Research Article Effect of Helicobacter pylori Eradication ...downloads.hindawi.com/journals/mi/2015/481972.pdf · Research Article Effect of Helicobacter pylori Eradication on TLR2

Research ArticleEffect of Helicobacter pylori Eradication on TLR2 and TLR4Expression in Patients with Gastric Lesions

Aline Cristina Targa Cadamuro1 Ana Flaacutevia Teixeira Rossi1 Joice Matos Biselli-Peacuterico1

Patriacutecia Fucuta Pereira2 Edla Polsinelli Bedin Mascarin Do Vale2 Ricardo Acayaba3

Kaacutetia Ramos Moreira Leite4 Eny Maria Goloni-Bertollo5 and Ana Elizabete Silva1

1Department of Biology Sao Paulo State University (UNESP) Sao Jose do Rio Preto Campus Rua Cristovao Colombo 226515054-000 Sao Jose do Rio Preto SP Brazil2Base Hospital Ambulatory of Gastrohepatology Avenida Brigadeiro Faria Lima 5544 15090-000 Sao Jose do Rio Preto SP Brazil3Joao Paulo II Hospital Rua Doutor Eduardo Nielsem 960 15030-070 Sao Jose do Rio Preto SP Brazil4School of Medicine Sao Paulo University (USP) Avenida Dr Arnaldo 455 01246-903 Sao Paulo SP Brazil5School of Medicine FAMERP Avenida Brigadeiro Faria Lima 5416 15090-000 Sao Jose do Rio Preto SP Brazil

Correspondence should be addressed to Ana Elizabete Silva anabeteibilceunespbr

Received 7 November 2014 Revised 4 March 2015 Accepted 6 March 2015

Academic Editor Chiara De Luca

Copyright copy 2015 Aline Cristina Targa Cadamuro et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Objective Helicobacter pylori (Hp) is recognized by TLR4 and TLR2 receptors which trigger the activation of genes involvedin the host immune response Thus we evaluated the effect of eradication therapy on TLR2 and TLR4 mRNA and proteinexpression in H pylori-infected chronic gastritis patients (CG-Hp+) and 3 months after treatmentMethods A total of 37 patientsCG-Hp+ were evaluated The relative quantification (RQ) of mRNA was assessed by TaqMan assay and protein expression byimmunohistochemistryResults Before treatment bothTLR2 andTLR4mRNA inCG-Hp+ patients were slightly increased (TLR2=132 TLR4 = 126) in relation to Hp-negative normal gastric mucosa (119875 le 005) After successful eradication therapy no significantchange was observed (TLR2 = 147 TLR4 = 153 119875 gt 005) In addition the cagA and vacA bacterial genotypes did not influencethe gene expression levels and we observed a positive correlation between the RQ values of TLR2 and TLR4 both before and aftertreatment Immunoexpression of the TLR2 and TLR4 proteins confirmed the gene expression results Conclusion In conclusionthe expression of both TLR2 and TLR4 is increased in CG-Hp+ patients regardless of cagA and vacA status and this expressionpattern is not significantly changed after eradication of bacteria at least for the short period of time evaluated

1 Introduction

The Helicobacter pylori (H pylori) bacterium is responsiblefor 55 of all infection-associated cancers [1] and is themajor cause of gastric cancer in consequence of chronicinflammation Persistent gastricmucosa inflammation resultsin chronic gastritis and progresses through a multistep pro-cess to gastric atrophy intestinal metaplasia dysplasia andfinally carcinoma [2] The clinical consequences of H pyloriinfection are determined by bacteria virulence genes as wellas by host genetic factors such as immune response genesbesides environmental factors [3ndash5] Among the bacterial

products the CagA (cytotoxin-associated gene A) and VacA(vacuolating cytotoxin) proteins are the major virulencefactors related to the severity of gastric lesions and cellresponses [6 7]

The gastric epithelium cells provide the first point ofcontact for H pylori adhesion through interaction withToll-like receptors (TLRs) responding to the infection byactivating various signaling pathways [8] TLRs are keyregulators of both innate and adaptive immune responsesrecognizing several microbial products such as lipoproteinspeptidoglycans and lipopolysaccharides (LPS) [9] The LPSof H pylori is recognized mainly not only by TLR4 [10] but

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 481972 9 pageshttpdxdoiorg1011552015481972

2 Mediators of Inflammation

also by TLR2 which recognizes other forms that are struc-turally different from those recognized by TLR4 [11] BothTLR2 and TLR4 are activated after the bacteria recognitionin cooperation with the adapter molecule MyD88 triggeringthe mitogen-activating protein kinase (MAPK) signalingpathway At this point there is a subsequent activation ofthe transcription factor NF-120581B which leads to the rapidexpression of inducible nitric oxide synthase (iNOS) andproinflammatory cytokines chemokines and their receptorsand interleukins [12 13] When these factors are stimulatedthey initiate a marked inflammatory response of the mucosacharacterized as chronically active gastritis and may acquireoncogenic potential [14 15]

So far the strategy for prevention of H pylori-associatedgastric cancer has been the eradication of these bacteriaregarded as a first-line therapy to reverse the preneoplasticlesions and prevent malignant progression [16] Howevertreatment is not adopted for asymptomatic carriers in devel-oping countries due to its high cost [17] H pylori issusceptible to most antibiotics although resistance has beencommon and triple or quadruple therapy consisting of twoantibiotics a proton pump inhibitor and bismuth has latelybeen used to eradicate the bacteria [18] Unfortunately theeradication is not always successful mainly due to chemore-sistance [19] Studies to evaluate changes in expression levelsof genes involved in the recognition of the bacteria andthe immune response of the host in patients infected by Hpylori are scarce both before and after eradication treatmentMoreover there are no reports about the expression ofTLR2 and TLR4 in gastric lesions before and after bacterialclearance Therefore the main goal of the present studywas to evaluate for the first time the mRNA and proteinexpression levels of TLR2 and TLR4 in H pylori-infectedchronic gastritis patients and the occurrence of changes in theexpression levels of these receptors after successful H pylorieradication therapy

2 Materials and Methods

21 Patients At first 59 patients scheduled for upperendoscopy with positive histological andmolecular diagnosisfor H pylori and not yet submitted to eradication therapywere enrolled prospectively betweenMay 2010 andDecember2012 from the Gastro-Hepatology Outpatient Clinic at theBase Hospital and the Joao Paulo II Hospital both at Sao Josedo Rio Preto SP Brazil

From each patient gastric biopsies of the antrum regionwere collected for histological analyses and molecular andimmunohistochemical studies None of the individuals hadtaken any antibiotics nonsteroidal anti-inflammatory drugsor corticosteroids during the twomonths prior to endoscopynor did they take proton pump inhibitors or H

2antagonists

in the 15 days preceding the procedure Patients with gastriccancer and infectious diseases were excluded from this studyGastric biopsy specimens were examined histologically by aspecialized pathologist for the presence of the bacteria andhistopathologically classified as superficial chronic gastritis(119899 = 45 mean age 44 years 19 females and 17males) atrophic

Table 1 Demographic and clinicopathological data of H pylori-positive patients with chronic gastritis

Patients Total119873 = 59AgeMean (SD) years 480 plusmn 159Range 21ndash82

GenderMale 26 (44)Female 33 (56)

DrinkingYes 19 (32)No 36 (61)Not available 4 (7)

SmokingYes 21 (36)No 36 (61)Not available 2 (3)

Histological diagnosisChronic gastritis 45 (76)Atrophic gastritis 8 (14)Atrophic gastritis-associated intestinal metaplasia 6 (10)

Eradication therapyCompleted treatment 3759 (63)Bacteria eradication 2337 (62)Bacteria noneradication 1437 (38)119873 number of individuals

gastritis (119899 = 8 mean age 50 years 3 females and 5 males)and atrophic gastritis with intestinal metaplasia (119899 = 6 meanage 50 years 4 females and 2 males) according to the Sydneysystem [20] constituting the so-called CG-Hp+ group Of the59 CG-Hp+ patients only 37 (63) concluded the treatmentand were called completed treatment group and 2337 (62)of them had the bacteria eradicated as evidenced by concor-dant histological and molecularH pylori-negative diagnosisHowever 1437 (38) remain infected showing histologicalor molecular H pylori-positive diagnosis (Table 1) Fourgastric biopsy specimens presented histologically normal Hpylori-negative gastric mucosa (normal Hp- group) and wereused as control (mean age 356 years 3 females and 1 male)Epidemiological data of patients and controls were collectedusing a standard interviewer-administered questionnairecontaining questions about smoking habits alcohol intakeprevious or ongoing treatment use of medications previoussurgeries and family history of cancer

TheCG-Hp+groupwas submitted to standard triple ther-apy consisting of amoxicillin (1 g) clarithromycin (500mg)and omeprazole (20mg) all given twice daily for seven daysThree months after treatment the individuals underwentanother endoscopy for collection of gastric biopsies of theantrum region Immediately after collection the biopsyspecimens were placed into RNA Later solution (AppliedBiosystems) and stored at minus20∘C until nucleic acid extraction

The study protocol was approved by the local ResearchEthics Committee (CEPIBILCEUNESP number 03010)

Mediators of Inflammation 3

and written informed consent was obtained from all partici-pating individuals

22 Molecular Diagnosis for H pylori and cagA and vacAGenotypes DNARNA extraction from the gastric biopsieswas performed according to the protocol accompanyingthe reagent Trizol (Invitrogen) and the concentrations weredetermined in a NanoDrop ND1000 spectrophotometer(Thermo Scientific) Firstly multiplex PCR was performedusing 100 ng of DNA in a final volume of 25 120583L containingspecific primers for H pylori genes such as UreA and tsaAbesides the constitutive human CYP1A1 gene according toour protocol which was described in previous study [21]Molecular diagnosis was considered positive when at leastone gene (UreA or tsaA) had been amplified The H pylori-positive samples were also subjected to PCR for investigationof polymorphisms in the sm regions of the gene vacA aspreviously described [22] Primers amplify s1 fragment of176 bp or s2 fragment of 203 bp while primers for ldquomrdquo allelesamplify m1 fragment of 400 bp or m2 fragment of 475 bpPositive and negative controls were used in all experiments

23 TaqMan Quantitative Real Time PCR (qPCR) for TLR2and TLR4 mRNA Reverse transcription (RT) of total RNAwas performed using a High Capacity cDNA Archive Kit(Applied Biosystems) in a total volume of 25 120583L accordingto the manufacturerrsquos protocol Then qPCR was carriedout in a StepOnePlus Real Time PCR System 222 (AppliedBiosystems) using specific TaqMan probes for target genesTLR2 (assay ID Hs00610101 m1 Applied Biosystems) andTLR4 (assay ID Hs01060206 m1 Applied Biosystems) andtwo reference genes ACTB (part number 4352935E AppliedBiosystems) and GAPDH (glyceraldehyde 3-phosphate dehy-drogenase) (part number 4352934E Applied Biosystems)used as endogenous controls according to the manufacturerrsquosinstructions All reactions were performed in triplicate in afinal volume of 20120583L using 100 ng120583L cDNA and a blank toensure the absence of contamination Relative quantification(RQ) of TLR2 and TLR4 mRNA was obtained accordingto the model proposed by Livak and Schmittgen [23] andnormalized to the ACTB and GAPDH reference genes and apool of normal Hp- samples

24 Immunohistochemical Assay for TLR2 and TLR4 ProteinsImmunohistochemical analysis was performed in 14 samplesfrom the CG-Hp+ group before and after bacteria eradicationand four samples from the normal Hp- group Consecutive4 120583m thick sections were cut from each trimmed paraffinblock Deparaffinized tissue slides were then submittedto antigen retrieval using a high-temperature antigen-unmasking technique The sections were incubated withspecific primary antibodies rabbit polyclonal antibodyanti-TLR2 (06-1119 1 50 dilution Millipore) and mousemonoclonal anti-TLR4 (76B3571 1 200 dilution Abcam)Then the slides were incubated with biotinylated secondaryantibody (Picture Max Polymer Detection Kit Invitrogen)for 30 minutes following the manufacturerrsquos protocolImmunostaining was done with 331015840-diaminobenzidine

tetrahydrochloride (DAB) containing 0005 H2O2 coun-

terstained with hematoxylin Placenta mucosa and appendixtissue were used respectively as positive controls forthe TLR2 and TLR4 proteins The immunostaining wasevaluated in the cytoplasm by densitometric analysis withan arbitrary scale going from 0 to 255 performed with AxioVision software under a Zeiss-Axioskop II light microscopeSixty equally distributed points were scored in each one ofthe regions and the results were expressed as mean plusmn SE

25 Statistical Analysis Data analysis was performed usingthe computer software GraphPad Prism 5 version 501 Thedistribution of continuous data was evaluated using theDrsquoAgostino and Pearson omnibus normality test or Shapiro-Wilk normality test Data are presented as median andrange as mean plusmn standard deviation (SD) or as frequenciesaccording to the data distribution Studentrsquos t-test for pairedand unpaired data or correspondent nonparametric testssuch as theMann-Whitney test and theWilcoxon signed ranktest were used for comparisons between groups To evaluatethe association between relative gene expression and riskfactors such as age gender smoking drinking and bacterialvirulence genotypes the Mann-Whitney test was performedThe correlation between TLR2 and TLR4 mRNA expressionbefore and after eradication therapy was analyzed usingSpearmanrsquos Correlation For protein expression the meansobtained from the densitometry analysis were comparedbefore and after treatment and with the normal Hp- groupusing ANOVA followed by the Bonferroni test The level ofsignificance was set at 119875 le 005

3 Results

31 The Relative Expression of TLR2 and TLR4 mRNA Is NotChanged after Successful Eradication Therapy Table 2 showsthe data regarding the relative expression levels of TLR2and TLR4 mRNA of 37 CG-Hp+ patients who concludedthe treatment (completed treatment group) 23 CG-Hp+patients in which the bacteria were eradicated allowingpaired analysis before and after eradication therapy and 14CG-Hp+ patients in which the bacteria were noneradicatedThe relative expression levels of TLR2 and TLR4mRNA afternormalization with the ACTB and GAPDH reference genesand comparison with normal mucosa H pylori-negative inall groups either before or after treatment were increasedsignificantly (119875 lt 005) Considering all patients that com-pleted the treatment no significant change was found aftertreatment in the relative expression levels of either TLR2 orTLR4mRNA (TLR2= 155 andTLR4= 164) in comparison tothe same cases before the treatment (TLR2 = 131 and TLR4 =145) In the group that eradicated the bacteria heterogeneityof relative expression levels for both TLR2 and TLR4mRNAscan be observed before and after the treatment (Figures 1(a)and 1(b)) However no significant differences were observedfor both genes comparing the expression levels in this groupbefore and after treatment (119875 = 0533 and 119875 = 0094 forTLR2 and TLR4 resp) (Figures 1(c) and 1(d)) Furthermorea positive correlation between the RQ values of TLR2 and


Table 2 Comparison of TLR2 and TLR4mRNA relative expressionlevels before and after H pylori eradication therapy

Before treatment After treatment119873 119873

TLR2Completed treatment 37 37RQ median 131 155Range 037ndash2305 034ndash4312119875 value 0291

Eradicated 23 23RQ median 132 147Range 037ndash1263 034ndash4312119875 value 0533

Noneradicated 14 14RQ median 123 183Range 066ndash2305 037ndash636119875 value 0357



Noneradicated 14 14RQ median 188 199Range 054ndash1109 056ndash975119875value 0626

119873 number of individuals 119875 probability RQ relative quantification statis-tical analysis by Wilcoxon signed rank test

TLR4mRNA before and after treatment considering only theeradicated patients was found (before 1199032 = 085 119875 lt 00001after 1199032 = 055 119875 = 0006)

The influence of cagA and vacA bacterial genotypeson the gene expression levels both before and after treat-ment (Table 3) showed no evidenced significant differencebetween cagA+ and cagAminus genotypes (119875 gt 005) forboth analyzed genes Similarly no significant difference wasobserved regardingVacA sm genotypeWe also evaluated theassociation between relative expression levels of TLR2 andTLR4mRNA and the risk factors such as age gender smok-ing drinking and histological type of gastric lesion None ofthe factors investigated showed significant differences (datanot shown)

32 TLR2 and TLR4 Protein and mRNA Relative ExpressionsAre Concordant In normal mucosa the TLR2 and TLR4protein expression was weak or absent mainly in the foveolarepithelium (Figures 2(a) and 2(b)) Nevertheless the CG-Hp+ samples collected before the treatment showed a cyto-plasmatic perinuclear and focal immunostaining pattern

mostly in the basal area of the foveolar epithelium A strongexpression in the inflammatory cells was also observed(Figures 2(c) and 2(d)) After the eradication of H pylori animmunostaining pattern similar to the one observed beforethe treatment was found for both TLR2 and TLR4 proteins(Figures 2(e) and 2(f))

The mean optical densitometry values observed in thenormal Hp- group for TLR2 and TLR4 were 1056 plusmn 27 and1014 plusmn 65 respectively While the CG-Hp+ group beforetreatment presented significantly increased mean values forboth TLR2 (1517plusmn61) and TLR4 (1322plusmn47) in comparisonwith the normal Hp- group (119875 = 0020 and 119875 = 0007 resp)After eradication of the bacteria both TLR2 and TLR4proteins showed a slight reduction in their mean opticaldensitometry values (1361 plusmn 61 and 1228 plusmn 58 resp)However there were no significant differences between thesevalues before and after treatment (119875 = 0064 and 119875 =0198 resp) (Figures 2(g) and 2(h)) confirming the findingsregarding the mRNA relative expression

4 Discussion

In this study we investigated for the first time the occur-rence of alterations in the TLR2 and TLR4 mRNA andprotein expression inH pylori-infected patients with chronicgastritis before and after successful bacteria eradicationtreatment Our results did not reveal significant changes inthe relative expression levels of either TLR2 or TLR4 mRNAafter treatment in eradicated patients which was confirmedby immunohistochemistry Moreover the mRNA expressionof both receptors remained increased after eradication ther-apy compared to the normal Hp- group showing that theeradication of the bacteria did not normalize the expressionof these receptors at least under the conditions evaluatedAdditionally we also observed a positive correlation betweenthe mRNA expression values of TLR2 and TLR4 confirmingthat H pylori activates both receptors

TLRs are transmembrane proteins that play a criticalrole in the recognition of pathogen components [24] LPS ofGram-negative bacteria are recognized mainly by TLR4 andalso TLR2 activating signaling pathways that culminate in aninflammatory response [25] It is believed that the interactionbetween bacterial virulence and a genetically susceptiblehost is associated with more severe chronic inflammationwhich may in the long run lead to cancer [26] Undernormal physiological conditions the expression of thesereceptors in the mucosa of the gastrointestinal tract is lowdue to the action of their antagonists such as TOLLIP (Toll-interacting protein) and PPAR120574 (Peroxisome proliferator-activated receptor) that show higher levels in order to preventinappropriate activation of nonpathogenic antigens [27ndash29]

In our study we observed a slightly increased expressionof both TLR2 and TLR4 in CG-Hp+ patients even aftersuccessfulH pylori eradication compared to the noninfectednormal mucosa In children infected withH pylori Lagunes-Servin et al (2013) [30] found an increase in the expres-sion of the TLR2 TLR4 TLR5 and TLR9 in the gastricepithelium compared with noninfected children and also


0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TLR2

16 17 18 19 20 21 22 23

BeforeAer

5101520253035404550

Rela

tive

gene

exp

ress

ion

(a)

TLR4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0

5

10

15

20

25

30

Rela

tive

gene

exp

ress

ion

BeforeAer

(b)

TLR2

RQ m

edia

n

0

1

2

3

4

Before Aer

(c)

TLR4

RQ m

edia

n

0

1

2

3

4

Before Aer

(d)

Figure 1 Relative expression levels of TLR2 and TLR4RNAm in the eradicated patients group with chronic gastritis before and afterH pyloritreatment Relative quantification (RQ) of the mRNA expression levels of (a) TLR2 and (b) TLR4 per individual evaluated RQ median of(c) TLR2 and (d) TLR4mRNA before and after H pylori eradication Data are presented as median and range for experiments performed intriplicate Statistical significance was determined using Wilcoxonrsquos signed rank test

an association with pro- and anti-inflammatory cytokines(IL-8 TNF-120572 and IL-10) These findings confirm that Hpylori has the ability to increase the in vivo expressionof TLRs by gastric epithelial cells early during infectionin children starting a chronic and balanced inflammatoryprocess that will continue for decades and so may contributeto the development of H pylori-associated diseases laterin adulthood Pimentel-Nunes et al (2013) [31] observedthat considering the different TLRs of normal H pylori-negative mucosa the mRNAof TLR5was the most expressedfollowed by those of TLR2 and TLR4 Furthermore theseauthors found TLR2 and TLR4 overexpression in intestinalmetaplasia independent of the H pylori status and in thedysplasiacancer sequence Moreover upregulation of TLR2and TLR4 mRNA was also observed in H pylori-associatednormal mucosa These results were confirmed by immuno-histochemical analyses which found an increase in proteinexpression in H pylori-infected normal mucosa furtherincreasing in intestinal metaplasia and dysplasiacarcinomaThese findings suggest that progressive activation of thesereceptors initially not only by H pylori but also by otherPAMPs (pathogen-associated molecular patterns) or DAMPs(damage-associated molecular patterns) at later stages may

play an important role in gastric carcinogenesis and tumorprogression [31]

Upregulation of TLR4 expression responsiveness to LPSand H pylori in gastric cell lines has also been reported[32 33] H pylori infection induced both TLR4 mRNAand protein expression in AGS cells that were dependenton bacterial load and infection duration However thetransfection of AGS cells with TLR4 siRNA followed bythe bacterial infection suppressed the expression of thisreceptor [32] Moreover LPS of H pylori upregulate TLR4expression via TLR2 signaling in MKN28 gastric cell linesby the MEK12-ERK12 MAP kinase pathway [34] leadingalso to an increase in cell proliferation Conversely previousstudies [35ndash37] did not observe any relevant role of TLR4in the cellular recognition of H pylori in AGC cells Thesecontroversial results may be due to differences in the lipidA structures produced by distinct H pylori strains [38ndash40]Therefore the interaction of the bacteria with TLR2 shouldalso be considered mainly after the first contact with thegastric mucosa triggering immunologic responses [41] suchas induction of IL-8 and subsequent activation of NF-120581B [11]

Our study revealed no reduction of the transcript levelsof TLR2 and TLR4 or their proteins 3 months after treatment


(a) (b)

(c) (d)

(e) (f)

TLR2

NM CG before CG after

Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

200

lowast

(g)

TLR4


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

lowast

(h)

Figure 2 Immunohistochemistry images of Toll-like receptors (TLRs) in normal gastric mucosa (NM) and chronic gastritis (CG) Normalmucosa ((a) TLR2 and (b) TLR4) normal glandswith no staining or low expression intensityH pylori-positive chronic gastritis ((c) TLR2 and(d) TLR4) foveolar epithelial cells and glands before treatment presenting moderate to strong TLR expression compared to normal mucosa((e) TLR2 and (f) TLR4) foveolar epithelial cells and glands after bacteria eradication (no significant reduction of protein expression wasdetected) Counterstain hematoxylin Bars 50 120583m ((g)-(h)) Densitometry analyses (mean plusmn SE) lowast119875 lt 005 au = arbitrary unit


Table 3 Comparisons of TLR2 and TLR4 mRNA expression levels according to cagA and vacA genotypes of H pylori in infected patientsbefore and after bacteria eradication treatment

TLR2 TLR4Samples () RQ median (range) 119875 value Samples () RQ median (range) 119875 value

Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109

cagAminus 1223 (520) 141 1223 (520) 180071ndash1263 097ndash717

vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717

vacA others 1325 (520) 131 1325 (520) 202066ndash2305 097ndash1109

After treatmentEradicated

cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299


vacAothers (s1m2 s2m2 s1 s2 and m1) 119875 value = Mann-Whitney test 119875 lt 005

showing that the successful eradication of H pylori doesnot change the expression of these receptors within a shortperiod after the treatment Similarly Garza-Gonzalez et al(2008) [42] found no quantitative differences in the TLR4and TLR5 mRNA levels either regardless of the presence orabsence of H pylori in gastric epithelial cells biopsies andAGS cells suggesting that the mRNA levels of both receptorsmay not be influenced by the infection process or at leastnot at the time points selected for analysis However in ourstudy we observed higher levels of TLR2 and TLR4 mRNAand of both proteins in H pylori-infected mucosa comparedto noninfected normal mucosa It should however be takeninto consideration that the posttreatment time elapsed untilbiopsy collection which may not have been sufficient formucosal renovation and transcription level normalizationMoreover alterations in mRNA expression levels after Hpylori infection eradication therapy have been demonstratedinvolving genes associated with cell damage inflammationproliferation apoptosis and intestinal differentiation [4344]

This study did not investigate the molecular mechanismsinvolved in the inflammatory cascade induced by H pyloriinfection triggered by TLR4 and TLR2 Therefore furtherinvestigations are needed to clarify the possible involvementof signaling pathway MyD88-MAPK-NF120581B as well as therole of PPARs (Peroxisome proliferator-activated receptors)on inhibition of pathway regulating expression of proinflam-matory genes and stress kinase pathways [31 45 46] whichsuppresses inflammation in H pylori infection

When we compared the expression levels of TLR2 andTLR4 mRNA with risk factors and bacterial virulence geno-types we did not find any association The studies that assessthe effects of cagA and vacA virulence factors on the gene andprotein expression are controversial Our results evidencedthat therewere no quantitative differences in themRNA levelsof these receptors regardless of cagA and vacA status Similarresults were reported by Garza-Gonzalez et al (2008) [42]which demonstrated that themRNA levels ofTLR4 andTLR5in gastric cells both in vivo and in vitrowere not influenced bythe vacA status suggesting that this virulence factor may not


be involved in the first steps of innate immune-recognition ofH pylori Another study evidenced downregulation of TLRs2 and 5 and upregulation of TLR9 by H pylori in humanneutrophils regardless of cagPAI status and the integrity ofT4SS [47]

In conclusion we report a discrete increase in TLR2 andTLR4 mRNA and protein expression in CG-Hp+ patientsbefore eradication therapy and the maintaining of thisexpression pattern even after treatment suggesting that thesereceptors remain expressed in the gastric mucosa even aftereradication of the bacteria at least for the period evaluatedTherefore considering the higher risk of malignant progres-sion in patients infected by H pylori for a long time furtherinvestigations are needed to clarify the changes in the expres-sion of other genes related with the inflammatory cascadeinduced by bacteria such as those encoding cytokines andmalignant transformation processes as well as the signalingpathways involved

Conflict of Interests

The authors declare that there are no competing interests

Acknowledgments

The authors are grateful to Dr Sebastiao Roberto Tabogaand Luiz Roberto Faleiros Junior for their help with thehistological sections Isaque Santana for technical assistanceduring the immunohistochemical assay and nurse GizeldaWarickMazzale for her help in contacting patientsThis studywas supported by grants from the Brazilian agencies FAPESP(201215036-8) and CNPq (3048702012-9)

References

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[2] P Correa ldquoHuman model of gastric carcinogenesisrdquo CancerResearch vol 48 no 13 pp 3554ndash3560 1988

[3] J G de Oliveira and A E Silva ldquoPolymorphisms of the TLR2and TLR4 genes are associated with risk of gastric cancer in aBrazilian populationrdquoWorld Journal of Gastroenterology vol 18no 11 pp 1235ndash1242 2012

[4] H Xue J Liu B Lin Z Wang J Sun and G Huang ldquoAmeta-analysis of interleukin-8-251 promoter polymorphismassociated with gastric cancer riskrdquo PLoS ONE vol 7 no 1Article ID e28083 2012

[5] A Lamb and L-F Chen ldquoRole of the Helicobacter pylori-induced inflammatory response in the development of gastriccancerrdquo Journal of Cellular Biochemistry vol 114 no 3 pp 491ndash497 2013

[6] S F Moss and M J Blaser ldquoMechanisms of disease inflam-mation and the origins of cancerrdquo Nature Clinical PracticeOncology vol 2 no 2 pp 90ndash97 2005

[7] R M Peek Jr and J E Crabtree ldquoHelicobacter infection andgastric neoplasiardquo The Journal of Pathology vol 208 no 2 pp233ndash248 2006

[8] K T Wilson and J E Crabtree ldquoImmunology of Helicobacterpylori insights into the failure of the immune response and

perspectives on vaccine studiesrdquo Gastroenterology vol 133 no1 pp 288ndash308 2007

[9] S Akira S Uematsu and O Takeuchi ldquoPathogen recognitionand innate immunityrdquo Cell vol 124 no 4 pp 783ndash801 2006

[10] M Matsuura ldquoStructural modifications of bacterial lipopoly-saccharide that facilitate gram-negative bacteria evasion of hostinnate immunityrdquo Frontiers in Immunology vol 4 article 1092013

[11] S-I Yokota T OhnishiMMuroi K-I Tanamoto N Fujii andK-I Amano ldquoHighly-purifiedHelicobacter pylori LPS prepara-tions induce weak inflammatory reactions and utilize Toll-likereceptor 2 complex but not Toll-like receptor 4 complexrdquo FEMSImmunology and Medical Microbiology vol 51 no 1 pp 140ndash148 2007

[12] F Re and J L Strominger ldquoHeterogeneity of TLR-inducedresponses in dendritic cells from innate to adaptive immunityrdquoImmunobiology vol 209 no 1-2 pp 191ndash198 2004

[13] S-Z Ding A M Torok M F Smith Jr and J B GoldbergldquoToll-like receptor 2-mediated gene expression in epithelial cellsduringHelicobocter pylori infectionrdquoHelicobacter vol 10 no 3pp 193ndash204 2005

[14] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[15] M Fukata and M T Abreu ldquoPathogen recognition receptorscancer and inflammation in the gutrdquo Current Opinion inPharmacology vol 9 no 6 pp 680ndash687 2009

[16] M F Tsan ldquoToll-like receptors inflammation and cancerrdquoSeminars in Cancer Biology vol 16 no 1 pp 32ndash37 2006

[17] M-S Wu L-P Chow J-T Lin and S-H Chiou ldquoProteomicidentification of biomarkers related to Helicobacter pylori-associated gastroduodenal disease challenges and opportuni-tiesrdquo Journal of Gastroenterology and Hepatology vol 23 no 11pp 1657ndash1661 2008

[18] M Asaka M Kato S-I Takahashi et al ldquoGuidelines for themanagement of Helicobacter pylori infection in Japan 2009revised editionrdquo Helicobacter vol 15 no 1 pp 1ndash20 2010

[19] P Malfertheiner F Megraud C A OrsquoMorain et alldquoManagement of Helicobacter pylori infectionmdashthe MaastrichtIVFlorence consensus reportrdquo Gut vol 61 no 5 pp 646ndash6642012

[20] M F Dixon RM Genta J H Yardley et al ldquoClassification andgrading of Gastritis the updated Sydney systemrdquoTheAmericanJournal of Surgical Pathology vol 20 no 10 pp 1161ndash1181 1996

[21] A F T Rossi M C Duarte A B Poltronieri et al ldquoDeregu-lation of annexin-A1 and galectin-1 expression in precancerousgastric lesions intestinal metaplasia and gastric ulcerrdquo Media-tors of Inflammation vol 2014 Article ID 478138 11 pages 2014

[22] L L Gatti E K Fagundes E Souza K R Leite et al ldquocagA vacAalelles and babA2 genotypes of Helicobacter pylori associatedwith gastric disease in Brazilian adult patientsrdquo DiagnosticMicrobiology and Infectious Disease vol 51 no 4 pp 231ndash2352005

[23] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔ119862119879 methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[24] R Medzhitov and C Janeway Jr ldquoinnate immunityrdquo The NewEngland Journal of Medicine vol 343 no 5 pp 338ndash344 2000

[25] M R Amieva and E M El-Omar ldquoHost-bacterial interactionsin Helicobacter pylori infectionrdquo Gastroenterology vol 134 no1 pp 306ndash323 2008


[26] J C Machado C Figueiredo P Canedo et al ldquoA proinflam-matory genetic profile increases the risk for chronic atrophicgastritis and gastric carcinomardquo Gastroenterology vol 125 no2 pp 364ndash371 2003

[27] P Pimentel-Nunes J B Soares R Roncon-Albuquerque Jr MDinis-Ribeiro and A F Leite-Moreira ldquoToll-like receptors astherapeutic targets in gastrointestinal diseasesrdquo Expert OpiniononTherapeutic Targets vol 14 no 4 pp 347ndash368 2010

[28] G Melmed L S Thomas N Lee et al ldquoHuman intestinalepithelial cells are broadly unresponsive to toll-like receptor2-dependent bacterial ligands Implications for host-microbialinteractions in the gutrdquoThe Journal of Immunology vol 170 no3 pp 1406ndash1415 2003

[29] F Y Liew D Xu E K Brint and L A J OrsquoNeill ldquoNegativeregulation of toll-like receptor-mediated immune responsesrdquoNature Reviews Immunology vol 5 no 6 pp 446ndash458 2005

[30] H Lagunes-Servin J Torres C Maldonado-Bernal et al ldquoToll-like receptors and cytokines are upregulated duringHelicobacterpylori infection in childrenrdquoHelicobacter vol 18 no 6 pp 423ndash432 2013

[31] P Pimentel-Nunes N Goncalves I Boal-Carvalho et al ldquoHeli-cobacter pylori induces increased expression of toll-like recep-tors and decreased toll-interacting protein in gastric mucosathat persists throughout gastric carcinogenesisrdquo Helicobactervol 18 no 1 pp 22ndash32 2013

[32] B Su P J M Ceponis S Lebel H Huynh and P M ShermanldquoHelicobacter pylori activates Toll-like receptor 4 expression ingastrointestinal epithelial cellsrdquo Infection and Immunity vol 71no 6 pp 3496ndash3502 2003

[33] D-Y Lu H-C Chen M-S Yang et al ldquoCeramide and toll-likereceptor 4 are mobilized into membrane rafts in response toHelicobacter pylori infection in gastric epithelial cellsrdquo Infectionand Immunity vol 80 no 5 pp 1823ndash1833 2012

[34] S-I Yokota T Okabayashi M Rehli N Fujii and K-I AmanoldquoHelicobacter pylori lipopolysaccharides upregulate toll-likereceptor 4 expression and proliferation of gastric epithelialcells via the MEK12-ERK12 mitogen-activated protein kinasepathwayrdquo Infection and Immunity vol 78 no 1 pp 468ndash4762010

[35] S Maeda M Akanuma Y Mitsuno et al ldquoDistinct mechanismofHelicobacter pylori-mediated NF-kappa B activation betweengastric cancer cells and monocytic cellsrdquoThe Journal of Biologi-cal Chemistry vol 276 no 48 pp 44856ndash44864 2001

[36] F Backhed B Rokbi E Torstensson et al ldquoGastric mucosalrecognition of Helicobacter pylori is independent of Toll-likereceptor 4rdquoThe Journal of Infectious Diseases vol 187 no 5 pp829ndash836 2003

[37] J-M Otte H M Neumann S Brand H Schrader W ESchmidt and F Schmitz ldquoExpression of beta-defensin 4 isincreased in human gastritisrdquo European Journal of ClinicalInvestigation vol 39 no 2 pp 126ndash138 2009

[38] T Kawahara S Teshima A Oka T Sugiyama K Kishi andK Rokutan ldquoType I Helicobacter pylori lipopolysaccharidestimulates toll-like receptor 4 and activates mitogen oxidase 1in gastric pit cellsrdquo Infection and Immunity vol 69 no 7 pp4382ndash4389 2001

[39] S Ishihara M A K Rumi Y Kadowaki et al ldquoEssential role ofMD-2 in TLR4-dependent signaling duringHelicobacter pylori-associated gastritisrdquoThe Journal of Immunology vol 173 no 2pp 1406ndash1416 2004

[40] Y Hirata T Ohmae W Shibata et al ldquoMyD88 and TNFreceptor-associated factor 6 are critical signal transducers in

Helicobacter pylori-infected human epithelial cellsrdquoThe Journalof Immunology vol 176 no 6 pp 3796ndash3803 2006

[41] M F Smith Jr AMitchell G Li et al ldquoToll-like receptor (TLR)2 and TLR5 but not TLR4 are required forHelicobacter pylori-induced NF-kappaB activation and chemokine expression byepithelial cellsrdquoThe Journal of Biological Chemistry vol 278 no35 pp 32552ndash32560 2003

[42] E Garza-Gonzalez V Bocanegra-Garcıa F J Bosques-PadillaJ P Flores-Gutierrez F Moreno and G I Perez-Perez ldquomRNAlevels of TLR4 and TLR5 are independent of H pylorirdquo WorldJournal of Gastroenterology vol 14 no 34 pp 5306ndash5310 2008

[43] C J Tsai R Herrera-Goepfert R J Tibshirani et al ldquoChangesof gene expression in gastric preneoplasia following Helicobac-ter pylori eradication therapyrdquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 2 pp 272ndash280 2006

[44] S S Kim PMeitner T A Konkin Y S ChoM B Resnick andS F Moss ldquoAltered expression of Skp2 c-Myc and p27 proteinsbut not mRNA after H pylori eradication in chronic gastritisrdquoModern Pathology vol 19 no 1 pp 49ndash58 2006

[45] M Muzio G Natoli S Saccani M Levrero and A MantovanildquoThe human toll signaling pathway divergence of nuclearfactor 120581b and jnksapk activation upstream of tumor necrosisfactor receptor-associated factor 6 (TRAF6)rdquo The Journal ofExperimental Medicine vol 187 no 12 pp 2097ndash2101 1998

[46] J-M Lee S S Kim and Y-S Cho ldquoThe role of PPAR120574 inHelicobacter pylori infection and gastric carcinogenesisrdquo PPARResearch vol 2012 Article ID 687570 6 pages 2012

[47] N A Sanchez-Zauco J Torres G E Perez-Figueroa et alldquoImpact of cagPAI and T4SS on the inflammatory response ofhuman neutrophils toHelicobacter pylori infectionrdquo PLoS ONEvol 8 no 6 Article ID e64623 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


also by TLR2 which recognizes other forms that are struc-turally different from those recognized by TLR4 [11] BothTLR2 and TLR4 are activated after the bacteria recognitionin cooperation with the adapter molecule MyD88 triggeringthe mitogen-activating protein kinase (MAPK) signalingpathway At this point there is a subsequent activation ofthe transcription factor NF-120581B which leads to the rapidexpression of inducible nitric oxide synthase (iNOS) andproinflammatory cytokines chemokines and their receptorsand interleukins [12 13] When these factors are stimulatedthey initiate a marked inflammatory response of the mucosacharacterized as chronically active gastritis and may acquireoncogenic potential [14 15]

So far the strategy for prevention of H pylori-associatedgastric cancer has been the eradication of these bacteriaregarded as a first-line therapy to reverse the preneoplasticlesions and prevent malignant progression [16] Howevertreatment is not adopted for asymptomatic carriers in devel-oping countries due to its high cost [17] H pylori issusceptible to most antibiotics although resistance has beencommon and triple or quadruple therapy consisting of twoantibiotics a proton pump inhibitor and bismuth has latelybeen used to eradicate the bacteria [18] Unfortunately theeradication is not always successful mainly due to chemore-sistance [19] Studies to evaluate changes in expression levelsof genes involved in the recognition of the bacteria andthe immune response of the host in patients infected by Hpylori are scarce both before and after eradication treatmentMoreover there are no reports about the expression ofTLR2 and TLR4 in gastric lesions before and after bacterialclearance Therefore the main goal of the present studywas to evaluate for the first time the mRNA and proteinexpression levels of TLR2 and TLR4 in H pylori-infectedchronic gastritis patients and the occurrence of changes in theexpression levels of these receptors after successful H pylorieradication therapy

2 Materials and Methods

21 Patients At first 59 patients scheduled for upperendoscopy with positive histological andmolecular diagnosisfor H pylori and not yet submitted to eradication therapywere enrolled prospectively betweenMay 2010 andDecember2012 from the Gastro-Hepatology Outpatient Clinic at theBase Hospital and the Joao Paulo II Hospital both at Sao Josedo Rio Preto SP Brazil

From each patient gastric biopsies of the antrum regionwere collected for histological analyses and molecular andimmunohistochemical studies None of the individuals hadtaken any antibiotics nonsteroidal anti-inflammatory drugsor corticosteroids during the twomonths prior to endoscopynor did they take proton pump inhibitors or H

2antagonists

in the 15 days preceding the procedure Patients with gastriccancer and infectious diseases were excluded from this studyGastric biopsy specimens were examined histologically by aspecialized pathologist for the presence of the bacteria andhistopathologically classified as superficial chronic gastritis(119899 = 45 mean age 44 years 19 females and 17males) atrophic

Table 1 Demographic and clinicopathological data of H pylori-positive patients with chronic gastritis

Patients Total119873 = 59AgeMean (SD) years 480 plusmn 159Range 21ndash82

GenderMale 26 (44)Female 33 (56)

DrinkingYes 19 (32)No 36 (61)Not available 4 (7)

SmokingYes 21 (36)No 36 (61)Not available 2 (3)

Histological diagnosisChronic gastritis 45 (76)Atrophic gastritis 8 (14)Atrophic gastritis-associated intestinal metaplasia 6 (10)

Eradication therapyCompleted treatment 3759 (63)Bacteria eradication 2337 (62)Bacteria noneradication 1437 (38)119873 number of individuals

gastritis (119899 = 8 mean age 50 years 3 females and 5 males)and atrophic gastritis with intestinal metaplasia (119899 = 6 meanage 50 years 4 females and 2 males) according to the Sydneysystem [20] constituting the so-called CG-Hp+ group Of the59 CG-Hp+ patients only 37 (63) concluded the treatmentand were called completed treatment group and 2337 (62)of them had the bacteria eradicated as evidenced by concor-dant histological and molecularH pylori-negative diagnosisHowever 1437 (38) remain infected showing histologicalor molecular H pylori-positive diagnosis (Table 1) Fourgastric biopsy specimens presented histologically normal Hpylori-negative gastric mucosa (normal Hp- group) and wereused as control (mean age 356 years 3 females and 1 male)Epidemiological data of patients and controls were collectedusing a standard interviewer-administered questionnairecontaining questions about smoking habits alcohol intakeprevious or ongoing treatment use of medications previoussurgeries and family history of cancer

TheCG-Hp+groupwas submitted to standard triple ther-apy consisting of amoxicillin (1 g) clarithromycin (500mg)and omeprazole (20mg) all given twice daily for seven daysThree months after treatment the individuals underwentanother endoscopy for collection of gastric biopsies of theantrum region Immediately after collection the biopsyspecimens were placed into RNA Later solution (AppliedBiosystems) and stored at minus20∘C until nucleic acid extraction

The study protocol was approved by the local ResearchEthics Committee (CEPIBILCEUNESP number 03010)









3 Results

















4 Discussion





0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TLR2

16 17 18 19 20 21 22 23

BeforeAer

5101520253035404550

Rela

tive

gene

exp

ress

ion

(a)

TLR4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0

5

10

15

20

25

30

Rela

tive

gene

exp

ress

ion

BeforeAer

(b)

TLR2

RQ m

edia

n

0

1

2

3

4

Before Aer

(c)

TLR4

RQ m

edia

n

0

1

2

3

4

Before Aer

(d)







(a) (b)

(c) (d)

(e) (f)

TLR2


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

200

lowast

(g)

TLR4


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

lowast

(h)





Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109


vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717



cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299











Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results

















4 Discussion





0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TLR2

16 17 18 19 20 21 22 23

BeforeAer

5101520253035404550

Rela

tive

gene

exp

ress

ion

(a)

TLR4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0

5

10

15

20

25

30

Rela

tive

gene

exp

ress

ion

BeforeAer

(b)

TLR2

RQ m

edia

n

0

1

2

3

4

Before Aer

(c)

TLR4

RQ m

edia

n

0

1

2

3

4

Before Aer

(d)







(a) (b)

(c) (d)

(e) (f)

TLR2


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

200

lowast

(g)

TLR4


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

lowast

(h)





Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109


vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717



cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299











Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































4 Discussion





0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TLR2

16 17 18 19 20 21 22 23

BeforeAer

5101520253035404550

Rela

tive

gene

exp

ress

ion

(a)

TLR4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0

5

10

15

20

25

30

Rela

tive

gene

exp

ress

ion

BeforeAer

(b)

TLR2

RQ m

edia

n

0

1

2

3

4

Before Aer

(c)

TLR4

RQ m

edia

n

0

1

2

3

4

Before Aer

(d)







(a) (b)

(c) (d)

(e) (f)

TLR2


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

200

lowast

(g)

TLR4


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

lowast

(h)





Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109


vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717



cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299











Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TLR2

16 17 18 19 20 21 22 23

BeforeAer

5101520253035404550

Rela

tive

gene

exp

ress

ion

(a)

TLR4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

0

5

10

15

20

25

30

Rela

tive

gene

exp

ress

ion

BeforeAer

(b)

TLR2

RQ m

edia

n

0

1

2

3

4

Before Aer

(c)

TLR4

RQ m

edia

n

0

1

2

3

4

Before Aer

(d)







(a) (b)

(c) (d)

(e) (f)

TLR2


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

200

lowast

(g)

TLR4


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

lowast

(h)





Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109


vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717



cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299











Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

(e) (f)

TLR2


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

200

lowast

(g)

TLR4


Mea

n op

tical

den

sitom

etry

(au

)

0

50

100

150

lowast

(h)





Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109


vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717



cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299











Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Before treatment

cagA+ 1123 (480) 132 1123 (480) 175

0689066ndash2305 0518 065ndash1109


vacA s1m1 1225 (480) 151 1225 (480) 197

0978067ndash1263 0849 065ndash717



cagA+ 511 (460) 155 512 (420) 161

0876050ndash721 0662 106ndash1201


vacA s1m1 713 (540) 071 713 (540) 142

0234034ndash721 0234 064ndash1201


Noneradicated

cagA+ 612 (500) 187 610 (600) 199

0762103ndash561 0699 056ndash392


vacA s1m1 512 (420) 136 411 (364) 166

0230065ndash561 0639 056ndash299











Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Acknowledgments


References
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Effect of Helicobacter pylori Eradication ...downloads.hindawi.com/journals/mi/2015/481972.pdf · Research Article Effect of Helicobacter pylori Eradication on TLR2