Infection with CagA-positiveHelicobacter pylori strain containing three EPIYA C phosphorylation sites is associated with more severe gastric lesions in experimentally infectedMongolian gerbils (Meriones unguiculatus)M. Ferreira Júnior,1 S.A. Batista,2P.V.T Vidigal,3 A.A.C. Cordeiro,1F.M.S. Oliveira,1 L.O. Prata,1A.E.T. Diniz,4 C.M. Barral,4R.C. Barbuto,4 A.D. Gomes,2I.D. Araújo,4 D.M.M. Queiroz,2* M.V. Caliari1*1Department of General Pathology,Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte2Laboratory of Research in Bacteriology,Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte3Department of Anatomical Pathology,Faculty of Medicine, Federal University of Minas Gerais, Belo Horizonte 4Surgery Department, Faculty ofMedicine, Federal University of MinasGerais, Belo Horizonte, MG, Brazil*Joint senior authors

Abstract

Infection with Helicobacter pylori strainscontaining high number of EPIYA-C phospho-rylation sites in the CagA is associated withsignificant gastritis and increased risk ofdeveloping pre-malignant gastric lesions andgastric carcinoma. However, these findingshave not been reproduced in animal modelsyet. Therefore, we investigated the effect onthe gastric mucosa of Mongolian gerbil(Meriones unguiculatus) infected with CagA-positive H. pylori strains exhibiting one orthree EPIYA-C phosphorilation sites.Mongolian gerbils were inoculated with H.pylori clonal isolates containing one or threeEPIYA-C phosphorylation sites. Control groupwas composed by uninfected animals chal-lenged with Brucella broth alone. Gastric frag-ments were evaluated by the modified SydneySystem and digital morphometry. Clonal relat-edness between the isolates was considered bythe identical RAPD-PCR profiles and sequenc-ing of five housekeeping genes, vacA i/d

region and of oipA. The other virulence mark-ers were present in both isolates (vacAs1i1d1m1, iceA2, and intact dupA). CagA ofboth isolates was translocated and phosphory-lated in AGS cells. After 45 days of infection,there was a significant increase in the numberof inflammatory cells and in the area of thelamina propria in the infected animals, notablyin those infected by the CagA-positive strainwith three EPIYA-C phosphorylation sites.After six months of infection, a high number ofEPIYA-C phosphorylation sites was associatedwith progressive increase in the intensity ofgastritis and in the area of the lamina propria.Atrophy, intestinal metaplasia, and dysplasiawere also observed more frequently in animalsinfected with the CagA-positive isolate withthree EPIYA-C sites. We conclude that infec-tion with H. pylori strain carrying a high num-ber of CagA EPIYA-C phosphorylation sites isassociated with more severe gastric lesions inan animal model of H. pylori infection.

Introduction

Helicobacter pylori is a Gram-negative spiralbacterium that chronically infects the gastricmucosa of more than 50% of the world popula-tion.1 The infection is associated with chronicgastritis, gastric and duodenal peptic ulcer,gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT)lymphoma.2 Inflammation, atrophy, intestinalmetaplasia, dysplasia, and gastric carcinomaare noteworthy3 among the histologicalchanges in the gastric mucosa caused by thecolonisation of H pylori.It is well demonstrated that there is a geo-

graphic variation among the H. pylori strainsthat exhibit a broad range of genetic diversity.A striking difference that characterises morevirulent strains is the presence of the cag PAI(cag pathogenicity island) with approximately40-Kb consisting of 27-31 genes.4 Among them,the cagA gene encodes the CagA protein thatpossesses carcinogenic properties. The islandis responsible for coding a type IV secretionsystem (T4SS), which allows the entrance ofCagA protein inside the gastric epithelial cells,followed by protein phosphorylation and theactivation of proinflammatory and antiapoptot-ic genes that increase the risk of developingprecancerous lesions.5,6 CagA phosphorylationoccurs on tyrosine residues in a repeated five-amino-acid sequences located in the carboxylterminus, denominated EPIYA (Glu, Pro, Ile,Tyr and Ala) A, B, C, and D according to differ-ent flanking amino acids. The D (Eastern) andC (Western) are the main sites for CagA phos-phorylation by multiple members of the Srckinase family. Phosphorylated CagA forms a

complex with SHP-2 phosphatase triggeringabnormal cellular signals that lead to deregula-tion of cell growth, cell to cell contact and cellmigration, elongation of epithelial cells andincrease of epithelial cell turnover, whichenhance the risk of acquiring precancerouschanges.5,7 Human infection with CagA-posi-tive H. pylori strains containing high numberof EPIYA-C phosphorylation sites is associatedwith a more severe chronic gastritis and anincreased risk of developing intestinal meta-plasia and gastric cancer.6,8 However, we are

European Journal of Histochemistry 2015; volume 59:2489

Correspondence: Prof. Marcelo Vidigal Caliari,Department of General Pathology, Institute ofBiological Sciences, Federal University of MinasGerais, Av. Antônio Carlos 6627, Belo Horizonte,MG, CEP 31 270-010, Brazil. Tel. +55.31.34092892 -Fax: +55.31.34092879. E-mail: caliari@icb.ufmg.br

Key words: Gastritis, atrophy, intestinal metapla-sia, dysplasia, Mongolian gerbil, cagA EPIYA Cmotif.

Contributions: SAB, ADG, DMMQ, DNA extrac-tion, RUT, analysis of CagA translocation andDNA sequencing; PVTV, IDA, classification andgrading of gastritis by the Sydney system andmacroscopic analysis of the gastric mucosa; MFJ,MVC, AETD, CMB, RCB, inoculation of the ani-mals, necropsy and histopathological techniques;MFJ, FMSO, AACC, LOP, immunohistochemicaltechniques; MVC, AACC, MFJ, morphometricanalysis of the inflammatory infiltrate and thearea of the antral lamina propria; DMMQ, Dataanalysis; DMMQ, MVC, MFJ, wrote the paper;DMMQ, MVC, IDA ,designed the study, supervisedthe laboratory works. All authors read andapproved the manuscript.

Conflict of interest: the authors declare no con-flict of interest.

Funding: this work was financially supported bythe Dean of Research of the Federal University ofMinas Gerais (Pró-Reitoria de Pesquisa daUniversidade Federal de Minas Gerais-PRPq/UFMG), The Minas Gerais State ResearchFoundation (Fundação de Amparo à Pesquisa doEstado de Minas Gerais - FAPEMIG), and theNational Council for Research and Development(Conselho Nacional de Pesquisa eDesenvolvimento - CNPq).

Received for publication: 4 February 2015.Accepted for publication: 4 April 2015.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright M. Ferreira Júnior et al., 2015Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2015; 59:2489doi:10.4081/ejh.2015.2489

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unaware of studies confirming, in animal mod-els, the associations observed in humanbeings.The aim of this study was therefore to eval-

uate qualitatively and through digital mor-phometry, the histological alterations inducedby the infection with CagA-positive H. pyloristrains with different numbers of EPIYA-C onthe gastric mucosa of Mongolian gerbil.

Materials and MethodsH. pylori strains Mongolian gerbil is more easily colonized by

H. pylori strain that was recently isolated,without many subcultures. Thus, fragments ofthe antral mucosa of the stomach obtained atsurgery from a 71-year-old patient with gastriccarcinoma was smeared on BHM agar platesand incubated in microaerophilic conditions at37ºC.9 After 48 h of growth, the entire popula-tion of bacterial cells recovered from the plateswas harvested and stored at -70ºC (H. pyloristock collection from the Laboratory ofResearch in Bacteriology, Faculty of Medicine,Federal University of Minas Gerais). Single-colonies of H. pylori, obtained from the origi-nal culture, were propagated as pools for lessthan 10 passages before freezing at -70ºC. Thegrowth of each colony was tested for EPIYA-Cpatterns by PCR according to Yamaoka et al.10

and the results were confirmed bysequencing.6 Two isolates, LPB 1034-14 andLPB 1034-6, containing one or three EPIYA-Cphosphorylation sites, were used to inoculatethe animals. Strains isolated from adifferentindividuals show substantial genomic diversi-ty, although isolates from single individual orfamily members are frequently clonal.11 Clonalrelatedness between the isolates was evaluat-ed by Random-amplified DNA (RAPD)-PCR byusing two sets of primers according toAkopyanz et al.12 and sequencing of the house-

keeping genes efp (564 kb), yphC (724 kb),atpA (829 kb), ureI (670 kb) and mutY (652kb)13 and the virulence genes involved in theinflammatory response to the infection; vacAi/d regions, CT dinucleotide repeated motifregion of oipA14,15 and intact dupA.16 The pres-ence of other cag PAI genes involved in thetranslocation of the cagA to the host cells (cagl,cagL, cagX, cagY and cagT) and cagE17 and thegenotype of vacA and iceA18 were also evaluat-ed. All primers and PCR conditions are listed inSupplementary Table 1. Two days culture of theisolates was prepared for the inocula. The cellswere harvested in Brucella broth (difco). Theconcentration was adjusted according to OD at550 nm and the cells were administered to theanimals immediately after harvesting.

Analysis of CagA translocation CagA translocation was confirmed by

Western blotting and immunoprecipitation asproposed by Liang et al.19 Cag PAI functionalitywas evaluated by investigating the transloca-tion followed by phosphorylation of the CagAEPIYA-C from the isolates into the human gas-tric epithelial AGS cells (ATCC® CRL 1739™).The cells were cultured in RPMI 1640 medium(Invitrogen, São Paulo, Brazil), containing10% FBS (Invitrogen), incubated at 37°C in afully humidified atmosphere with 5% CO2 inair. On the day of the experiment, confluentcells were incubated overnight in fresh serumand antibiotic-free media. AGS cells were co-incubated with H. pylori up to multiplicities ofinfection of 100:1. After six hours the cellswere washed four times with PBS and lysedwith a lysis buffer containing 50 mM Tris (pH7.5), 5 mM EDTA, 100 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, andprotease inhibitors (Roche Applied Science).To confirm the CagA translocation, proteins inthe whole cell extracts (30 μg) were separatedby SDS-PAGE (4-12% gels) and electrotrans-ferred to nitrocellulose membranes(Invitrogen). After, blotting was blocked in a

solution of 3% skim milk, 0.05% Tween 20, andTBS, the membrane-bound proteins wereprobed with primary monoclonal antibodyagainst CagA (A-10, Santa Cruz Biotechnology,Santa Cruz, CA, USA). The membranes werewashed and then incubated with horseradishperoxidase-conjugated secondary antibody for60 min. Antibody-bound protein bands weredetected using enhanced chemiluminescencereagents (Millipore, Billerica, MA, USA) andcaptured with ImageQuant 350 (GEHealthcare, Uppsala, Sweden).Immunoprecipitation was performed using

1 mg of the lysate protein obtained from theAGS cells co-incubated with the H. pylori iso-lates. Each sample was incubated overnight at4ºC with monoclonal anti-H.pylori CagA anti-body (A-10, Santa Cruz Biotechnology) fol-lowed by four hours at 4ºC with 30 µL aliquotof protein G Sepharose beads (Invitrogen).The beads were washed five times with coldlysis buffer, and proteins were eluted by boil-ing for 10 min in 2x electrophoresis samplebuffer containing 5 mmol/L Tris, 10% sodiumdodecyl sulfate, 12% 2-mercaptoethanol, 20%glycine, and 1% Bromophenol blue.Immunoprecipitated proteins were subjectedto SDS-PAGE, transferred to nitrocellulosemembranes and incubated with monoclonalanti p-tyr antibody (pY-99, Santa CruzBiotechnology). The bands were identified byusing a chemiluminescence reagent(Millipore) and captured with ImageQuant 350(GE Healthcare).

DNA extraction DNA was extracted with the QIAamp kit

(QIAGEN, Hilden, Germany) according to themanufacturer’s instructions with slight modi-fications.20 The DNA concentration was deter-mined by spectrophotometry using theUltrospec® 2100 Pro apparatus (AmershamBiosciences, Uppsala, Sweden), and theextracted DNA was kept at -20°C until use.

Original Paper

Table 1. Gastric inflammation, inflammatory activity and the number of lymphoid follicles in Mongolian gerbils infected withHelicobacter pylori 45 days and six months post-inoculation.

Groups Inflammation (MN cells) Activity (PMN cells) LF 0 + ++ +++ 0 + ++ +++

45 days CTRL 8 0 0 0 8 0 0 0 0 CagA1Ep 0 6 2 0 2 6 0 0 3 CagA3Ep 0 3 5 0 0 5 3 0 12Six months CTRL 8 0 0 0 8 0 0 0 0 CagA1Ep 0 2 6 0 1 4 3 0 13 CagA3Ep 0 1 4 3 0 2 3 3 15

MN, mononuclear cells; PMN, polymorphonuclear cells; LF, lymphoid follicles; CTRL, control group consisting of uninfected animal; CagA1Ep, CagA-positive with one EPIYA-C group; CagA3Ep, CagA-pos-itive with three EPIYA-C group; 0, absent; +, mild; ++ moderate; +++, severe. Significant differences were observed when the controls were compared with the CagA3Ep and with the CagA1Ep animalsregarding the scores of MN (P<0.001) and of PMN (P≤0.003) cells after 45 days and six months of infection. Data were analysed by the two tailed Mann-Whitney U test.

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H. pylori specific urea, cagA andcagA 3’ variable region in the gastric mucosa The primers and conditions used for the

amplification of urea and cagA were previouslydescribed by Clayton et al.21 and Peek et al.22

and the cagA 3’ region was amplified accordingto Yamaoka et al.10 (Supplementary Table 1).

Sequencing of the cagA 3’ variableregion, vacA i/d region, intactdupA, and CT dinucleoptiderepeat motif region of oipAIn general, PCR products were purified

with the Wizard SV Gel and PCR Clean-UpSystem (Promega, Madison, MI, USA) accord-ing the manufacturer’s recommendations andsequenced using an ABI Prism BigDyeTerminator Cycle Sequencing kit in an ABIPrism 310 automatic sequencer (AppliedBiosystems, Foster City, CA, USA). Thesequences obtained were aligned using theCAP3 Sequence Assembly Program (http://pbil.univ-lyon1.fr/cap3.php). Regarding thedupA, the primer walking methodology usingfive primer sets was employed(Supplementary Table 1).16 After alignment,nucleotide sequences of the cagA 3’ variableregion and vacA i/d region were transformedinto aminoacid sequences using the Blastxprogram (http://blast.ncbi.nlm.nih. gov/Blast.cgi). The nucleotide sequences of thegene loci of the gene strains were depositedin GenBank under the following accessionnumbers: efp GenBank accession numbersKP723366, KP723367, KP723368; yphCGenBank accession numbers KP723369,KP723370, KP723371; atpA GenBank acces-sion numbers KP723372, KP723373,KP723374; ureI GenBank accession numbersKP723375, KP723376, KP723377; mutYGenBank accession numbers KP723378,KP723379, KP723380.

AnimalsForty-eight three-month-old female

Mongolian gerbils obtained from the animalhouse of the Institute of BiologicalSciences/Universidade Federal de MinasGerais were maintained with appropriatenutrition, ventilation and lighting conditionsand handled in accordance with the ethicalstandards of the UFMG. They were dividedinto three experimental groups: a controlgroup consisting of uninfected animals chal-lenged with Brucella broth alone (CTRL), ani-mals inoculated with H. pylori CagA-positivestrain with one EPIYA-C segment (CagA1Ep),and animals inoculated with H. pylori CagA-positive strain with three EPIYA-C segments(CagA3Ep). Each group consisted of 16 ani-mals; eight animals from each group were

sacrificed 45 days after the inoculation, andthe remaining eight animals were sacrificedsix months after the infection. Prior to the inoculation, the animals were

fasted for eight hours and slightly sedatedwith isoflurane. The inocula of 0.8 mL of thebacterial suspensions containing 109 CFU/mLin sterile Brucella broth or Brucella brothalone (control group) were administered byintragastric gavage three times at intervals of48 h. Four h after the inoculation, the animalsreceived standard diet for rats (Labina®,Purina, Brazil) and water ad libitum.

Rapid urease test The rapid urease test (RUT) was per-

formed by incubating one gastric antral frag-ment into a semi-solid agar containing phe-nol red 2% and urea 0.01M at 37°C for 24 h.

Histopathological analysis andsemi-quantitative evaluation of spiral bacteriumAfter abdominal incision, the stomach was

removed and opened along the greater curva-ture. Mucosa fragments were obtained alongthe lesser and greater curvatures, includingthe regions of the gastric body and gastricantrum for DNA, RUT and histology. For histo-logical processing, the fragments were fixedin 10% buffered formalin for 48 h. Four µmthick tissue sections were stained withHematoxylin and Eosin (H&E) for histologi-cal analyses and Silver staining confirmed byCarbolfuchsin staining for the semi quantita-tive evaluation of the spiral bacteria load inscores (from 0 to 3) in the gastric glands.23

Single organism or no more than two smallclusters of stainable organisms were scoredas 1, three to six clusters were scored as 2 andmore than six as 3.24 The H&E stained sec-tions were used for the histopathologicalanalysis and the classification of gastritisusing the Sydney system,3 as well as for themorphometric analysis of the inflammatoryinfiltrate and calculation of the antral laminapropria area. The presence of intestinal meta-plasia was confirmed by using the PAS-AlcianBlue technique, pH 2.5, which stains acidmucins.

Classification and grading of thegastritis by the modified SydneysystemThe degree of gastric inflammation

(mononuclear cells; MN) and activity (poly-morphonuclear cells; PMN) was evaluated bythe modified Sydney system3 and classified asfollows: absent (no inflammatory cells), mild,moderate, and severe, according to the num-ber of inflammatory cells. The presence ofatrophy, intestinal metaplasia, and dysplasia

as well as lymphoid follicles were also investi-gated.

Morphometric analysis of theinflammatory infiltrate and antrallamina propria areaAll histological sections were analysed with

a 40x objective, and 15 images of the gastricantral mucosa were captured using a Q-Color3microcamera (Olympus, Tokyo, Japan). A totalarea of 1.37¥106 µm2 of mucosa was analysedfor each case. Algorithms from the KS300 soft-ware (Carl Zeiss, Oberkochen, Germany) wereused for image processing and segmentation,definition of the morphometric conditions, cellcounting, and calculation of the area of thelamina propria. Segmentation allowed toselect the nuclei of all cell types present in thelamina propria of the antral mucosa and toexclude the cell cytoplasm and other structurespresent in the histological sections. By thisprocess, a binary image was created, and thenuclei of all cell types presented in the antralmucosa were counted. The result obtained inthe control animals was considered the expect-ed cellular pattern of the antral mucosa with-out inflammatory infiltration. In the infectedanimals, the nuclei of the cell types normallypresent in the lamina propria of the antralmucosa and nuclei of leukocytes recruited inthe inflammatory process were counted, allow-ing a quantitative assessment of inflammationby a method that we previously used in otherstudies.25 Subsequently, all pixels of the lami-na propria were selected to obtain anotherbinary image to calculate the area in μm2.

Statistical analysisThe comparison between the degree of

inflammation and activity of the gastritis aswell as the scores of the spiral microorganismamong the groups were evaluated by the twotailed Mann-Whitney U test. Because the num-ber of inflammatory cells and the area of theantral lamina propria were not normally dis-tributed, even after log transformation, themorphometric data were compared among thedifferent groups and periods of infection by thetwo tailed Mann-Whitney U test. P values ≤0.05were considered significant.

ResultsComparison of the two H. pyloriisolatesThe isolates yielded cagA PCR products with

different size of 640 and 850 bp (Figure 1a).The sequence analyses demonstrated an inser-tion of 204-bp in the isolate LPB1034-6 com-pared with the isolate LPB1034-14. The cagA

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sequences were 99.2% identical (Figure 1c).Translation of the nucleotide sequences iden-tified an insertion that corresponds to twoidentical EPYIA-C motifs and multimerizationsites in the LPB1034-6 isolate (Figure 1d). The strains were considered closely related,

by the identical RAPD-PCR profiles (Figure 1b)and identical oipA, with 9 CT (SupplementaryFigure 1a), vacA i/d (Supplementary Figure1b), and five housekeeping genes sequences(efp GenBank accession number 1794575;yphC GenBank accession number 1794676;atpA GenBank accession number 1794697;ureI GenBank accession number 1794705;mutY GenBank accession number 1796141).Since the cagE, cagl, cagL, cagX, cagY and cagTgenes are involved in the induction of IL-8secretion and the lack of one of them mayimpair the CagA translocation, their presencewas also evaluated and confirmed in both iso-lates. Furthermore, the genotypes of the otherbacterium virulence markers involved in gas-tric inflammation (vacA s1i1d1m1, iceA2, andintact dupA with 1884 bp) were identical inboth isolates.

Translocation of CagA into AGS cellThe lysates obtained after co-incubation of

the isolates and AGS cells were subjected toimmunoprecipitation and Western blot usinganti-CagA and anti-pY99 monoclonal antibod-ies. The results in Figure 2 show that the pre-cipitate protein reacted with both anti-CagAand anti-phosphotyrosine demonstrating thatCagA is the tyrosine phosphorylated protein.

H. pylori status The animals were considered H. pylori

infected when two were positive among thetests. RUT was positive in 84.4% of the infect-ed animals and negative in all control animals.The ureA as well cagA genes were present inthe gastric mucosa of all infected animals, butnot in the gastric mucosa of the control ani-mals. PCR to detect the number of CagA EPIYA-C motifs was positive in 45 days and sixmonths post-infection in all infected animals.The EPIYA-C PCR results were confirmed bysequencing without differences before andafter inoculation (Figure 1 c,d). The virulencegenes remained present as detected by PCR.In the inoculated animals, the degree of

stainable spiral organism varied from 1 to 3(median=2) in both groups infected withstrains containing one or three EPIYA-C seg-ments either 45 days or six months post-infec-tion without differences between the groups(P=0.53). The bacterial load was higher in theantral (median=2) than in the oxyntic (medi-an=1) gastric mucosa in both infected animalgroups (P=0.04).

Macroscopic analysis of the gastricmucosaForty-five days after the infection, none of

the control animals exhibited macroscopicalterations in the gastric mucosa. Six monthspost-infection, macroscopic lesion continuedto be absent in the control group (Figure 3a).Among the eight animals from the CagA1Epgroup, one exhibited a non-ulcerative flatred/purple haemorrhagic lesion in the oxynticmucosa (Figure 3b). A reduction of the oxynticmucosa as a consequence of an expansion ofthe antral mucosa area was observed in thestomach of another animal from the CagA1Epgroup and of four animals from the CagA3Epgroup. In one animal from the CagA3Ep group,the expansion of the antral mucosa was more

Original Paper

Figure 1. Paired H. pylori isolates from a single patient that differ in the cagA 3' region. a) Polymerase chain reaction showing differentcagA allele size of the isolate 1034-14 and 1034-6. b) Identical RAPD-PCR profiles of the isolates 1034-14 and 1034-6 with two setsof primers, 1281 and 1254. c) The sequencing of the PCR products demonstrated an insertion of 204-bp in the isolate 1034-6 com-pared with the isolate 1034-14. d) Translation of the nucleotide sequences identified an insertion that corresponds to two identical mul-timerization sites and EPYIA-C motifs in the 1034-6 isolate.

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marked, resulting in almost complete loss ofthe oxyntic mucosa (Figure 3c).

Classification and grading of gastri-tis by the Sydney systemThe results regarding the classification and

grading of the gastritis according to theSydney system are shown in Table 1. Only fewsparsely distributed MN cells were observed inthe antral mucosa of the control animals either45 days or six months after the inoculation,which was considered the expected cellularitypattern of the antral lamina propria of gerbil inthe age ranges studied. Forty-five days post-inoculation, all animals

from the CagA1Ep and CagA3Ep groups exhib-ited gastric inflammatory infiltrate composedby lymphocytes, macrophages, plasmocytes,and neutrophils, predominantly in the basalmucosa (Figure 4a). These cells were also

found in the middle third of the gastric mucosaand in the glandular epithelium in some ani-mals from the CagA3Ep group (Figure 4b). Theinflammation was mild in the stomach of mostanimals of the CagA1Ep group, whereas, mod-erate degree of gastric inflammation wasobserved in five gerbils of the CagA3Ep group.A higher degree of gastritis activity (P=0.03)and a tendency of higher degree of MN cells(P=0.1) were observed in the CagA3Ep groupwhen compared with the CagA1Ep group(Table 1). Significant differences wereobserved in respect to all parameters when thecontrols were compared with the CagA3Ep ani-mals in relation to the scores of MN and PMNcells (P<0.001) and with the CagA1Ep animalsregarding to MN (P<0.001) and PMN(P=0.003) cells (Table 1).Six months post-inoculation, the degree of

gastric mucosa MN (P=0.01) and PMN

(P=0.04) cells was more pronounced in theanimals at six months of infection than in the45 days post-infection animals (Figure 4 c-f,Table 1). The inflammatory infiltrate was moreintense at the base, but expanding to the mid-dle third of the mucosa in most animals, espe-cially in those from the CagA3Ep group. Theinflammatory infiltrate also reached the apicalthird of the mucosa in animals from theCagA3Ep group (Figure 4 e,f). There was a sig-nificant higher degree of neutrophils (P=0.05)in the gastric mucosa of the CagA3Ep whencompared to CagA1Ep group. There was also atrend towards an increase score of MN cells inthe CagA3Ep group compared with theCagA1Ep group (P=0.06) (Table 1). The lym-phoid follicles were abundant in both groups ofinfected animals, with no difference betweenthem. Other lesions such as atrophy, intestinalmetaplasia and dysplasia were also observed in

Original Paper

Table 2. Lesions in the gastric mucosa of Mongolian gerbils of the control group and the groups with six months of infection withHelicobacter pylori.

Groups Intestinal metaplasia Atrophy Displasia 0 + ++ +++ 0 + ++ +++ 0 + ++ +++

CTRL 8 0 0 0 8 0 0 0 8 0 0 0CagA1Ep 6 2 0 0 4 3 1 0 6 2 0 0CagA3Ep 4 4 0 0 1 4 3 0 5 3 0 0

CTRL, control group consisting of uninfected animal; CagA1Ep, CagA-positive with 1 EPIYA-C group; CagA3Ep, CagA-positive with 3 EPIYA-C group; 0, absent; +, mild; ++ moderate; +++, severe.Significant differences were observed between the control group and the CagA3Ep group with respect to atrophy (P<0.001) and intestinal metaplasia (P<0.03) and a trend toward dysplasia (P=0.06).The degree of atrophy was also significantly higher (P=0.03) in the group of animals infected by the strain containing one EPIYA-C motif when compared with the controls. Data were analysed by thetwo tailed Mann-Whitney U test.

Figure 2. Immunoblotting analysis of CagA protein and tyrosine phosphorylation of H. pylori isolates with CagA possessing one orthree EPIYA-C segments in human gastric epithelial cells (AGS). Total lysates of AGS cells after co-incubation with H. pylori harbouringCagA with one or three EPIYA-C segments (MOI: 100; for 6 h) were immunoblotted (IB) with anti-CagA (A-10) monoclonal antibody.To assess tyrosine phosphorylation, the total lysates were immunoprecipitated (IP) with anti-CagA antibody and then immunoblotted(IB) with the anti-phosphotyrosine monoclonal antibody (PY99). Uninfected AGS cells were used as control. The GAPDH blot servedas loading control in each sample.

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the infected animals (Figure 5), but not in thecontrol group. Significant differences wereobserved between the control group and theCagA3Ep group with respect to atrophy(p<0.001), intestinal metaplasia (P<0.03) anda trend toward dysplasia (P=0.06). The degreeof atrophy was also significantly higher(P=0.03) in the group of animals infected bythe strain containing one EPIYA-C motif whencompared with the controls. The scores of atro-phy were higher (P=0.05) in the CagA3Ep thanin the CagA1Ep group (Table 2).

Morphometric analysis of the inflammatory infiltrateForty-five days post-infection, there was a

significant increase in the number of inflam-matory cells when compared both infectedgroups with the non-infected animals(P≤0.003). The number of inflammatory cellswas significantly higher in the CagA3Ep thanin the CagA1Ep group, (P=0.02) (Figure 6a).Confirming these results, the area of the lami-na propria occupied by the inflammatory infil-

trate after the same period of infection wasalso significantly larger in both infectedgroups than in the control group (P≤0.003).CagA3Ep animals showed a larger area thanthe CagA1Ep animals (P=0.01) (Figure 6b).Six months post-infection, there were signifi-cantly higher number of inflammatory cellsand larger area of the antral lamina propria inboth infected groups compared with the CTRLgroup (P≤0.004). Additionally, there was a sta-tistically significant higher inflammatory infil-trate (P=0.01) and a larger area of lamina pro-

Original Paper

Figure 3. Stomach of Mongolian gerbilsinfected with H. pylori 6 months after theinoculation. a) Normal gastric mucosa ofan animal from the control group. b) aflat area with a haemorrhagic aspect(arrowhead) in the antral mucosa of ananimal from the CagA1Ep group. c)Expansion of the antral mucosa (*) on theoxyntic mucosa (#) of an animal from theCagA3Ep group. Scale bars: 2 cm.

Figure 4. Antral mucosa of Mongolian gerbils infected with Helicobacter pylori: muscularismucosae (m). a) Antral mucosa of an animal from the CagA1Ep 45 days after the infection;discreet inflammation observed on the basal third of the gastric mucosa. b) Antral mucosaof an animal from the CagA3Ep group 45 days after the infection; inflammation was moresevere, also affecting the middle third. c) Antral mucosa of an animal from the CagA1Epgroup 6 months after the inoculation. d) Higher magnification of the previous image;insert: detail of the inflammatory infiltrate. e) Antral mucosa of an animal from theCagA3Ep group 6 months after the infection; severe inflammation was observed withincrease in the area of the lamina propria. f ) Magnification of panel e); insert: detail of theinflammatory infiltrate; H&E. Scale bars: a,b,d,f ), 50 µm; c,e), 100 µm.

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pria (P=0.03) in the CagA3Ep than in theCagA1Ep group (Figure 6 c,d). A higher num-ber of cells (Pp=0.007) and a larger area oflamina propria (P=0.05) were also observed inthe gastric mucosa of infected animals at sixmonths post-infection when compared withthe gastric mucosa of animals at 45 days post-infection (Figure 6 e,f).

Discussion

There are studies demonstrating that infec-tion with CagA-positive H. pylori strains con-taining more EPYIA-C phosphorylation sites isassociated with more severe gastritis, pre-can-cerous gastric lesions and gastric cancer.5,6,9,21

However, the associations observed in humanbeings have not been confirmed experimental-ly in animal models. The most important find-ing of this study was the presence of moresevere lesions in the gastric mucosa ofMongolian gerbils experimentally infectedwith H. pylori strain carrying three EPIYA-Cmotifs in the CagA protein and a functionalcagPAI. There are several studies in the litera-ture that have evaluated both humans andexperimental animals infected by H. pyloristrains containing or lacking the Cag PAI genein different parts of the world. However, todate, this is the only study investigating H.pylori infection of the gastric mucosa with H.pylori strains containing different CagA phos-phorylation sites in a classic experimentalmodel for studying this type of infection. It isworthy noticing that CagA from both isolateswere translocated and tyrosine phosphorylatedin AGS cells and that the results we observeddid not depend on either the bacterium load oron great differences between the strains oth-ers than the EPIYA-C motif number. In fact, thestrains, isolated from a single patient, are fre-quently clonal.11,26 In agreement, all sequencedgenes were ~100% identical when the isolateswere compared. Furthermore, both isolateshave the same genotypes of H. pylori virulencegenes involved in gastric inflammation.27

Therefore, the difference in the number ofEPIYA-C segments of the strains results fromintragenomic rearrangement and not fromhorizontal acquisition from another H. pyloristrain that would differ in nucleotide sequenceby ~6% on average.28 One advantage of thisstudy was to use for inoculation isolates withfew passages, which colonize the gastricmucosa of gerbil better than manipulatedstrains or collection strains. The histopatho-logical results evaluated by the semi-quantita-tive Sydney system and the quantitative mor-phometric methods were in accordance; how-ever, the last approach allowed a better dis-crimination between strains containing one or

three EPIYA-C segments in the CagA proteinwith respect to the number of the inflammato-ry cells. When crossing the gastric mucouslayer, H. pylori adheres to epithelial cells,releases toxins, and triggers an inflammatoryprocess characterised as gastritis, which startsin the antral mucosa. This inflammatoryprocess leads to the accumulation of a superfi-cial acute inflammatory infiltrate composed of

lymphocytes, monocytes, plasmocytes, andneutrophils which causes oedema in the lami-na propria. Forty-five days after the inocula-tion, a predominantly antral inflammatoryinfiltrate, an increase in the area of the laminapropria and the presence of lymphoid folliclesthat are characteristic markers of the gastritiscaused by H. pylori3 were observed in theinfected animals, especially in those carrying

Original Paper

Figure 5. Gastric mucosa of Mongolian gerbils infected with H. pylori for six months: mus-cularis mucosae (m). a) Moderate atrophy of the gastric oxyntic mucosa with the gastricglands replaced by connective tissue in an animal from the CagA3Ep group (*); H&E; scalebar: 100 µm. b) Mild intestinal metaplasia in the oxyntic mucosa of an animal from theCagA3Ep group; note the presence of goblet cells in the mucosa (arrowheads); H&E; scalebar: 20 µm. c) Gastric mucosa of an animal from the CagA3Ep group with usual glandularepithelium (arrowheads) consisting of mucus producing cells and areas of incompleteintestinal metaplasia (arrows) stained by PAS-Alcian Blue; pH 2.5; scale bar: 50 μm. d)Detail of the previous figure; Goblet cells positive for PAS-Alcian Blue (arrows); scale bar:20 µm. e) Gastric mucosa of an animal from CagA3Ep group with tubular glands contain-ing well-formed goblet cells (arrows) stained by PAS-Alcian Blue; scale bar: 20 µm. f )Presence of mild dysplasia in the gastric oxyntic mucosa with nuclear pleomorphism andevident nucleoli (arrowheads) as well as mitotic figure (arrow) in an animal from theCagA3Ep group; H&E; scale bar: 50 μm.

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the isolate with cagA containing three EPYIA-Csegments. The follicles were also more abun-dant in the gastric mucosa of the CagA3Ep ani-mals. Six months after infection, the inflam-matory infiltrate was more intense and com-posed predominantly of mononuclear cells andneutrophils, characterised as active chronicgastritis. The inflammatory infiltrate was morefrequent in the antral mucosa and expanded tothe oxyntic mucosa. These results are inagreement with other authors, who demon-strated in gerbils infected with H. pylori thatthe intensity of the parasitism and gastritisare initially greater in the antral mucosa,expanding later to the oxyntic mucosa.28 Inaddition, we observed a significant and pro-gressive increase in the number of inflamma-tory cells in the groups infected with H. pylori(CagA1Ep and CagA3Ep), and this increasewas most pronounced in the CagA3Ep group,as demonstrated by the quantitative digitalmorphometric analysis. A direct relationshipbetween the presence of the CagA protein andmore severe forms of the gastric diseasescaused by H. pylori infection is reported byother authors.5,6,7,29 Álvares et al. found inpatients infected with different H. pyloristrains that the inflammation, activity, atrophy,and IM were significantly more severe inpatients infected with cagA-positive samplescompared with the negative ones.30 Moreover,H. pylori strains with higher numbers ofEPIYAs exhibit higher levels of CagA phospho-rylation and, therefore, are associated withmore severe forms of gastric diseases. In ourexperiment, in addition to increased inflam-mation, we also observed significant differ-ences in the inflammatory activity between theCagA1Ep and CagA3Ep groups.Intestinal metaplasia was also often

observed in the animals from the CagA3Epgroup 6 months after infection, and in someanimals, its development was concomitantwith the development of atrophy and dysplasia.This type of lesion is a characteristic finding ofchronic atrophic gastritis in the human stom-ach and is considered the most important riskfactor for the development of intestinal carci-noma. The development of intestinal metapla-sia has not been observed in any other experi-mental model of H. pylori infection. In the gas-tric mucosa of gerbils infected with H. pyloristrains CagA+, gastric mucosa lesions arereportedly more severe, including chronicatrophic gastritis, incomplete-type IM and/oran increased risk of gastric cancer comparedwith animals infected with CagA-negative H.pylori strains or with a mutant form ofCagA.29,31 Thus, the gerbil was an ideal modelto investigate the histogenesis of inflamma-tion and metaplasia and its relationship withcarcinogenesis. The morphometric analysisallowed us to quantify the level of inflamma-

tion, providing information that was impossi-ble to obtain through semi-quantitative analy-sis. The inflammation induced by H. pyloriinfection is a chronic active gastritis, charac-terised by lymphocytic and neutrophilic infil-tration in the gastric mucosa. From the inflam-matory infiltrate, foci of intestinal metaplasiawith subsequent atrophy of the mucosa, aggre-gates, and lymphoid follicles occur concurrent-ly, and the foveolar epithelium is replaced by aregenerative epithelium with reduced mucinsecretion.21 In the present study, the atrophy

of the gastric mucosa also occurred concur-rently with the development of intestinal meta-plasia and lymphoid follicles. Using gerbilsinfected with H. pylori, Murakami et al.demonstrated a reduction in the intercellularcadherin content with the subsequent loss ofthe parietal cells and its replacement bymucous cells.32 It is convincingly reported indifferent articles that some proinflammatorycytokines, such as tumour necrosis factoralpha (TNF-α), induce apoptosis of parietalcells;33 the loss of these cells reduces the secre-

Original Paper

Figure 6. Box plots representing the total number of inflammatory cells and the laminapropria area in 15 fields of the gastric mucosa of Mongolian gerbils infected withH. pyloristrains containing one or three CagA EPIYA-C phosphorylation C sites and uninfected ani-mals as a control group at 45 days (a, b) or six months (c, d) after the inoculation; eachgroup is composed by 8 animals. e, f ) Represent the higher number of inflammatory cellsand increased lamina propria area respectively in infected animals with six months of infec-tion compared with those with 45 days of infection; the upper and lower limits of the boxesrepresent the 75th and 25th percentiles, respectively; the horizontal bar across the box indi-cates the median and capped bars indicate the minimum and maximum data values. Datawere analyzed by the two tailed Mann-Whitney U Test.

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tion of the signal peptide that modulates thegrowth and differentiation of the gastric pro-genitor cells, resulting in an increased prolif-eration of undifferentiated cells. On the otherhand, the atrophy induces a hypochlorhydriacondition in the gastric mucosa, with conse-quent stimulation of gastrin-producing cells,inducing hypergastrinemia, which also resultsin increased proliferation of gastric mucosalcells.34 In fact, some studies reported a higherfrequency of malignant gastric lesions in indi-viduals over 50 years of age with chronicatrophic gastritis.35 Thus, for several authors,atrophic gastritis is more important thanintestinal metaplasia as a risk factor for thedevelopment of gastric cancer.36 Like atrophy,dysplasia occurred 6 months after infection.This lesion was characterised by an increasein the proliferation, loss of cell differentiation,and cellular atypia, mainly karyomegaly. Weproperly demonstrated the entire sequence ofthe lesions in the gastric mucosa caused by H.pylori in an experimental model that is mostsimilar to human infection. However, to assessthe effects of different H. pylori strains on thelikelihood of developing gastric carcinoma, anevaluation of the infection for periods longerthan 6 month is required.

Our results may be explained by the higherSHP-2 activity induced by the isolate withthree EPIYA-C phosphorylation sites. It hasbeen demonstrated that phosphorylated CagAprotein forms a physical complex with SHP-2phosphatase that activates MAPK/ERK 1/2 sig-naling pathway. ERK regulates inflammationby stimulating neutrophil adhesion and super-oxide generation, and mediates pro-inflamma-tory cytokine secretion.37 Furthermore, ERKleads to activation of genes that encode anti-apoptotic proteins, deregulation of the cellgrowth and differentiation with an increase ofthe epithelial cell turnover, which enhance therisk of damaged cells acquire pre-cancerousgenetic changes. Thus, H. pylori strain carry-ing EPIYA with multiple C repeats leads tomore SHP-2 activity and consequently higherdegree of gastric inflammation, atrophic gas-tritis, gastric metaplasia and increased risk ofgastric cancer.6,38

Concluding, we reproduced in Mongoliangerbil the pattern of gastric lesions observed inhuman beings infected with H. pylori strainwith a greater number of EPIYA-C segments inthe CagA protein. The animals developed gas-tric histopathological lesions that followed thesteps of the carcinogenesis model proposed byPelayo-Correa for intestinal gastric carcinoma,including chronic gastritis, atrophy, intestinalmetaplasia and dysplasia.31

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