Clinical Study Cytokine Polymorphisms, Their Influence and

Hindawi Publishing CorporationTuberculosis Research and TreatmentVolume 2013 Article ID 285094 13 pageshttpdxdoiorg1011552013285094

Clinical StudyCytokine Polymorphisms Their Influence and Levelsin Brazilian Patients with Pulmonary Tuberculosis duringAntituberculosis Treatment

Eliana Peresi12 Larissa Ragozo Cardoso Oliveira1 Weber Laurentino da Silva3

Eacuterika Alessandra Pellison Nunes da Costa1 Joatildeo Pessoa Araujo Jr4

Jairo Aparecido Ayres5 Maria Rita Parise Fortes6 Edward A Graviss7

Ana Carla Pereira3 and Sueli Aparecida Calvi1

1 Tropical Disease Department Botucatu School of Medicine UNESP Sao Paulo State University Botucatu SP Brazil2 Departamento de Doencas Tropicais e Diagnostico por Imagem Faculdade de Medicina de Botucatu UNESP Rubiao Junior SNBotucatu 18618-970 SP Brazil

3 Lauro de Souza Lima Institute Bauru SP Brazil4Microbiology and Immunology Department Bioscience Institute UNESP Sao Paulo State University Botucatu SP Brazil5 Nursing Department Botucatu School of Medicine UNESP Sao Paulo State University Botucatu SP Brazil6Dermatology and Radiotherapy Department Botucatu School of Medicine UNESP Sao Paulo State UniversityBotucatu SP Brazil

7The Methodist Hospital Research Institute Houston TX USA

Correspondence should be addressed to Eliana Peresi elianaperesiyahoocombr

Received 19 December 2012 Revised 19 February 2013 Accepted 26 February 2013

Academic Editor Carlo Garzelli

Copyright copy 2013 Eliana Peresi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cytokines play an essential role during active tuberculosis disease and cytokine genes have beendescribed in associationwith alteredcytokine levels Therefore the aim of this study was to verify if IFNG IL12B TNF IL17A IL10 and TGFB1 gene polymorphismsinfluence the immune response of Brazilian patients with pulmonary tuberculosis (PTB) at different time points of antituberculosistreatment (T1 T2 and T3) Our results showed the following associations IFNG +874 T allele and IFNG +2109 A allele with higherIFN-120574 levels IL12B +1188 C allele with higher IL-12 levels TNF minus308 A allele with higher TNF-120572 plasma levels in controls andmRNA levels in PTB patients at T1 IL17A A allele at rs7747909 with higher IL-17 levels IL10 minus819 T allele with higher IL-10 levelsand TGFB1 +29 CC genotype higher TGF-120573 plasma levels in PTB patients at T2 The present study suggests that IFNG +874TAIFNG +2109AG IL12B +1188AC IL10 minus819CT and TGFB1 +21CT are associated with differential cytokine levels in pulmonarytuberculosis patients and may play a role in the initiation and maintenance of acquired cellular immunity to tuberculosis and inthe outcome of the active disease while on antituberculosis treatment

1 Introduction

Mycobacterium tuberculosis (M tuberculosis) is an intracel-lular obligate aerobic pathogen which has a predilection forthe lungs [1] Macrophages initiate phagocytosis ofM tuber-culosis bacilli and regulate immune responses mediated byproinflammatory cytokines such as TNF-120572 Effector T lym-phocytes (T cells) and natural killer (NK) cells secrete IFN-120574 which activate alveolar macrophages to produce reactive

intermediates from nitrogen and oxygen inhibiting growthand promoting mycobacteria death [2] IL-12 producedmainly by macrophages and dendritic cells has a key rolein the immune response to M tuberculosis bridging innateand adaptive immunity Moreover IL-12 induces T cells andNK cells to produce proinflammatory cytokines such as IFN-120574 and TNF-120572 while also regulating the production of IL-17[2 3] A synergistic response of IFN-120574 IL-12 TNF-120572 and IL-17 activates macrophage stimulating these cells to eliminate

2 Tuberculosis Research and Treatment

the intracellular pathogen acting as a major effector mecha-nism of the cellular immune response [4]

Despite the protective effect of Th1 responses againstM tuberculosis certain cytokines such as TNF-120572 are cor-related with the immunopathogenesis of the disease [5]To prevent tissue damage active tuberculosis is associatedwith decreased Th1 and increased production and actionof suppressing cytokines produced by Th2 and T regula-tory (Treg) cells IL-10 and TGF-120573 respectively which actby deactivating macrophages modulating proinflammatorycytokines and reducing the antigen presenting function of Tcells [6] TGF-120573 also participates in the induction of fibrosisa hallmark presentation of tuberculosis disease [7]

The dynamics of tuberculosis (TB) disease is complexas various aspects of the parasite-host interaction contributeto the occurrence and presentation of the outcome In thisscenario there is an important contribution of human geneticsusceptibility to disease after exposure to M tuberculosis[8ndash10] Cytokines have a key role in the defense againstmycobacteria and their genesmight be considered candidatesfor host susceptibility to the onset of active TB disease

The association between cytokines polymorphisms andhuman TB susceptibility has been reported in studies withex vivo cytokine production in response to mycobacterialantigens and their correlation with variant genotypes how-ever few studies have investigated their role in modulatingthe overall cytokine response during PTB treatment [11ndash19]We hypothesized that studying the actual overall immunecytokine pattern of PTB patients could be important to ourunderstanding of active disease and possible establishmentof biomarkers of recovery and anti-TB treatment efficiencyTherefore the aim of this study was to verify the influenceof IFNG IL12B TNF IL17A IL10 and TGFB1 gene polymor-phisms on the overall cytokine response of Brazilian patientswith PTB under anti-TB treatment

2 Material and Methods

21 Study Population The study group enrolled 31 Brazil-ian patients attending the Infectious and Parasitic DiseasesServices at Botucatu Medical School University HospitalUNESP Botucatu Teaching Health Centre and PrimaryHealthcare Units of Botucatu and surrounding region withPTBdiagnosis confirmed by sputum smear or culture positiv-ity forM tuberculosis or by clinical-epidemiologic data withlaboratory and image exams (radiography or computerizedtomography (CT)) compatible with active TB Patients withPTB concurrent with other active granulomatous diseases orHIVwere excluded All patients diagnosedwith PTB receivedtreatment for six months using the four first-line drugsisoniazid ethambutol pyrazinamide and rifampicin For theevaluation of immunologic function patientsrsquo samples werecollected based on the anti-TB treatment timeline defined asT1 after diagnosis and with nomore than onemonth of treat-ment T2 with three months of treatment and T3 with sixmonths of treatment Patient-specimen distribution for eachtime point included T1 (119899 = 5) T2 (119899 = 1) T3 (119899 = 2) withtwo time points T1 and T2 (119899 = 4) and with the three time

points T1 T2 and T3 (119899 = 19) For normal controls (C) westudied 20 health care workers fromBotucatuMedical School(Botucatu SP Brazil) 9 males (mean age 404 years) and11 females (mean age 341 years) without clinical complaintsand with no history of TB disease autoimmune disease andother infectious disease All controls were BCG vaccinated inchildhood and tuberculin skin test (TST) positive (indurationge 5mm) All patients and controls agreed to participate in thestudy after study clarification and written informed consent

22 Single Nucleotide Polymorphism (SNP) Genotyping Inthe current study seven SNPs were analyzed IFNG +874TAIFNG +2109AG IL12B +1188AC TNF minus308GC IL17Ars7747909 IL10 minus819CT and TGFB1 +29CT These poly-morphisms were selected after verifying their presence inhigh frequency (5) in the Brazilian population

For the current study 5mL of peripheral blood wasdrawn in an EDTA blood tube (by standard phlebotomyprocedures) from PTB patients (119899 = 31) and controls(119899 = 20) at base line (T

0) and genomic DNA was extracted

from leukocytes employing a DNAzol commercial reagent(Invitrogen Carlsbad CA USA) according to the manufac-turerrsquos instructionsQuantification andpurity of the extractedDNA were determined on a spectrophotometer (NanoDrop2000Thermo Fisher Scientific) Amplification of the genomicregions of interest was performed by PCR using 20 to 50 ngof DNA recombinant Taq DNA polymerase 02mM ofeach dNTP (deoxy-nucleotide-adenine guanine thymine orcytosine-triphosphate) 03 to 1mM concentration of eachof the specific primers appropriate buffer and ultrapurewater The IFNG +874TA was genotyped by PCR-ARMSas described by Pravica et al [20] The variation at IFNG+2109AG creates a restriction site for the enzyme AciI andwas genotyped by a previously reported PCR-RFLP method[21] The IL12B +1188 genotyping was performed using aPCR-RFLP method in accordance with Garcıa-Gonzalez etal [22] Fluorescence-based TaqMan technology (AppliedBiosystems Foster City CA USA) was applied to producegenotypes of IL17A and TGFB1 polymorphisms according tothemanufacturerrsquos instructionsThe TNF minus308AG genotypewas defined using a PCR-RFLP method in accordance withWilson et al [23]

23 Cytokines Gene Expression by Reverse Transcriptase RealTime PCR (RT-qPCR) To evaluate IFN-120574 IL-12 TNF-120572 IL-17 IL-10 and TGF-120573mRNA expressions 20mL of peripheralblood was drawn by a standard procedure in heparinizedblood tubes at a single time point from controls (119899 = 20) andat three serial time points for PTB patients (119899 = 31) basedon the antituberculosis treatment timeline (T1 T2 and T3)as previously defined Peripheral blood mononuclear cells(PBMCs) were isolated by a Histopaque gradient separationmethod [24] The layer rich in lymphocytes and monocyteswas aseptically removed and washed twice with PBS for15min at 1500 RPM The cell suspension was resuspended in1mL of PBS and the identification and viability of cells weredetermined by counting with Turk solution (50120583L aliquotsof cell suspension with 50 120583L of the dye solution at 5)

Tuberculosis Research and Treatment 3

Table 1 Primers sequence for qPCR

Primers Forward sequence Reverse sequenceIFN-120574 51015840-AAAAGAGTTCCATTATCCGCTACATC-31015840 51015840-GTTTTGGGTTCTCTCTTGGCTGTTA-31015840

IL-12 51015840-ACCTCCACCTGCCGAGAAT-31015840 51015840-CATGGTGGATGCCGTTCA-31015840

TNF-120572 51015840-GGTTTGCTACAACATGGGCTACA-31015840 51015840-CCCCAGGGACCTCTCTCTAATC-31015840

IL-17 51015840-TTAGGC ACATGGTGGACAATCGG-31015840 51015840-ATGACTCCTGGGAAGACCTCA TTG-31015840

IL-10 51015840-CTTGATGTCTGGGTCTTGGTTCT-31015840 51015840-GCTGGAGGACTTTAAGGGTTAACCT-31015840

TGF-120573 51015840-AGGGCCAGGACCTTGCTG-31015840 51015840-CAAGGGCTACCATGCCAACT-31015840

120573-actin 51015840-GCTGGAAGGTGGACAGCGA-31015840 51015840-GGCATCGTGATGGACTCCG-31015840

Total RNA extraction from PBMC (2 times 106 cellsmL) wasprepared using Trizol Reagent (Invitrogen Carlsbad CAUSA) according to manufacturerrsquos instructions The relativepurity concentration and quality of the isolated RNA weredetermined by spectrophotometry and the ratio of A260ndashA280 nm exceeded 18 for all preparations (NanoDrop1 1000Spectrophotometer Thermo Scientific) To ensure completeremoval of traces of genomic DNA 1 120583g of total RNA wasincubated with DNase I (Amp Grade)

First-strand cDNA synthesis was performed with 1 120583g oftotal RNA per 60 120583L of reaction using Reverse TranscriptaseSuper Script II (Invitrogen Carlsbad CA USA) and randomprimers (3 120583g120583L) (Invitrogen Carlsbad CA USA) accord-ing to manufacturerrsquos instructions Within 5 minutes afterRNAse H (Invitrogen Carlsbad CA USA) was added andincubated at 37∘C for 20 minutes

Relative quantification of each target mRNA was per-formed using a standard curve-based method for relativereal-time PCR data processing with a 7300 Real-Time PCRSystems (Applied Biosystems USA) and Power SYBR GreenPCR Master Mix [25] Primers sets used in the qPCR(amplifying cytokine fragments of mRNA and of the human120573-actinmRNA endogenousmRNA control) are presented inTable 1 Each q-PCR was set in duplicate in a total of 20mLeach which contained 02mM of each forward and reverseprimer 2mL of template cDNA 10 120583L qPCR master mixand 72mL nuclease-free water In addition a ldquono templaterdquocontrol was included in duplicate on each plate to verifythat amplicon contamination was absent PCR conditionswere as follows initial denaturation at 95∘C for 10min and40 cycles at 95∘C for 15 s and 60∘C for 60 s followed bya melting curve Amplification of specific transcripts wasconfirmed by melting curve profiles generated at the endof each run Control samples expression mean received therelative value of 10 and concentrations in all other sampleswere normalized proportionately

24 Plasma Cytokine Levels Plasma samples were obtainedfrom the same peripheral blood used for the genetic expres-sion of cytokines from controls (119899 = 20) and at three serialtime points from patients with PTB based on the antitu-berculosis treatment timeline (T1 T2 and T3) as previouslydefined Samples were maintained frozen (minus80∘C) until useand then thawed at room temperature on the day they wereused Quantikine ELISA kits (R amp D Systems) were usedaccording to manufacturerrsquos instructions to measure IFN-120574

IL-12 TNF-120572 IL-17 IL-10 and TGF-120573 plasma levels andmethod sensitivity was in accordance with each kit Cytokineanalysis was not possible in all 31 PTB patients becausesamples were also used in other experiments therefore thedistribution of individuals among cytokines was IFN-120574 (119899 =30) T1 (119899 = 5) T2 (119899 = 1) T3 (119899 = 2) with two time pointsT1 and T2 (119899 = 4) with two time points T1 and T3 (119899 = 1)and with the three time points T1 T2 and T3 (119899 = 17) IL-12IL-17 and IL-10 (119899 = 30) T1 (119899 = 7) T2 (119899 = 1) T3 (119899 = 2)with two time points T1 and T2 (119899 = 4) and with the threetime points T1 T2 and T3 (119899 = 16) TNF-120572 (119899 = 30) T1(119899 = 5) T2 (119899 = 1) T3 (119899 = 2) with two time points T1 andT2 (119899 = 4) and with the three time points T1 T2 and T3(119899 = 18) TGF-120573 (119899 = 29) T1 (119899 = 5) T2 (119899 = 1) T3 (119899 = 2)with two time points T1 and T2 (119899 = 4) and with the threetime points T1 T2 and T3 (119899 = 17)

25 Statistical Analysis Comparisons between different gen-otypes in the control and patient groups were made usingMann-Whitney 119880 Test with two-tail 119875 value For the ana-lytical comparison between the three time points of thetreatment in the PTB case group (T1 T2 and T3) a Friedmantest was used to verify which time point differed from theother a Dunnrsquos multiple comparisons test was applied as aposttest Results were considered significant when 119875 lt 005Tests were performed using GraphPad Prism version 500for Windows GraphPad Software (San Diego CA USA)httpwwwgraphpadcom

3 Results

31 Demographic Characteristics of PTBPatients Gender andage distribution among PTB patients was 23 males (mean ageof 488 years) and 8 females (mean age of 389 years) andall patients were BCG (Bacillus Calmette-Guerin) vaccinatedin childhood Despite the large differences of age and sexin controls and PTB patients groups these variables had noeffect on the cytokines expression and plasma levels (data notshown)

PTB patientsrsquo diagnosis was confirmed by sputum smearor culture positivity for M tuberculosis (119899 = 2) sputumsmear or culture positive forM tuberculosis and image exams(radiography or CT) compatible with active TB (119899 = 19)or by image exams (radiography or CT) compatible withactive TB (119899 = 10) alone with diagnosis confirmation withclinical response after the beginning of the anti-TB treatment


0

50

100

150

C T1 T2 T3

AAATTT

IFN

-120574(p

g120583

L) lowast

lowast

+874

(a)

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1000

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4000

AAATTT

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

lowast

lowast

+874

(b)

Figure 1 Influence of IFNG +874TA gene SNP on IFN-120574 plasma levels (a) and mRNA expression (b) in the control group (C) and PTBpatients group at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data areshown as median levels lowast119875 lt 005

All patients had respiratory symptoms consistent with dys-pnea cough and ventilation-dependent pain and mostpresented with constitutional symptoms consistent withweight loss fever and weakness at the beginning of the anti-TB treatment (T1) The symptoms were less frequent alongtreatment timeline (T2) and at the end (T3) all patients wereconsidered recovered Studies classify pulmonary tuberculo-sis by its severity as minimal moderate or advanced diseasedepending on the characteristics of clinical-epidemiologicdata andor image exams (radiography or CT) [16 19] In ourstudy medical evaluation of these characteristics showed thatall of our patients had a moderate form of active TB disease

32 General Immune Response during Anti-TB TreatmentIn general when compared to controls plasma levels ofIFN-120574 IL-12 and IL-10 were similar among PTB patientsduring anti-TB treatment IL-17 and TGF-120573 plasma levelswere increased among PTB patients only at the beginningof anti-TB treatment (T1) and plasma levels for TNF-120572 werelower among PTB patients during anti-TB treatment (T1T2 and T3) (data not shown) When compared to controlsmRNA expression levels of IL-12 TNF-120572 and IL-17 weresimilar among PTB patients during anti-TB treatment (T1T2 and T3) IFN-120574 and IL-10 were increased among PTBpatients during anti-TB treatment (T1 T2 and T3) and TGF-120573was increased only at the T2 time point among PTBpatients(data not shown)

33 Influence of IFNG +874TA and +2109AG Gene Polymor-phisms on IFN-120574 Plasma Level and mRNA Expression The Tallele carriers for IFNG +874TA (ATTT) in PTB patientswere associated with significantly higher plasma and mRNAexpression levels of IFN-120574 when compared to individualswith AA genotype at T2 (119875 = 004 119875 = 003 resp) andT3 (119875 = 004 119875 = 003) time points of the treatment

(Figures 1(a) and 1(b)) When we compared the three timepoints the AA genotype in PTB patients at T1 presentedsignificant higher plasma levels than T2 (119875 lt 005) andT3 (119875 lt 005) (Figure 8(a)) and no differences for mRNAexpression (Figure 8(b)) T allele carriers at T2 presentedwithsignificantly higher mRNA expression levels than at T1 (119875 lt005) (Figure 8(b)) and no differences at the IFN-120574 plasmalevel (Figure 8(a)) were seen There was no influence of thispolymorphism on the control group (Figures 1(a) and 1(b))

Results for the IFNG +2109AG SNP showed that individ-uals with AA genotype presented higher levels of IFN-120574 thantheAGgenotype in controls (119875 lt 005) and in PTB patients atT2 (119875 = 004) and T3 (119875 = 002) (Figure 2(a)) Comparisonsbetween the heterozygous AG genotype of PTB patientstime points using the same individuals showed that at T1significantly higher levels of IFN-120574 were seen than at T2 (119875 lt005) and T3 (119875 lt 005) There were no differences betweentreatment time points for PTB patients with the AA genotype(Figure 8(c)) No significant differences in mRNA expressionwere seen between genotypes in the control group and inPTB patients although patients with the AA genotype tendedto present with higher expression levels when compared toindividuals with the AG genotype (Figure 2(b)) There werealso no differences between treatment time points of PTBpatients for mRNA expression within both genotypes (datanot shown) In our study group we did not find individualswith the GG genotype

34 Influence of IL12B +1188AC Gene Polymorphism on IL-12Plasma Level and mRNA Expression TheC allele carriers forIL12B +1188 (ACCC) had significantly higher plasma levelsof IL-12 than individuals with the AA genotype in the controlgroup (119875 = 004) and at the T2 (119875 = 003) time point ofPTB patients (Figure 3(a)) IL-12 mRNA expression analysisshowed no difference whenAA genotype andC allele carriers


0

50

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150+2109

C T1 T2 T3

AAAG

IFN

-120574(p

g120583

L) lowast

lowast

lowast

(a)

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3000

4000

C T1 T2 T3

AAAG

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+2109

(b)

Figure 2 Influence of IFNG +2109AG gene SNP on IFN-120574 plasma levels (a) and mRNA expression (b) in the control group (C) and PTBpatients group at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data areshown as median levels lowast119875 lt 005

0

2

4

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8

10

IL-12

(pg120583

L)

+1188

C T1 T2 T3

AAACCC

lowastlowast

(a)

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4000

5000

Gen

ic ex

pres

sion

IL-12120573

-act

in

C T1 T2 T3

AAACCC

lowast

+1188

(b)

Figure 3 Influence of IL12B +1188AC gene SNP on IL-12 plasma levels (a) and mRNA expression (b) in the control group (C) and PTBpatients group at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data areshown as median levels lowast119875 lt 005

were compared in the control group However at the T2 timepoint for PTB patients C allele carriers expressed more IL-12mRNA expression levels than individuals with AA genotype(119875 lt 005) (Figure 3(b)) There were also no differencesbetween time points of PTB patients for IL-12 plasma andmRNA expression levels within both genotypes (data notshown)

35 Influence of TNF minus308GC Gene Polymorphism on TNF-120572 Plasma Level and mRNA Expression Control individualswith the AG genotype had significantly higher plasma levels

of TNF-120572 than those with GG genotype (119875 = 004) Therewas no difference between genotypes in the PTB patientsgroup (Figure 4(a))The analyses between time points for theGG genotype in PTB patients showed higher levels of TFN-120572 at T1 when compared with T3 (119875 lt 005) No differenceswere seen between time points for the AG genotype PTBpatients (Figure 8(d)) The TNF-308GC gene SNP did notinfluence TNF-120572mRNA expression in controls (Figure 4(b))At T1 PTB patients with the AG genotype had significantlyhigher mRNA expression levels than those patients with theGG genotype (119875 = 002) (Figure 4(b)) There were also nodifferences between time points of PTB patients for mRNA


0

50

100

150TN

F-120572

(pg120583

L)minus308

C T1 T2 T3

AGGG

lowast

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

TNF-120572

120573-a

ctin

AGGG

C T1 T2 T3

minus308

lowast

(b)

Figure 4 Influence of TNF minus308GC gene SNP on TNF-120572 plasma levels (a) and mRNA expression (b) in the control group (C) and PTBpatients group at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data areshown as median levels lowast119875 lt 005

0

50

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200

IL-1

7 (p

gm

L)

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(a)

0

500

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Gen

ic ex

pres

sion

IL-17

120573-a

ctin

lowast

lowast

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(b)

Figure 5 Influence of IL17 SNP rs7747909 on IL-17 plasma levels (a) and mRNA expression (b) in the control group (C) and PTB patientsgroup at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data are shown asmedian levels lowast119875 lt 005

expression with either genotype (data not shown) In ourstudy group we did not find individuals carrying the AAgenotype

36 Influence of IL17A rs7747909 Gene Polymorphism onIL-17A Plasma Level and mRNA Expression The markerrs7747909 at IL17 gene had no influence on IL-17 controlplasma levels PTB patients who were A allele carriers(AAAG) had significantly higher IL-17 plasma levels onlyat the T3 time point (119875 = 004) (Figure 5(a)) In controlsthe A allele carriers had significantly higher IL-17 mRNAexpression levels than individuals with the GG genotype

(119875 = 004) In PTB patients who were A allele carriers wefound significantly higher IL-17 mRNA expression levels atthe T2 (119875 lt 005) and T3 (119875 = 004) time points thanpatients with the GG genotype (Figure 5(b)) There were nodifferences between time points of PTB patients for IL-17plasma and mRNA expression levels within both genotypegroups (data not shown)

37 Influence of IL10 minus819CT Gene Polymorphism on IL-10 PLasma Level and mRNA Expression There was no dif-ference in IL-10 plasma levels in the controls between CCgenotype andT allele carriers PTBpatients whowere carriers


0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)

Figure 6 Influence of IL10 minus819CT gene SNP on IL-10 plasma levels (a) andmRNA expression (b) in the control group (C) and PTB patientsgroup at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data are shown asmedian levels lowast119875 lt 005

lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)

Figure 7 Influence of TGF1205731 +29CT gene SNP on TGF-120573 plasma levels (a) and mRNA expression (b) in the control group (C) and PTBpatients at three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Data are shownas median levels lowast119875 lt 005

of the T allele (TCTT) had significantly higher levels of IL-10when compared with PTB patients with the CC genotype inall time points of treatment T1 (119875 lt 005) T2 (119875 = 003)and T3 (119875 lt 005) (Figure 6(a)) IL-10 minus819CT SNP hadno influence on IL-10 mRNA expression in the control groupand PTB patients when the CC genotype and T allele carrierswere compared (Figure 6(b)) There were also no differencesbetween time points in PTB patients for IL-10 plasma andmRNA expression levels within both genotypes (data notshown)

38 Influence of TGFB1 +29CT Gene Polymorphism on TGF-120573 Plasma Level and mRNA Expression No differences inTGF-120573 plasma levels were seen in the control group betweenboth genotypes PTB patients with CC genotype had higherTGF-120573 plasma levels when compared with T allele carriersonly at the T2 time point (119875 = 002) (Figure 7(a)) Analysesbetween time points of PTB patients with CC genotypeshowed higher TGF-120573 plasma levels at T1 when comparedto T3 (119875 lt 005) (Figure 8(e)) No differences in mRNAexpression were seen in both controls and PTB patients


lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

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(a)

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AA ATTT0

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-120574120573

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(b)

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-120574(p

g120583

L)

AA AG0

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TNF-120572

(pg120583

L)

AG GG0

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(d)

CC CTTT0

200

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T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)

Figure 8 Influence of IFNG +874TA gene SNP on IFN-120574 plasma levels (a) and mRNA expression (b) IFNG +2109AG gene SNP on IFN-120574plasma levels (c) TNF minus308GC gene SNP on TNF-120572 plasma levels (d) and TGFB1 +29CT gene SNP on TGF-120573 plasma levels (e) in PTBpatients that have all three time points of the treatment T1 (after diagnosis and first month) T2 (three months) and T3 (six months) Dataare shown as median levels lowast119875 lt 005


within both genotypes (Figure 7(b)) There were also nodifferences between time points of PTB patients for mRNAexpression within both genotypes (data not shown)

4 Discussion

It is well known that cytokines play an essential role duringthe immune response to active TB disease and changesin cytokine levels may lead to abnormal or ineffectiveimmune responses as seen in human infections caused byM tuberculosis [6] The genetic component that contributesto susceptibility and progression of PTB most certainlyinvolves an interaction between multiple alleles located ondifferent genes [26] As a number of cytokine genes have beendescribed in association with altered cytokine levels [13 20]we evaluated the influence of SNPs at cytokine genes on theoverall cytokine response of PTBpatients undergoing anti-TBtreatment When pertinent controls results were consideredto indicate an effect of the polymorphism under normalconditions without the interference of active TB or anti-TBtreatment in other words an independent genetic effect

Our results showed that PTB patients who were T allelecarriers had higher plasma and mRNA expression levels ofIFN-120574 at the middle and end of anti-TB treatment Studieshave shown that the IFNG +874AT SNP influences IFN-120574 production by providing a binding site for NF-120581B and isassociated with TB susceptibility [14 15 20] This fact couldhave functional consequences for the transcription of IFN-120574production [27 28]

A recent study in Spain showed that the IFNG +874AA genotype had the lowest IFN-120574 production in PBMCculture after stimulation with PPD in PTB patients at thetime of diagnosis and after completion of therapy [14] whichagrees with our results at the end of anti-TB treatmentStill in agreement with our results 2 additional studieshave shown that the homozygous TT genotype of normaland PTB individuals produces higher IFN-120574 in response tomycobacterial antigens [15 16] In another functional studypatients with tuberculosis carrying the genotype +874AAshowed significantly lower IFN-120574 plasma levels than thosewith the +874AT and +874TT genotypes [11] This trend wasalso seen in the plasma of Indians patients with active PTB[12]

Other studies that evaluated IFN-120574 serum levels inHBV infection and IFN-120574 production in PBMC culturesin acutely ill patients cutaneous leishmaniasis and normalindividuals stimulated with LPS (Lipopolysaccharide) PHA(Phytohemagglutinin) or Mycobacterium leprae (M leprae)antigens also support our findings of higher IFN-120574 levelsin PTB patients that were T allele carriers (TATT) [29ndash31] Another study has shown the relation between an IFNGpolymorphic microsatellite marker (allele 2) polymorphismand the IFN-120574 gene transcription Since this region showsan absolute correlation with the IFNG +874 T allele thisstudyrsquos findings are in agreementwith our results that showedthat the IFNG +874 T allele was related to a higher IFN-120574 expression from the middle to the end of the anti-TBtreatment [20]

Some studies evaluating tuberculosis patients in theinitial stages of TB disease using normal controls have foundno differences between genotypes of IFNG +874 SNP in IFN-120574 production in PBMC cultures of M tuberculosis H37Rvculture filtrate antigen (CFA) of M tuberculosis nor PHAstimulus [17 18] An additional study found no influence ofIFNG +874 locus on mRNA expression during Helicobacterpylori (H pylori) infection [32] These different findings maybe due to ethnic differences in genotypic frequencies amongvarious population studiesThis result however is in completeagreement with genetic epidemiologic data that the T allele isassociated with protection against TB [33]This same allele isalso associated with resistance to leprosy [34]

Another polymorphism in the INFG gene located2109 bp downstream from the translation start site in thethird intron has been reported to be involved in tran-scriptional regulation of IFN-120574 gene of this polymorphismalthough the contribution is still unclear [35 36] To ourknowledge our study is the first report regarding the func-tional effect of INFG +2109AG on IFN-120574 plasma andmRNAexpression levels as our results showed that controls and PTBpatients with the AG genotype had lower plasma levels at theend of the treatment

IFN-120574 is a key cytokine in activation of macrophagesfor mycobacterial stasis and killing [2] the persistence oflow IFN-120574 production from the middle to end of ther-apy in patients with IFNG +874 AA genotype and INFG+2109 AG genotype could result in a worse prognosis tothe resolution of the active disease and efficiency of theanti-TB treatment and may also underlie their increasedrisk for reactivation of a latent PTB focus In agreementwith our results decreased production of IFN-120574 in PTBpatients when compared with healthy controls have beenreported and it may be due to the initial T cell anergyseen in the disease [18 37ndash39] Such an inadequate IFN-120574production may result in failure of macrophage activationwhich could lead to active disease progression and couldplay a role affecting TB diagnostic tests as IFG alleles wereassociated with microscopy-positivenegative and bacterialculture-positivenegative forms of disease [18 40]

IL-12 is important in mediating protective immunityagainst TB An SNP at 3rsquoUTR region of the IL12B gene(+1188AC) coding for IL-12p40 is known to modulate IL-12p40 levels The present study showed that IL12B +1188AA genotype is associated with lower IL-12 plasma levelsin normal controls and in TB patients after 3 months ofanti-TB treatment Our results agree with another study thatevaluated PTB patients and normal controls [17]

Several studies have found that the IL12B +1188 C alleleis associated with lower IL-12p40 production of PBMC fromhealthy individuals stimulated with C3 binding glycoproteinLPS or PPD [41ndash43] Arababadi et al [30] did not find asignificant difference in IL-12 serum level between AA andAC genotypes in occult HBV infection Discordance betweenresults may be due to differences in ethnicity diseases anddesigns of the studies

IL-12 production is induced following phagocytosis ofM tuberculosis by macrophages and dendritic cells whichleads to development of a Th1 response with production


of IFN-120574 [1] Since IL-12p40 is a component of both IL-12p70 and IL-23 and regulates initiation and maintenance ofacquired cellular responses to TB low IL-12p40 levels in AAgenotype individuals might have a role in limiting chronicinflammation [44] which could result in more difficulties inresolving active disease and dampen the efficiency of the anti-TB treatment

TNF-120572 plays an important role in granuloma formationin tuberculosis [19] The promoter region of the TNF gene ishighly polymorphic and our evaluation of the TNF minus308GAlocus showed that normal individuals with the AG genotypehad higher TNF-120572 plasma levels PTB patients carrying thesame genotype tended to have higher levels of TNF-120572 thoughnot significant There are conflicting results regarding thefunctional effect of the TNF minus308AG SNP Some authorshave demonstrated increased TNF-120572 production related toTNF minus308 A allele carriers in LPS stimulated PBMC andwhole blood cultures of leprosy patients after LPS and Mleprae stimulation and of paracoccidioidomycosis patientswhile others failed to show any effect of this SNP on TNF-120572production after LPS stimulation in vivo or in vitro in HCVpatients and in tuberculosis patients [19 44ndash50]

An in vitro expression study has indicated that the TNFminus308GA SNP has a direct effect on TNF-120572 gene regulationand the A allele at this locus may lead to a higher expressionlevel [51] Our results agree with this study that demonstratedthat PTB patients with the AG genotype at the beginningof anti-TB treatment have higher TNF-120572 mRNA expressionthan GG genotype individuals

Since TNF-120572 is important for walling off infections andpreventing dissemination by granuloma formation low levelsof TNF-120572 as seen in our PTB patients could impair thecontainment of theM tuberculosis bacilli leading to difficultresolution of active disease dampening the efficiency of anti-TB treatment and increasing the reactivation risk as shownin rheumatoid arthritis patients who were undergoing anti-TNF-120572 therapy [52 53]

IL-17 is a potent inflammatory cytokine induced by Mtuberculosis infection [54] To our knowledge this is the firststudy to verify the functional effect of the IL17A rs7747909polymorphism on IL-17A plasma and mRNA expressionlevels Our results showed that A allele carriers (at rs7747909)produce higher plasma levels of IL-17A at the end of therapyand in general PTB patients produced higher levels thannormal controls

Although Th17 cells are not as important as Th1 cellsin mediating protection against primary M tuberculosisinfection IL-17 appears to be critical to the induction of Mtuberculosis-specific memory response and the mediation ofprotection against challenge infections and during vaccina-tions [55ndash57] Our results suggest that PTB patients have noimpairment to IL-17A production

IL-10 is known to have deactivating properties andundermines humanTh1 immunologic responses About 50of the observed variability of IL-10 secretion can be explainedby genetic factors [58] Our results showed that IL10 minus819 Tallele carriers had higher plasma levels of IL-10 during the6 months of anti-TB treatment A previous study evaluatingPTB patients showed no influence of this SNP on IL-10 levels

[17] Additionally no impact of the IL10 minus819 locus was foundon IL-10 serum level of controls and patients with HCVinfection or on normal individuals PBMCcultures stimulatedwith LPS and PPD [13 48] A study on patients withleprosy showed that IL10 minus819 T allele carriers produce lowerlevels of IL-10 when compared with non-T allele carriers[59]

In a recent study evaluatingH pylori patients with SNPsin the promoter region of IL10 were shown to have higherIL-10 mRNA expression with the GCC haplotype carriersat IL10 minus1082GAminus819CTminus592CA and low IL-10 mRNAexpression levels in ATA haplotype carriers [32] In our studythe different IL10 minus819 genotypes had no influence on IL-10mRNA expression

Published data suggests that IL-10 inhibits synthesis ofIFN-120574 by T cells and that production of IL-10 has beenassociated with anergy in tuberculosis [60 61] Our resultssuggest that PTB patients T allele carriers could have moredifficulties in building a protective response towards activePTB since they are present with higher levels of IL-10 duringanti-TB treatment These results suggest that in cases withadvanced forms of PTB higher IL-10 production in T allelecarries could lead to a worse recovery from active disease andpoor anti-TB treatment efficiency

TGF-120573 is present in the granulomatous lesions of TBpatients [62] At low concentrations TGF-120573 is a chemotacticfactor formonocytes and acts to induce secretion of IL-1120572 andTNF-120572 and in high concentration inactivates macrophagesinhibits the expression and function of receptors for IFN-120574 IL-1120572 and IL-2 and decreases the production of TNF-120572parallel events related to increase of intracellular mycobac-terial growth [63 64] Our results showed that PTB patientswith the TGFB1 +21CC genotype have higher TGF-120573 plasmalevels at T2 of the anti-TB treatment These results areconsistent with other studies focused on cancer and myocar-dial infarction [65ndash67] Our results revealed that TGF-120573plasma levels were higher at the beginning of the anti-TBtreatment in all PTB patients when compared to the controlswhich could result in the impairment of the initial protectiveimmune response for the resolution of the active disease andin depressing the efficiency of anti-TB treatment The factthat PTB patients with the TGFB1 +21 CC genotype alsomaintained higher levels of the cytokine at T2 could resultin a more persistent active disease

Our work lacks an association between demographiccharacteristics and cytokine levels In addition the influenceof cytokine SNPs on TB outcomes may be driven by asmall sample size and by the fact that all patients hada moderate presentation of PTB We also had differencesbetween the plasma levels patterns and mRNA expressionlevels patterns of the cytokines evaluated in the controlsand TB patients during anti-TB treatment This fact couldbe explained by mRNA stability and transcription rate andby factors of translational regulation which could directlyaffect the expression and production of mediators involvedin immune response [68]

The present study suggests that IFNG +874TA IFNG+2109AG IL12 +1188AC IL10 minus819CT and TGFB1 +21CTare associated with different cytokine levels in PTB patients


and may play a role in the initiation and maintenance ofacquired cellular immunity to TB and in the outcome of theactive disease and antituberculosis treatment As cytokinesplay a major role in TB immunity studying the overallcytokine profile determined by the respective functionalSNPs andor other closely linked genes could demonstrate theactual pattern of the cytokine response against the mycobac-teria and may provide a better understanding of active TBdisease progression and response to the anti-TB treatmentand may serve as genetic risk markers of TB susceptibility

5 Conclusion

In this study we demonstrated that cytokine SNPs can inducedifferent overall cytokine levels in PTB patients during anti-TB treatment and these levels could be important to the out-come of the treatment Future studieswith a larger populationand different forms and severity stages of tuberculosis willhelp to better understand why many individuals are infectedby the mycobacterial bacilli but only 10 develop activedisease

Conflict of Interests

The authors have no conflict of interests in connection withthis paper

Acknowledgment

This study was funded by grants from ldquoFundacao de Amparoa Pesquisa do Estado de Sao Paulordquo (FAPESP) Brazil

References

[1] A Raja ldquoImmunology of tuberculosisrdquo Indian Journal of Medi-cal Research vol 120 pp 213ndash232 2004

[2] S H E Kaufmann ldquoProtection against tuberculosis cytokinesT cells and macrophagesrdquo Annals of the Rheumatic Diseasesvol 61 no 2 pp 1154ndash1158 2002

[3] M A Hoeve N D Savage T de Boer et al ldquoDivergent effectsof IL-12 and IL-23 on the production of IL-17 by human T cellsrdquoEuropean Journal of Immunology vol 36 pp 661ndash670 2006

[4] E Sahiratmadja B Alisjahbana T de Boer et al ldquoDynamicchanges in pro- and anti-inflammatory cytokine profiles andgamma interferon receptor signaling integrity correlate withtuberculosis disease activity and response to curative treat-mentrdquo Infection and Immunity vol 75 no 2 pp 820ndash829 2007

[5] R van Crevel T H M Ottenhoff and J W M van der MeerldquoInnate immunity to Mycobacterium tuberculosisrdquo ClinicalMicrobiology Reviews vol 15 no 2 pp 294ndash309 2002

[6] J l Flynn and J Chan ldquoImmunology of tuberculosisrdquo AnnualReview of Immunology vol 19 pp 93ndash129 2001

[7] Z Toossi and J J Ellner ldquoThe role of TGF beta in thepathogenesis of human tuberculosisrdquo Clinical Immunology andImmunopathology vol 87 pp 107ndash114 1998

[8] R Bellamy ldquoSusceptibility to mycobacterial infections theimportance of host geneticsrdquoGenesamp Immunity vol 4 pp 4ndash112003

[9] J L Casanova and L Abel ldquoGenetic dissection of immunity tomycobacteria the human modelrdquo Annual Review of Immunol-ogy vol 20 pp 581ndash620 2002

[10] N Remus A Alcais and L Abel ldquoHuman genetics of commonmycobacterial infectionsrdquo Immunologic Research vol 28 pp109ndash129 2003

[11] A C Vallinoto E S Graca M S Arajo et al ldquoIFNG +874TApolymorphism and cytokine plasma levels are associated withsusceptibility toMycobacterium tuberculosis infection and clin-ical manifestation of tuberculosisrdquoHuman Immunology vol 71no 7 pp 692ndash696 2010

[12] Abhimanyu I R Mangangcha P Jha et al ldquoDifferential serumcytokine levels are associated with cytokine gene polymor-phisms in north Indians with active pulmonary tuberculosisrdquoInfection Genetics and Evolution vol 11 no 5 pp 1015ndash10222011

[13] V Yilmaz S P Yentour and G Saruhan-Direskeneli ldquoIL-12 andIL-10 polymorphisms and their effects on cytokine productionrdquoCytokine vol 30 no 4 pp 188ndash194 2005

[14] D Lopez-Maderuelo F Arnalich R Serantes et al ldquoInterferon-gamma and interleukin-10 gene polymorphisms in pulmonarytuberculosisrdquo American Journal of Respiratory and Critical CareMedicine vol 167 no 7 pp 970ndash975 2003

[15] N Sallakci M Coskun Z Berber et al ldquoInterferon-120574gene+874T-A polymorphism is associated with tuberculosisand gamma interferon responserdquo Tuberculosis vol 87 no 3 pp225ndash230 2007

[16] A Ansari N Talat B Jamil et al ldquoCytokine gene poly-morphisms across tuberculosis clinical spectrum in Pakistanipatientsrdquo PLoS ONE vol 4 no 3 Article ID e4778 2009

[17] P Selvaraj K Alagarasu M Harishankar M Vidyarani DNisha Rajeswari and P R Narayanan ldquoCytokine gene poly-morphisms and cytokine levels in pulmonary tuberculosisrdquoCytokine vol 43 no 1 pp 26ndash33 2008

[18] M Vidyarani P Selvaraj S P Anand M S Jawahar A RAdhilakshmi and P R Narayanan ldquoInterferon gamma (IFN120574)amp interleukin-4 (IL-4) gene variants amp cytokine levels inpulmonary tuberculosisrdquo Indian Journal of Medical Researchvol 124 no 4 pp 403ndash410 2006

[19] S Sharma J Rathored B Ghosh and S K Sharma ldquoGeneticpolymorphisms in TNF genes and tuberculosis in North Indi-ansrdquo BMC Infectious Diseases vol 10 no 165 pp 1ndash9 2010

[20] V Pravica C Perrey A Stevens J H Lee and I V HutchinsonldquoA single nucleotide polymorphism in the first intron of thehuman IFN-120574 gene absolute correlation with a polymorphicCA microsatellite marker of high IFN-120574 productionrdquo HumanImmunology vol 61 no 9 pp 863ndash866 2000

[21] S Henri F Stefani D Parzy C Eboumbou A Dessein andC Chevillard ldquoDescription of three new polymorphisms in theintronic and 31015840 UTR regions of the human interferon gammagenerdquo Genes and Immunity vol 3 no 1 pp 1ndash4 2002

[22] M A Garcıa-Gonzalez A Lanas J Wu et al ldquoLack ofassociation of IL-12 p40 gene polymorphism with peptic ulcerdiseaserdquo Human Immunology vol 66 no 1 pp 72ndash76 2005

[23] AGWilson F S di Giovine A I F Blakemore andGWDuffldquoSingle base polymorphism in the human Tumour NecrosisFactor alpha (TNF120572) gene detectable byNcoI restriction of PCRproductrdquo Human Molecular Genetics vol 1 no 5 p 353 1992

[24] A Boyum ldquoSeparation of leukocytes from blood and bonemarrow Introductionrdquo Scandinavian Journal of Clinical ampLaboratory Investigation vol 97 article 7 1968


[25] A Larionov A Krause and W R Miller ldquoA standard curvebased method for relative real time PCR data processingrdquo BMCBioinformatics vol 6 article 62 2005

[26] A Larionov A Krause and W Miller ldquoA standard curvebased method for relative real time PCR data processingrdquo BMCBioinformatics vol 6 article 62 2005

[27] A V Hill ldquoThe immunogenetics of human infectious diseasesrdquoAnnual Review of Immunology vol 16 pp 593ndash617 1998

[28] T Heinemeyer E Wingender I Reuter et al ldquoDatabases ontranscriptional regulation TRANSFAC TRRD and COMPELrdquoNucleic Acids Research vol 26 pp 362ndash367 1998

[29] V Pravica A Asderakis C Perrey A Hajeer P J Sinnott andI V Hutchinson ldquoIn vitro production of IFN-120574 correlates withCA repeat polymorphism in the human IFN-120574 generdquo EuropeanJournal of Immunogenetics vol 26 no 1 pp 1ndash3 1999

[30] MKArababadi AA Pourfathollah A Jafarzadeh et al ldquoNon-association of IL-12 +1188 and IFN-120574 +874 polymorphisms withcytokines serum level in occult HBV infected patientsrdquo SaudiJournal of Gastroenterology vol 17 no 1 pp 30ndash35 2011

[31] U Vollmer-Conna B F Piraino B Cameron et al ldquoCytokinepolymorphisms have a synergistic effect on severity of the acutesickness response to infectionrdquo Clinical Infectious Diseases vol47 no 11 pp 1418ndash1425 2008

[32] G I Matos C J Covas R C Bittar et al ldquoIFNG +874TApolymorphism is not associated with American tegumen-tary leishmaniasis susceptibility but can influence Leishmaniainduced IFN-gamma productionrdquo BMC Infectious Diseases vol7 article 33 2007

[33] R Rad A Dossumbekova B Neu et al ldquoCytokine genepolymorphisms influence mucosal cytokine expression gastricinflammation and host specific colonisation during Helicobac-ter pylori infectionrdquo Gut vol 53 no 8 pp 1082ndash1089 2004

[34] A G Pacheco C C Cardoso and M O Moraes ldquoIFNG+874TA IL10 -1082GA and TNF -308GA polymorphismsin association with tuberculosis susceptibility a meta-analysisstudyrdquo Human Genetics vol 123 no 5 pp 477ndash484 2008

[35] C C Cardoso A C Pereira V N Brito-de-Souza et al ldquoIFNG+874 TgtA single nucleotide polymorphism is associated withleprosy among Braziliansrdquo Human Genetics vol 128 no 5 pp481ndash490 2010


[37] M Liu B Cao H Zhang Y Dai X Liu and C Xu ldquoAssociationof interferon-gamma gene haplotype in the Chinese populationwith hepatitis B virus infectionrdquo Immunogenetics vol 58 no 11pp 859ndash864 2006

[38] Y Lin M Zhang F M Hofman J Gong and P F BarnesldquoAbsence of a prominent Th2 cytokine response in humantuberculosisrdquo Infection and Immunity vol 64 no 4 pp 1351ndash1356 1996

[39] M Zhang Y Lin D V Iyer J Gong J S Abrams and PF Barnes ldquoT-cell cytokine responses in human infection withMycobacterium tuberculosisrdquo Infection and Immunity vol 63no 8 pp 3231ndash3234 1995

[40] G E Etokebe L Bulat-Kardum M S Johansen et alldquoInterferon-120574 gene (T874A and G2109A) polymorphisms areassociated with microscopy-positive tuberculosisrdquo Scandina-vian Journal of Immunology vol 63 no 2 pp 136ndash141 2006

[41] M K Balcewicz-Sablinska J Keane H Kornfeld and HG Remold ldquoPathogenic Mycobacterium tuberculosis evadesapoptosis of host macrophages by release of TNF-R2 resultingin inactivation of TNF-120572rdquo Journal of Immunology vol 161 no5 pp 2636ndash2641 1998

[42] A Davoodi-Semiromi J J Yang and J X She ldquoIL-12p40 isassociated with type 1 diabetes in Caucasian-American fami-liesrdquo Diabetes vol 51 no 7 pp 2334ndash2336 2002

[43] S Stanilova and L Miteva ldquoTaq-I polymorphism in 31015840UTRof the IL-12 and association with IL-12p40 production fromhuman PBMCrdquo Genes amp Immunity vol 6 pp 364ndash366 2005

[44] A M Cooper A Solache and S A Khader ldquoInterleukin-12and tuberculosis an old story revisitedrdquo Current Opinion inImmunology vol 19 pp 441ndash447 2006

[45] G Bouma J B A Crusius M Oudkerk Pool et al ldquoSecretionof tumour necrosis factor 120572 and lymphotoxin 120572 in relation topolymorphisms in the TNF genes and HLA-DR alleles Rele-vance for inflammatory bowel diseaserdquo Scandinavian Journal ofImmunology vol 43 no 4 pp 456ndash463 1996

[46] A Bozzi P P Pereira B S Reis et al ldquoInterleukin-10 and tumornecrosis factor-alpha single nucleotide gene polymorphismfrequency in paracoccidioidomycosisrdquo Human Immunologyvol 67 no 11 pp 931ndash939 2006

[47] C C Cardoso A C Pereira V N Brito-de-Souza et al ldquoTNF-308G gt A single nucleotide polymorphism is associated withleprosy among Brazilians a genetic epidemiology assessmentmeta-analysis and functional studyrdquo The Journal of InfectiousDiseases vol 204 no 8 pp 1256ndash1263 2011

[48] T Y Chen Y S Hsieh T T Wu et al ldquoImpact of serum levelsand gene polymorphism of cytokines on chronic hepatitis Cinfectionrdquo Translational Research vol 150 no 2 pp 116ndash1212007

[49] E Louis D Franchimont A Piron et al ldquoTumour necrosisfactor (TNF) gene polymorphism influences TNF-120572 productionin lipopolysaccharide (LPS)-stimulatedwhole blood cell culturein healthy humansrdquoClinical and Experimental Immunology vol113 no 3 pp 401ndash406 1998

[50] S Taudorf K S Krabbe R M G Berg K Moslashller B K Ped-ersen and H Bruunsgaard ldquoCommon studied polymorphismsdo not affect plasma cytokine levels upon endotoxin exposurein humansrdquo Clinical amp Experimental Immunology vol 152 no1 pp 147ndash152 2008

[51] AGWilson J A Symons T LMcDowell HOMcDevitt andG W Duff ldquoEffects of a polymorphism in the human tumornecrosis factor alpha promoter on transcriptional activationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 94 no 7 pp 3195ndash3199 1997

[52] M Feldmann and R N Maini ldquoAnti-TNF-alpha therapy ofrheumatoidarthritis what have we learnedrdquo Annual Review ofImmunology vol 19 pp 163ndash196 2001

[53] RMaini EW St Clair F Breedveld et al ldquoInfliximab (chimericanti-tumour necrosis factor 120572 monoclonal antibody) versusplacebo in rheumatoid arthritis patients receiving concomitantmethotrexate a randomised phase III trialrdquoTheLancet vol 354no 9194 pp 1932ndash1939 1999

[54] S A Khader G K Bell J E Pearl et al ldquoIL-23 and IL-17 in the establishment of protective pulmonary CD4+ Tcell responses after vaccination and during Mycobacteriumtuberculosis challengerdquoNature Immunology vol 8 pp 369ndash3772007

[55] S A Khader J E Pearl K Sakamoto et al ldquoIL-23 compensatesfor the absence of IL-12p70 and is essential for the IL-17


response during tuberculosis but is dispensable for protectionand antigen-specific IFN-120574 responses if IL-12p70 is availablerdquoJournal of Immunology vol 175 no 2 pp 788ndash795 2005

[56] M Umemura A Yahagi S Hamada et al ldquoIL-17-mediatedregulation of innate and acquired immune response againstpulmonary Mycobacterium bovis bacille Calmette- Guerininfectionrdquo The Journal of Immunology vol 178 pp 3786ndash37962007

[57] TMWozniak A A Ryan J A Triccas andW J Britton ldquoPlas-mid interleukin-23 (IL-23) but not plasmid IL-27 enhances theprotective efficacy of a DNA vaccine against Mycobacteriumtuberculosis infectionrdquo Infection and Immunity vol 74 no 1pp 557ndash565 2006

[58] S H Opdal ldquoIL-10 gene polymorphisms in infectious diseaseand SIDSrdquo FEMS Immunology and Medical Microbiology vol42 no 1 pp 48ndash52 2004

[59] A C Pereira V N Brito-de-Souza C C Cardoso etal ldquoGenetic epidemiological and biological analysis ofinterleukin-10 promoter single-nucleotide polymorphismssuggests a definitive role for -819CT in leprosy susceptibilityrdquoGenes and Immunity vol 10 no 2 pp 174ndash180 2009

[60] V A Boussiotis E Y Tsai E Yunis et al ldquoIL-10 producingT cells suppress immune responses in anergic tuberculosispatientsrdquoThe Journal of Clinical Investigation vol 105 pp 1317ndash1325 2000

[61] F O Sanchez J I Rodriguez G Agudelo and L F Gar-cia ldquoImmune responsiveness and lymphokine production inpatients with tuberculosis and healthy controlsrdquo Infection andImmunity vol 62 pp 5673ndash5678 1994

[62] F Ruscetti L Varesio A Ochoa and J Ortaldo ldquoPleiotropiceffects of transforming growth factor-120573 on cells of the immunesystemrdquo Annals of the New York Academy of Sciences vol 685pp 488ndash500 1993

[63] R P Numerof F R Aronson and J W Mier ldquoIL-2 stimulatesthe production of IL-1120572 and IL-1120573 by human peripheral bloodmononuclear cellsrdquo Journal of Immunology vol 141 no 12 pp4250ndash4257 1988

[64] T M Lasco L Cassone H Kamohara T Yoshimura and DN McMurray ldquoEvaluating the role of tumor necrosis factor-alpha in experimental pulmonary tuberculosis in the guineapigrdquo Tuberculosis vol 85 pp 254ndash258 2005

[65] D J Grainger K Heathcote M Chiano et al ldquoGenetic controlof the circulating concentration of transforming growth factortype beta1rdquo Human Molecular Genetics vol 8 pp 93ndash97 1999

[66] A M Dunning P D Ellis S McBride et al ldquoA transforminggrowth factor1205731 signal peptide variant increases secretion invitro and is associated with increased incidence of invasivebreast cancerrdquo Cancer Research vol 63 no 10 pp 2610ndash26152003

[67] M Yokota S Ichihara T L Lin N Nakashima and Y YamadaldquoAssociation of a T29rarrC polymorphism of the transforminggrowth factor- 1205731 gene with genetic susceptibility to myocardialinfarction in Japaneserdquo Circulation vol 101 no 24 pp 2783ndash2787 2000

[68] J S Chang J F Huggett K Dheda L U Kim A Zumla and GA W Rook ldquoMyobacterium tuberculosis induces selective up-regulation of TLRs in the mononuclear leukocytes of patientswith active pulmonary tuberculosisrdquo Journal of Immunologyvol 176 no 5 pp 3010ndash3018 2006

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the intracellular pathogen acting as a major effector mecha-nism of the cellular immune response [4]

Despite the protective effect of Th1 responses againstM tuberculosis certain cytokines such as TNF-120572 are cor-related with the immunopathogenesis of the disease [5]To prevent tissue damage active tuberculosis is associatedwith decreased Th1 and increased production and actionof suppressing cytokines produced by Th2 and T regula-tory (Treg) cells IL-10 and TGF-120573 respectively which actby deactivating macrophages modulating proinflammatorycytokines and reducing the antigen presenting function of Tcells [6] TGF-120573 also participates in the induction of fibrosisa hallmark presentation of tuberculosis disease [7]

The dynamics of tuberculosis (TB) disease is complexas various aspects of the parasite-host interaction contributeto the occurrence and presentation of the outcome In thisscenario there is an important contribution of human geneticsusceptibility to disease after exposure to M tuberculosis[8ndash10] Cytokines have a key role in the defense againstmycobacteria and their genesmight be considered candidatesfor host susceptibility to the onset of active TB disease

The association between cytokines polymorphisms andhuman TB susceptibility has been reported in studies withex vivo cytokine production in response to mycobacterialantigens and their correlation with variant genotypes how-ever few studies have investigated their role in modulatingthe overall cytokine response during PTB treatment [11ndash19]We hypothesized that studying the actual overall immunecytokine pattern of PTB patients could be important to ourunderstanding of active disease and possible establishmentof biomarkers of recovery and anti-TB treatment efficiencyTherefore the aim of this study was to verify the influenceof IFNG IL12B TNF IL17A IL10 and TGFB1 gene polymor-phisms on the overall cytokine response of Brazilian patientswith PTB under anti-TB treatment

2 Material and Methods

21 Study Population The study group enrolled 31 Brazil-ian patients attending the Infectious and Parasitic DiseasesServices at Botucatu Medical School University HospitalUNESP Botucatu Teaching Health Centre and PrimaryHealthcare Units of Botucatu and surrounding region withPTBdiagnosis confirmed by sputum smear or culture positiv-ity forM tuberculosis or by clinical-epidemiologic data withlaboratory and image exams (radiography or computerizedtomography (CT)) compatible with active TB Patients withPTB concurrent with other active granulomatous diseases orHIVwere excluded All patients diagnosedwith PTB receivedtreatment for six months using the four first-line drugsisoniazid ethambutol pyrazinamide and rifampicin For theevaluation of immunologic function patientsrsquo samples werecollected based on the anti-TB treatment timeline defined asT1 after diagnosis and with nomore than onemonth of treat-ment T2 with three months of treatment and T3 with sixmonths of treatment Patient-specimen distribution for eachtime point included T1 (119899 = 5) T2 (119899 = 1) T3 (119899 = 2) withtwo time points T1 and T2 (119899 = 4) and with the three time

points T1 T2 and T3 (119899 = 19) For normal controls (C) westudied 20 health care workers fromBotucatuMedical School(Botucatu SP Brazil) 9 males (mean age 404 years) and11 females (mean age 341 years) without clinical complaintsand with no history of TB disease autoimmune disease andother infectious disease All controls were BCG vaccinated inchildhood and tuberculin skin test (TST) positive (indurationge 5mm) All patients and controls agreed to participate in thestudy after study clarification and written informed consent

22 Single Nucleotide Polymorphism (SNP) Genotyping Inthe current study seven SNPs were analyzed IFNG +874TAIFNG +2109AG IL12B +1188AC TNF minus308GC IL17Ars7747909 IL10 minus819CT and TGFB1 +29CT These poly-morphisms were selected after verifying their presence inhigh frequency (5) in the Brazilian population

For the current study 5mL of peripheral blood wasdrawn in an EDTA blood tube (by standard phlebotomyprocedures) from PTB patients (119899 = 31) and controls(119899 = 20) at base line (T

0) and genomic DNA was extracted

from leukocytes employing a DNAzol commercial reagent(Invitrogen Carlsbad CA USA) according to the manufac-turerrsquos instructionsQuantification andpurity of the extractedDNA were determined on a spectrophotometer (NanoDrop2000Thermo Fisher Scientific) Amplification of the genomicregions of interest was performed by PCR using 20 to 50 ngof DNA recombinant Taq DNA polymerase 02mM ofeach dNTP (deoxy-nucleotide-adenine guanine thymine orcytosine-triphosphate) 03 to 1mM concentration of eachof the specific primers appropriate buffer and ultrapurewater The IFNG +874TA was genotyped by PCR-ARMSas described by Pravica et al [20] The variation at IFNG+2109AG creates a restriction site for the enzyme AciI andwas genotyped by a previously reported PCR-RFLP method[21] The IL12B +1188 genotyping was performed using aPCR-RFLP method in accordance with Garcıa-Gonzalez etal [22] Fluorescence-based TaqMan technology (AppliedBiosystems Foster City CA USA) was applied to producegenotypes of IL17A and TGFB1 polymorphisms according tothemanufacturerrsquos instructionsThe TNF minus308AG genotypewas defined using a PCR-RFLP method in accordance withWilson et al [23]

23 Cytokines Gene Expression by Reverse Transcriptase RealTime PCR (RT-qPCR) To evaluate IFN-120574 IL-12 TNF-120572 IL-17 IL-10 and TGF-120573mRNA expressions 20mL of peripheralblood was drawn by a standard procedure in heparinizedblood tubes at a single time point from controls (119899 = 20) andat three serial time points for PTB patients (119899 = 31) basedon the antituberculosis treatment timeline (T1 T2 and T3)as previously defined Peripheral blood mononuclear cells(PBMCs) were isolated by a Histopaque gradient separationmethod [24] The layer rich in lymphocytes and monocyteswas aseptically removed and washed twice with PBS for15min at 1500 RPM The cell suspension was resuspended in1mL of PBS and the identification and viability of cells weredetermined by counting with Turk solution (50120583L aliquotsof cell suspension with 50 120583L of the dye solution at 5)
















3 Results




0

50

100

150

C T1 T2 T3

AAATTT

IFN

-120574(p

g120583

L) lowast

lowast

+874

(a)

C T1 T2 T30

1000

2000

3000

4000

AAATTT

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

lowast

lowast

+874

(b)









0

50

100

150+2109

C T1 T2 T3

AAAG

IFN

-120574(p

g120583

L) lowast

lowast

lowast

(a)

0

1000

2000

3000

4000

C T1 T2 T3

AAAG

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+2109

(b)


0

2

4

6

8

10

IL-12

(pg120583

L)

+1188

C T1 T2 T3

AAACCC

lowastlowast

(a)

0

1000

2000

3000

4000

5000

Gen

ic ex

pres

sion

IL-12120573

-act

in

C T1 T2 T3

AAACCC

lowast

+1188

(b)






0

50

100

150TN

F-120572

(pg120583

L)minus308

C T1 T2 T3

AGGG

lowast

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

TNF-120572

120573-a

ctin

AGGG

C T1 T2 T3

minus308

lowast

(b)


0

50

100

150

200

IL-1

7 (p

gm

L)

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

IL-17

120573-a

ctin

lowast

lowast

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(b)







0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)


lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)





lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results




0

50

100

150

C T1 T2 T3

AAATTT

IFN

-120574(p

g120583

L) lowast

lowast

+874

(a)

C T1 T2 T30

1000

2000

3000

4000

AAATTT

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

lowast

lowast

+874

(b)









0

50

100

150+2109

C T1 T2 T3

AAAG

IFN

-120574(p

g120583

L) lowast

lowast

lowast

(a)

0

1000

2000

3000

4000

C T1 T2 T3

AAAG

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+2109

(b)


0

2

4

6

8

10

IL-12

(pg120583

L)

+1188

C T1 T2 T3

AAACCC

lowastlowast

(a)

0

1000

2000

3000

4000

5000

Gen

ic ex

pres

sion

IL-12120573

-act

in

C T1 T2 T3

AAACCC

lowast

+1188

(b)






0

50

100

150TN

F-120572

(pg120583

L)minus308

C T1 T2 T3

AGGG

lowast

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

TNF-120572

120573-a

ctin

AGGG

C T1 T2 T3

minus308

lowast

(b)


0

50

100

150

200

IL-1

7 (p

gm

L)

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

IL-17

120573-a

ctin

lowast

lowast

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(b)







0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)


lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)





lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

C T1 T2 T3

AAATTT

IFN

-120574(p

g120583

L) lowast

lowast

+874

(a)

C T1 T2 T30

1000

2000

3000

4000

AAATTT

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

lowast

lowast

+874

(b)









0

50

100

150+2109

C T1 T2 T3

AAAG

IFN

-120574(p

g120583

L) lowast

lowast

lowast

(a)

0

1000

2000

3000

4000

C T1 T2 T3

AAAG

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+2109

(b)


0

2

4

6

8

10

IL-12

(pg120583

L)

+1188

C T1 T2 T3

AAACCC

lowastlowast

(a)

0

1000

2000

3000

4000

5000

Gen

ic ex

pres

sion

IL-12120573

-act

in

C T1 T2 T3

AAACCC

lowast

+1188

(b)






0

50

100

150TN

F-120572

(pg120583

L)minus308

C T1 T2 T3

AGGG

lowast

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

TNF-120572

120573-a

ctin

AGGG

C T1 T2 T3

minus308

lowast

(b)


0

50

100

150

200

IL-1

7 (p

gm

L)

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

IL-17

120573-a

ctin

lowast

lowast

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(b)







0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)


lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)





lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150+2109

C T1 T2 T3

AAAG

IFN

-120574(p

g120583

L) lowast

lowast

lowast

(a)

0

1000

2000

3000

4000

C T1 T2 T3

AAAG

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+2109

(b)


0

2

4

6

8

10

IL-12

(pg120583

L)

+1188

C T1 T2 T3

AAACCC

lowastlowast

(a)

0

1000

2000

3000

4000

5000

Gen

ic ex

pres

sion

IL-12120573

-act

in

C T1 T2 T3

AAACCC

lowast

+1188

(b)






0

50

100

150TN

F-120572

(pg120583

L)minus308

C T1 T2 T3

AGGG

lowast

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

TNF-120572

120573-a

ctin

AGGG

C T1 T2 T3

minus308

lowast

(b)


0

50

100

150

200

IL-1

7 (p

gm

L)

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

IL-17

120573-a

ctin

lowast

lowast

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(b)







0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)


lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)





lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150TN

F-120572

(pg120583

L)minus308

C T1 T2 T3

AGGG

lowast

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

TNF-120572

120573-a

ctin

AGGG

C T1 T2 T3

minus308

lowast

(b)


0

50

100

150

200

IL-1

7 (p

gm

L)

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(a)

0

500

1000

1500

Gen

ic ex

pres

sion

IL-17

120573-a

ctin

lowast

lowast

lowast

C T1 T2 T3

AAGAGG

rs 7747909

(b)







0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)


lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)





lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

IL-10

(pg120583

L)

lowast

lowastlowast

C T1 T2 T3

CCTC TT

minus819

(a)

0

500

1000

1500

2000

Gen

ic ex

pres

sion

IL-10120573

-act

in

C T1 T2 T3

CCTC TT

minus819

(b)


lowast

C T1 T2 T30

500

1000

1500

TGF-120573

(pg

mL)

CCCTTT

+29

(a)

CCCTTT

0

1000

2000

3000

4000

Gen

ic ex

pres

sion

TGF-120573

120573-a

ctin

+29

C T1 T2 T3

(b)





lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowast

+874

IFN

-120574(p

g120583

L)

AA ATTT0

50

100

150

(a)

lowast

AA ATTT0

1000

2000

3000

4000

Gen

ic ex

pres

sion

IFN

-120574120573

-act

in

+874

(b)

lowast

lowast

+2109

IFN

-120574(p

g120583

L)

AA AG0

50

100

150

(c)

lowast

minus308

TNF-120572

(pg120583

L)

AG GG0

10

20

30

40

(d)

CC CTTT0

200

400

600

800

1000

T2T3

T1

TGF-120573

(pg

mL)

+29

lowast

(e)




4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Discussion



























5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































5 Conclusion




Acknowledgment


References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion




Acknowledgment


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





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Clinical Study Cytokine Polymorphisms, Their Influence and