin The biopsy specimens wereanalyzed by an experienced pathologist who was unaware ofthe clinical data of each patientHistopathological parameterswere graded from 0ndash3 (corresponding to absent mild mod-erate and intense) using the criteria of the updated Sydneyclassification system [16] for chronic inflammation polymor-phonuclear activity and intestinal metaplasia Gastric car-cinoma cases were classified using the Lauren classification[17]

23 Extraction of H pylori Genomic DNA Genomic DNAwas extracted from the antral biopsy specimens using thePureLink Genomic DNA Mini kit (Invitrogen Sao PauloBrazil)

24 Detection of H pylori PCR amplification for the detec-tion of H pylori DNA in gastric mucosa was performed aspreviously described [18] Briefly one set of primers (p1-F andp2-R) that amplify a fragment of 298 bp of the 26-kDa antigengene present in all H pylori strains was used for detection ofthe bacterium The study included samples from patients inwhich bacterial DNA was isolated

25 Amplification of the Constant Region of the cagA GeneThe constant region of the cagA gene was analyzed in samplespositive forH pylori All patients positive for this region werethen submitted to investigation of variable region (EPIYA)polymorphisms The constant region of the cagA gene wasamplified by the polymerase chain reaction (PCR) using theCagAConF 51015840-GTGCCTGCTAGTTTGTCAGCG-31015840 andCagAConR 51015840 TTGGAAACCACCTTTTGTATTAGC-31015840primers [14] Negative and positive controls were includedin all reactions The PCR products were separated by elec-trophoresis on 2 agarose gel that was stained with ethidiumbromide and visualized under a UV transilluminator

26 Amplification of the 31015840-Variable Region of the cagA Geneand Determination of the EPIYA Pattern The followingprimers described by Yamaoka et al [19] were used foramplification of the 31015840-variable region of the cagA geneCAG1 51015840-ACCCTAGTCGGTAATGGGTTA-31015840 and CAG251015840-GTAATTGTCTAGTTTCGC-31015840The reaction consisted of05mM of each primer 1X PCR buffer 15mMMgCl2 sterilewater 02mM of each deoxynucleotide 125120583L Taq DNApolymerase (Invitrogen) and 2 120583L DNA in a final volume of25 120583LThe amplification conditions were initial denaturationat 95∘C for 2min followed by 35 cycles of denaturation at95∘C for 1min annealing at 56∘C and extension at 72∘Cfor 1min with a final extension at 72∘C for 10min PCRwas carried out in a thermocycler GeneAmp PCR system9700 (Applied Biosystems) Products of 500 to 850 bp wereobtained depending on the type and number of repeats ofthe EPIYA-C motif in the cagA gene The PCR productswere separated by electrophoresis on 2 agarose gel that wasstained with ethidium bromide and visualized under a UVtransilluminator Positive controls of the different patterns of

International Journal of Bacteriology 3

the EPIYA motif were used to confirm the PCR results Thismethodology also allows the detection of mixed infection

27 Sequencing of the 31015840-Variable Region of cagA PCRproducts were purified with the Wizard SV Gel and PCRClean-Up System (Promega Madison MI) according tothe manufacturerrsquos recommendations Purified productswere sequenced using a BigDye Terminator v31 CycleSequencing Kit in an ABI 3130 Genetic Analyzer (AppliedBiosystems Foster City CA) The sequences obtainedwere aligned using the CAP3 Sequence Assembly Pro-gram (available from httppbiluniv-lyon1frcap3php)After alignment nucleotide sequences were transformedinto aminoacid sequences using the Blastx program (avail-able from httpblastncbinlmnihgovBlastcgi) and com-pared to sequences deposited into the GenBank (httpwwwncbinlmnihgovGenbank)

28 Statistical Analysis Odds ratios were calculated to eval-uate the association of the cagA gene and EPIYA motifs withgastric diseases and histological parameters and the MannWhitney 119880 test was used to compare frequencies adopting alevel of significance of 95 Statistical analysis was performedusing the Biostat 50 program [20]

3 Results

A total of 384 patients with gastric symptoms and infectedby Hpylori participated in the study According to the histo-logical report 5052 (194384) of the patients had chronicgastritis and 4948 (190384) had gastric adenocarcinomaincluding 3211 (61190) with the diffuse type and 6789(129190) with the intestinal type

Age ranged from 18 to 63 years (mean 372 years) forpatients with chronic gastritis and from 27 to 90 years (mean599 years) for patients with gastric cancer With respect togender there was a predominance of men in the two groupswith a frequency of 6316 (120190) in the cancer group andof 5258 (102194) in the gastritis group

Investigation of the cagA gene in gastric biopsies showedthat 256384 (667) of the patients harbored strains carryingthis gene This frequency was higher among patients withadenocarcinoma 159190 (821) than among those withgastritis 100194 (515) (OR = 431 95IC (270ndash687) 119875 lt001)

To examine the association between different patterns ofEPIYA with gastric diseases and histopathological data wasused only samples monoinfected

31 Determination of the EPIYA Pattern of the cagA Geneand Its Association with Gastric Diseases The distributionof the EPIYA genotypes is shown in Table 1 The sequencingconfirmed the results obtained by PCR and was not foundEPIYA-D sites in the H pylori strains studied

Analysis of the different EPIYA motifs in the 31015840-regionof the cagA gene revealed that 58 (150256) of the patientsinfected with cagA-positive H pylori harbored strains withonly one cagA EPIYA genotype (monoinfected) and 42

Table 1 Distribution of Helicobacter pylori cagA EPIYA genotypesin patients with chronic gastritis and gastric carcinoma

EPIYA pattern Gastritis119899 ()

Cancer119899 ()

Monoinfection

EPIYA-AB 4 (540) 4 (526)EPIYA-ABC 42 (5676) 19 (25)EPIYA-ABCC 26 (3514) 25 (3290)EPIYA-ABCCC 2 (270) 28 (3684)

Mixedinfection

EPIYA-ABCABCC 20 (7692) 33 (4125)EPIYA-ABCABCCC mdash 2 (25)EPIYA-ABCCABCCC 2 (77) 14 (175)

EPIYA-ABCABCCABCCC 4 (1538) 31 (3875)

(106256) presented mixed infection with strains carryingdifferent cagA EPIYA genotypes (Table 1) The frequency ofmixed infection was higher among patients with adenocarci-noma compared with those with gastritis (OR = 299 95 IC(173ndash513) 119875 lt 001)

The analysis of the distribution of the EPIYA patterns inmonoinfected patients showed that colonization by H pyloriCagA-positive with two or three EPIYA C motifs was moreprevalent among patients with gastric adenocarcinoma (OR= 378 95 IC (192ndash746) 119875 = 0002)

32 Association between Presence of the cagA Gene andHistopathological Findings When the histopathologicalfindings of the patients colonized by CagA-positive strains(256384) with those harboring CagA-negative strains(128384) were compared a higher degree of gastric inflam-mation neutrophil activity and intestinal metaplasia wasobserved in the former (Table 2)

33 Association between the Numbers of EPIYA-C Segmentsand Histopathological Findings The increased number ofEPIYA-C segments was associated with the presence ofintestinal metaplasia (Table 3) but not with the other his-tological parameters such as degree of inflammation andneutrophil activity (Table 4)

4 Discussion

TheCagA protein is an importantH pylori virulence markerthat is associatedwith diseases such as peptic ulcer and gastriccarcinoma in Western countries [21 22] Studies conductedin different Brazilian states including the State of Para havedemonstrated a high prevalence of strains carrying the cagAgene in the Brazilian population as well as an association ofthese strains with peptic ulcer disease and gastric carcinoma[3 12 23] Similar results were observed in the present studyin which the prevalence of strains carrying the cagA genewas higher among patients with gastric adenocarcinoma thanamong those with gastritis

In addition to the presence of the cagA gene the typeof EPIYA motif in the carboxy-terminal region of the gene


Table 2 Association between the presence or absence of the cagA gene and the histopathological findings of the study participants

Histopathological finding cagA gene OR (95 CI) 119875Positive () Negative ()

Degree of inflammationMild 39 (1523) 50 (3906) 357 (218ndash584) 00001Moderate to intense 217 (8477) 78 (6094)

Neutrophil activityMild 51 (20) 94 (7344) 1111 (548ndash2230) 00001Moderate to intense 205 (80) 34 (2656)

MetaplasiaMild 39 (1523) 6 (469) 365 (150ndash888) 0004Moderate to intense 217 (8477) 122 (9531)

Table 3 Association between EPIYA polymorphisms and the presence or absence of metaplasia in monoinfected patients with chronicgastritis and gastric carcinoma

Gastritis CancerMetaplasia OR (95 CI) 119875 Metaplasia OR (95 CI) 119875

Present Absent Present AbsentEPIYA (AB or ABC) 1 46 1314 (149ndash11615) 001 2 20 500 (10509ndash23789) 005EPIYA (ABCC or ABCCCC) 6 21 18 36Total 7 67 20 56

Table 4 Association between EPIYA polymorphisms in the cagA gene and degree of inflammation and neutrophil activity in monoinfectedpatients

Degree of inflammation Neutrophil activityEPIYA pattern 119880 (119901) EPIYA pattern 119880 (119901)

AB or ABC ABCC or ABCCCC AB or ABC ABCC or ABCCCCGastritis 2 (1ndash3) 2 (1ndash3) 21750 (052) 2 (1ndash3) 2 (1ndash3) 28050 (041)Cancer 2 (1ndash3) 2 (1ndash3) 59250 (088) 2 (1ndash3) 2 (1ndash3) 56900 (067)Mild (1) moderate (2) intense (3)

has recently been shown to influence the biological activityof CagA and the aggressiveness of H pylori strains [11 24]A study conducted in Western countries demonstrated thatthe presence of bacterial strains with multiple repeats ofthe EPIYA-C motif predisposes to precancerous lesions andgastric cancer [24]

All patients in this study were natives of the stateof Para and based on the study of Santos et al 1999the population of this state has an ethnic background ofapproximately 50 Portuguese 40 Amerindian and 10African In Brazil a continental country there are few studieson this subject this study being the first to evaluate thepattern of CagA EPIYA segments in the North region of thecountry

In the present study strains with two or three repeatsof the EPIYA-C motif were more frequent in patientswith gastric adenocarcinoma The risk of gastric cancerwas approximately 4-fold higher among patients infectedwith cagA-positive strains carrying two or three EPIYA-C motifs compared to patients infected with cagA-positivestrains carrying no or only one EPIYA-C motif Further-more a higher frequency of colonization with mixed strains

harboring different types of EPIYA motifs was observedin patients with adenocarcinoma Similar results have beenreported byBatista et al [13]who studied patients fromMinasGerais Brazil

Although several studies have demonstrated an associa-tion between infection with H pylori strains harboring twoor three EPIYA-C motifs and gastric cancer no associationcould be established between the number of EPIYA-C repeatsand increased gastric inflammation [24 25]

In the present study a positive association was observedbetween cagA status and neutrophil activity degree of inflam-mation and intestinal metaplasia However there was noassociation between the number of EPIYA-C repeats and thedegree of lymphocyte or neutrophil infiltration

CagA has been associated with increased gastric inflam-mation characterized by high-grade leukocyte infiltrationwhich is a long-term risk factor for carcinogenesis since theintense oxidative process triggered by the disintegration ofcells in the gastric mucosa releases potentially mutagenicsubstances [26 27] Some studies suggest that the numberof EPIYA-C repeats is not associated with proinflammatorycytokine production but rather increases the binding to


SHP-2 with consequent morphological changes in the cellthat induce mucosal damage [25 28]

5 Conclusions

H pylori strains harboring two or three EPIYA-C motifswhich are a risk factor for gastric cancer predominated inpatients with adenocarcinoma and were associated with thedevelopment of intestinal metaplasia in the gastric mucosa

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] J GKusters AHMVanVliet andE J Kuipers ldquoPathogenesisof Helicobacter pylori infectionrdquo Clinical Microbiology Reviewsvol 19 no 3 pp 449ndash490 2006

[2] K M Fock and T L Ang ldquoEpidemiology of Helicobacter pyloriinfection and gastric cancer inAsiardquo Journal of Gastroenterologyand Hepatology vol 25 no 3 pp 479ndash486 2010

[3] R M F Vinagre T C D O Corvelo V C Arnaud A C KLeite K A D S Barile and L C Martins ldquoDeterminationof strains of Helicobacter pylori and of polymorphism in theinterleukin-8 gene in patients with stomach cancerrdquo Arquivosde Gastroenterologia vol 48 no 1 pp 46ndash51 2011

[4] M Q F Cavalcante C I S Silva M B B Neto et alldquoHelicobacter pylori vacA and cagA genotypes in patientsfrom northeastern Brazil with upper gastrointestinal diseasesrdquoMemorias do Instituto Oswaldo Cruz vol 107 no 4 pp 561ndash5632012

[5] M Selbach S Moese C R Hauck T F Meyer and S BackertldquoSrc is the kinase of theHelicobacter pyloricagA protein in vitroand in vivordquo Journal of Biological Chemistry vol 277 no 9 pp6775ndash6778 2002

[6] H Higashi R Tsutsumi A Fujita et al ldquoBiological activity ofthe Helicobacter pylori virulence factor cagA is determined byvariation in the tyrosine phosphorylation sitesrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 22 pp 14428ndash14433 2002

[7] Y Furuta K Yahara M Hatakeyama and I KobayashildquoEvolution of cagA oncogene of Helicobacter pylori throughrecombinationrdquoPLoSONE vol 6 no 8 Article ID e23499 2011

[8] L A Sicinschi P Correa R M Peek Jr et al ldquocagA C-terminal variations in Helicobacter pylori strains from Colom-bian patients with gastric precancerous lesionsrdquo Clinical Micro-biology and Infection vol 16 no 4 pp 369ndash378 2010

[9] P Correa and M B Piazuelo ldquoEvolutionary history of theHeli-cobacter pylori genome implications for gastric carcinogenesisrdquoGut and Liver vol 6 no 1 pp 21ndash28 2012

[10] H Higashi R Tsutsumi S Muto et al ldquoSHP-2 tyrosinephosphatase as an intracellular target ofHelicobacter pyloricagAproteinrdquo Science vol 295 no 5555 pp 683ndash686 2002

[11] M Naito T Yamazaki R Tsutsumi et al ldquoInfluence of EPIYA-repeat polymorphism on the phosphorylation-dependent bio-logical activity of Helicobacter pyloricagArdquo Gastroenterologyvol 130 no 4 pp 1181ndash1190 2006

[12] L C Martins T C De Corvelo Oliveira S Demachki et alldquoClinical and pathological importance of vacA allele hetero-geneity and cagA status in peptic ulcer disease in patients fromNorth BrazilrdquoMemorias do Instituto Oswaldo Cruz vol 100 no8 pp 875ndash881 2005

[13] S A Batista G A Rocha A M C Rocha et al ldquoHighernumber of Helicobacter pyloricagA EPIYA C phosphorylationsites increases the risk of gastric cancer but not duodenal ulcerrdquoBMCMicrobiology vol 11 p 61 2011

[14] C A Rota J C Pereira-Lima C Blaya and N B NardildquoConsensus and variable region PCR analysis of Helicobacterpriori 31015840 region of cagA gene in isolates from individuals withor without peptic ulcerrdquo Journal of ClinicalMicrobiology vol 39no 2 pp 606ndash612 2001

[15] S S E Batista J D Rodrigues A K Ribeiro-dos-Santos andM A Zago ldquoDifferential contribution of indigenous men andwomen to the formation of an urban population in the Amazonregion as revealed by mtDNA and Y-DNArdquo American Journalof Physical Anthropology vol 109 no 2 pp 175ndash180 1999

[16] M F Dixon R M Genta J H Yardley et al ldquoClassificationand grading of Gastritis the updated Sydney systemrdquoAmericanJournal of Surgical Pathology vol 20 no 10 pp 1161ndash1181 1996

[17] P Lauren ldquoThe two histological main types of gastric car-cinoma diffuse and so-called intestinal-type carcinoma Anattempt at a histo-clinical classificationrdquo Acta pathologica etmicrobiologica Scandinavica vol 64 pp 31ndash49 1965

[18] M Hammar T Tyszkiewicz T Wadstrom and P W OrsquoTooleldquoRapid detection ofHelicobacter pylori in gastric biopsymaterialby polymerase chain reactionrdquo Journal of Clinical Microbiologyvol 30 no 1 pp 54ndash58 1992

[19] Y Yamaoka T Kodama K Kashima D Y Graham and AR Sepulveda ldquoVariants of the 3rsquo region of the cagA genein Helicobacter pylori isolates from patients with different Hpylori-associated diseasesrdquo Journal of Clinical Microbiology vol36 no 8 pp 2258ndash2263 1998

[20] M Ayres M J Ayres D L Ayres and A S Santos Bioestat30-Aplicacoes Estatısticas nas areas das Ciencias Biologicas eMedicas SociedadeCivilMamirauaMCT-CNPq Belem Brazil2003

[21] C Nogueira C Figueiredo F Carneiro et al ldquoHelicobacterpylori genotypes may determine gastric histopathologyrdquo Amer-ican Journal of Pathology vol 158 no 2 pp 647ndash654 2001

[22] M J Blaser G I Perez-Perez H Kleanthous et al ldquoInfectionwith Helicobacter pylori strains possessing cagA is associatedwith an increased risk of developing adenocarcinoma of thestomachrdquo Cancer Research vol 55 no 10 pp 2111ndash2115 1995

[23] D M Queiroz E N Mendes G A Rocha et al ldquocagA positiveHelicobacter pylori and risk for developing gastric carcinoma inBrazilrdquo International Journal of Cancer vol 78 no 2 pp 135ndash139 1998

[24] D Basso C Zambon D P Letley et al ldquoClinical relevanceof Helicobacter pyloricagA and vacA gene polymorphismsrdquoGastroenterology vol 135 no 1 pp 91ndash99 2008

[25] R M Ferreira J C Machado M Leite F Carneiro and CFigueiredo ldquoThe number of Helicobacter pyloricagA EPIYA Ctyrosine phosphorylation motifs influences the pattern of gas-tritis and the development of gastric carcinomardquo Histopathol-ogy vol 60 no 6 pp 992ndash998 2012

[26] P Correa ldquoHelicobacter pylori and gastric carcinogenesisrdquoAmerican Journal of Surgical Pathology vol 19 supplement 1pp S37ndashS43 1995


[27] C M Thomazini N A Pinheiro M I Pardini L E Naresseand M A M Rodrigues ldquoInfeccao por Helicobacter pylori ecancer gastrico frequencia de cepas patogenicas cagA e vacAempacientes com cancer gastricordquo Jornal Brasileiro de Patologiae Medicina Laboratorial vol 42 no 1 pp 25ndash30 2006

[28] R H Argent M Kidd R J Owen R J Thomas M CLimb and J C Atherton ldquoDeterminants and consequences ofdifferent levels of cagA phosphorylation for clinical isolates ofHelicobacter pylorirdquo Gastroenterology vol 127 no 2 pp 514ndash523 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


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GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


have been described (EPIYA-A -B -C and -D) [7 8] CagAproteins always possess the EPIYA-A and EPIYA-B sites butsome proteins also contain one ormore repeats of the EPIYA-C site This pattern is found normally in strains circulatingin Western countries such as Europe North America andAustralia (western CagA) whereas the presence of EPIYA-Aand EPIYA-B sites followed by the EPIYA-D site has beendescribed for H pylori strains isolated in Asian countries[6 8 9]

The EPIYA-C and -D motifs are the main sites forphosphorylation of CagA Phosphorylated CagA forms aphysical complex with SHP-2 phosphatase and triggersabnormal cellular signals that lead to the deregulation of cellgrowth cell-to-cell contact and cell migration elongationof epithelial cells and increased epithelial cell turnoverincreasing the risk of acquiring precancerous genetic changesThe presence of the EPIYA-D motif or of multiple EPIYA-C repeats is associated with increased SHP-2 phosphataseactivity induced by CagA [10 11] In Western countriesinfection with strains carrying EPIYA-C repeats has beenshown to predispose to the development of precancerouslesions and gastric cancer [8 11]

Studies conducted in Para state have demonstrated ahigh frequency of the cagA gene among circulating strainsIn addition CagA was found to be associated with pepticulcers and gastric cancer [3 12] However there are no studiesof polymorphism of CagA in bacterial strains isolated in theAmazon region And even in Brazil such studies are scarce[13 14]

The objective of the present study was to determine theprevalence of variants of the 31015840-region of the cagA gene amongstrains isolated from patients with chronic gastritis andgastric carcinoma and to investigate the association betweenthese variants and histopathological features of the diseases

2 Materials and Methods

21 Patients Participated in the study were a total of 384patients infected with H pylori (222 men and 162 women)seen between May 2010 and June 2011 at Hospital OphirLoyola Belem Para Brazil All subjects included in thestudy were of the same socioeconomic level had similarcultural habits were born in the State of Para and had thesame ethnic background (approximately 50 Portuguese40 Amerindian and 10 African) [15]

The study was approved by the Ethics Committee of theTropical Medicine Center Belem Para Brazil All patientsgave their informed consent to participate in the study

During endoscopy two biopsies of the area adjacent to thelesion (perilesion) were obtained from patients with a sus-picion of carcinoma for histological analysis and two antralbiopsy specimens were obtained for analysis by molecularmethods Four antral biopsies (two for histological analysisand two for molecular analysis) were obtained from patientswith gastritis

22 Histological Analysis The two antral biopsy specimens(one from the greater curvature and one from the incisura

angularis) were fixed in 10 buffered formalin embeddedin paraffin cut into sequential 04 120583m sections and stainedwith hematoxylin and eosin The biopsy specimens wereanalyzed by an experienced pathologist who was unaware ofthe clinical data of each patientHistopathological parameterswere graded from 0ndash3 (corresponding to absent mild mod-erate and intense) using the criteria of the updated Sydneyclassification system [16] for chronic inflammation polymor-phonuclear activity and intestinal metaplasia Gastric car-cinoma cases were classified using the Lauren classification[17]

23 Extraction of H pylori Genomic DNA Genomic DNAwas extracted from the antral biopsy specimens using thePureLink Genomic DNA Mini kit (Invitrogen Sao PauloBrazil)

24 Detection of H pylori PCR amplification for the detec-tion of H pylori DNA in gastric mucosa was performed aspreviously described [18] Briefly one set of primers (p1-F andp2-R) that amplify a fragment of 298 bp of the 26-kDa antigengene present in all H pylori strains was used for detection ofthe bacterium The study included samples from patients inwhich bacterial DNA was isolated

25 Amplification of the Constant Region of the cagA GeneThe constant region of the cagA gene was analyzed in samplespositive forH pylori All patients positive for this region werethen submitted to investigation of variable region (EPIYA)polymorphisms The constant region of the cagA gene wasamplified by the polymerase chain reaction (PCR) using theCagAConF 51015840-GTGCCTGCTAGTTTGTCAGCG-31015840 andCagAConR 51015840 TTGGAAACCACCTTTTGTATTAGC-31015840primers [14] Negative and positive controls were includedin all reactions The PCR products were separated by elec-trophoresis on 2 agarose gel that was stained with ethidiumbromide and visualized under a UV transilluminator

26 Amplification of the 31015840-Variable Region of the cagA Geneand Determination of the EPIYA Pattern The followingprimers described by Yamaoka et al [19] were used foramplification of the 31015840-variable region of the cagA geneCAG1 51015840-ACCCTAGTCGGTAATGGGTTA-31015840 and CAG251015840-GTAATTGTCTAGTTTCGC-31015840The reaction consisted of05mM of each primer 1X PCR buffer 15mMMgCl2 sterilewater 02mM of each deoxynucleotide 125120583L Taq DNApolymerase (Invitrogen) and 2 120583L DNA in a final volume of25 120583LThe amplification conditions were initial denaturationat 95∘C for 2min followed by 35 cycles of denaturation at95∘C for 1min annealing at 56∘C and extension at 72∘Cfor 1min with a final extension at 72∘C for 10min PCRwas carried out in a thermocycler GeneAmp PCR system9700 (Applied Biosystems) Products of 500 to 850 bp wereobtained depending on the type and number of repeats ofthe EPIYA-C motif in the cagA gene The PCR productswere separated by electrophoresis on 2 agarose gel that wasstained with ethidium bromide and visualized under a UVtransilluminator Positive controls of the different patterns of





3 Results









Cancer119899 ()

Monoinfection


Mixedinfection







4 Discussion
























5 Conclusions




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results









Cancer119899 ()

Monoinfection


Mixedinfection







4 Discussion
























5 Conclusions




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















5 Conclusions




References





































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

iuuq ey epj psh - downloads.hindawi.comdownloads.hindawi.com/archive/2014/418063.pdf · research...

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