Immunohistochemical
analysis
of
FoxP3
+
cells
in
periapical
granulomas
and
radicular
cysts
Raniel
Fernandes
Peixoto
a,
Joabe
dos
Santos
Pereira
b,
Cassiano
Francisco
Weege
Nonaka
c,
E´ricka
Janine
Dantas
da
Silveira
b,
Ma´rcia
Cristina
da
Costa
Miguel
b,*
a
FederalUniversityofRioGrandedoNorte,Natal,RN,Brazil
b
OralPathologyPostgraduateProgram,DepartmentofDentistry,FederalUniversityofRioGrandedoNorte,Natal,RN,Brazil
cOralPathology,DepartmentofDentistry,StateUniversityofParaı´ba,CampinaGrande,PB,Brazil
1.
Introduction
Periapicallesions,whichincludeperiapicalgranulomas(PGs) andradicularcysts(RCs),canariseafterpulpnecrosisasa consequenceoftheimmuneresponsetocontinuousantigen stimulationbymicroorganismspresentintherootcanals.1–3
Persistentinflammationattheperiapicaltissuesisassociated with resorption of adjacent bone, which is replaced with fibrovascularconnectivetissue,formingaPG.Thelatterisa
secondaryanddefensivereactionofthehostinanattemptto control progressionof the infectious process.4 As a
conse-quenceoftheseimmunopathologicalresponses,theepithelial restsofMalassezarestimulatedtoproliferate,aneventthat canresultinthedevelopmentofaRC.2,5
Factorssuchastheamountandtypeofcytokines,antigen nature and the adequate expression of receptors and costimulatory molecules, as well as the type of antigen-presentingcellsandgeneticconstitutionofthehost,modulate theprogressionandseverityofinflammatoryprocessesand
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Accepted3February2012 Keywords: Periapicalgranuloma Radicularcyst ForkheadboxP3 RegulatoryTcells Immunohistochemistrya
b
s
t
r
a
c
t
Objectives: TocomparethenumberofFoxP3+cellsbetweenperiapicalgranulomas(PGs)and
radicularcysts(RCs),andtocorrelatethisnumberwiththeintensityoftheinflammatory infiltrateintheselesionsandwithepithelialthicknessofRCs.
Studydesign: ThirtyPGsand30RCsweresubmittedtoimmunohistochemicalanalysisusing ananti-FoxP3polyclonalantibody.FoxP3+cellswerecountedundera lightmicroscope
(400magnification)infivefieldsandthemeanvaluewascalculatedforeachspecimen. StatisticaltestswereusedtoevaluatedifferencesinthenumberofFoxP3+cellsaccordingto
typeoflesion(PGvs.RC),intensityoftheinflammatoryinfiltrate(gradeI/IIvs.gradeIII),and epithelialthicknessofRCs(atrophicvs.hyperplastic).
Results: FoxP3+cellsweredetectedinmostPGs(93.3%)andRCs(93.3%).Themediannumber
ofFoxP3+cellswas2.40inPGsand 1.00in RCs,with thisdifferencebeingstatistically
significant(P=0.005).NosignificantdifferencesinthenumberofFoxP3+cellswereobserved
intermsoftheintensityoftheinflammatoryinfiltrate(P=0.465)orepithelialthicknessof RCs(P=0.737).
Conclusions: ThepresentresultssuggestagreaterparticipationofregulatoryTcellsinthe modulationoftheinflammatoryresponseinPGs.Inaddition,thepresenceofalesseffective regulatoryenvironmentinRCs,togetherwiththehighlevelsofinflammatorymediatorsas reportedintheliterature,maycontributetothegreatergrowthpotentialoftheselesions.
#2012ElsevierLtd.
*Correspondingauthorat:UniversidadeFederaldoRioGrandedoNorte,DepartamentodeOdontologia,ProgramadePo´s-Graduac¸a˜oem PatologiaOral,Av.SenadorSalgadoFilho,1787LagoaNova,Natal,RN,Brazil,CEP59056-000.Tel.:+558432154138;fax:+558432154138.
E-mailaddress:[email protected](M.C.daC.Miguel).
Available
online
at
www.sciencedirect.com
journalhomepage:http://www.elsevier.com/locate/aob
0003–9969 #2012ElsevierLtd. doi:10.1016/j.archoralbio.2012.02.005
Open access under the Elsevier OA license.
influencethe typeofhostresponse(T-helper1[Th1]or T-helper2[Th2]).6,7AlthoughTh1/Th2responsesareinduced by cytokines, the two types of effector responses are regulated by a heterogeneous family of cells, known as regulatoryT(Treg)cells.Thesecellsplayamajorroleinthe modulation of immune responses, induction and mainte-nanceofimmunetolerance,andalsointhe preventionof autoimmunediseases.4,8,9ThemechanismofactionofTreg cellsinvolvesdirectsuppressionoftheactivationofTandB lymphocytesand naturalkiller(NK) cellsthrough cellular mechanisms mediated by surface molecules such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), or by the synthesis of immunosuppressive cytokines such as interleukin 10 (IL-10) and transforming growth factor beta (TGF-b).9–13 At present, the forkhead box P3
(FoxP3) transcription factor is the best and most reliable immunohistochemicalmarkerforTregcells.14–17Thebasic
functionofthisproteinistoregulatethe activityofthese cells.18–20
Studies investigatingthe presenceand function ofTreg cells in periapical lesions are scarce in the literature. Therefore,theobjectiveofthepresentstudywastocompare the number of FoxP3+ cells between PGs and RCs, and to
correlatethisnumberwiththeintensityoftheinflammatory infiltrateintheselesionsandwithepithelialthicknessofRCs. Thesedatashouldcontributetoabetterunderstandingofthe immunopathogenesis and the consequent progressionand severityofperiapicallesions.
2.
Materials
and
methods
Sixtyspecimens,including30PGsand30RCs,obtainedfrom theOralPathologyDepartmentoftheFederalUniversityofRio GrandedoNorte(UFRN)wererandomlyselectedforthisstudy. All PGs andRCs wereobtained fromhuman teethwithout endodontictreatment.Pathologicaldiagnoseswereconfirmed onthebasisofclinical,radiographicandhistologicalcriteria. Only PGs without odontogenic epithelium were selected. Moreover,allRCspresentedunequivocalcysticcavitylinedby odontogenicepithelium.Thecaseswerenotmatchedforage, gender,oranatomiclocation.Thestudywasapprovedbythe ResearchEthics CommitteeofUFRN, Natal,Brazil(protocol number:056/10).
2.1. Morphologicalanalysis
Forthemorphological analysis,5-mm-thicktissuesections werestainedwithhaematoxylinandeosin.Theintensityof the inflammatoryinfiltrate was evaluatedaccording to an adaptationofthemethodproposedbyTsaietal.21Grading of each specimen was recorded on the inflammatory condition in 1 microscopic field, starting from the inner portion of the specimen and proceeding deeper into connective tissue. Briefly, each specimen was graded at 200 magnification as: grade I, inflammatory cells less thanone-third; grade II,inflammatory cellsbetween one-third and two-thirds; and grade III, inflammatory cells more than two-thirds. Analysis of the thickness of the epithelial liningin RCswas performed consideringallthe
cystlining.Thethicknessoftheepithelialliningwasdefined as atrophic (2–10cell layers and flat epithelial/capsule boundary) or hyperplastic (>10cell layers and undulating epithelial/capsuleboundary,oftenarrangedinto proliferat-ing arcades)22 based on the predominant pattern in each
case.
2.2. Immunohistochemicalmethods
For the immunohistochemical study, 3-mm-thick sections were obtained from paraffin-embedded tissue blocks. The tissue sections were deparaffinized and immersed in 3% hydrogenperoxidetoblockendogenousperoxidaseactivity. AntigenretrievalwasperformedinaPascalpressurecooker with citrate buffer, pH 6.0. After treatment with normal serum,the sectionswereincubatedwiththeprimary anti-FoxP3polyclonalantibodydiluted1:150(cloneH190,Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 18h. The tissuesectionswerethenwashedtwiceinPBSandtreated with immunoperoxidase-basedkit(EnVisionTM+DualLink
System-HRP;Dako,Carpinteria,CA,USA)atroom tempera-ture to bind the primary antibody. Peroxidase activity was visualized by immersing tissue sections in diamino-benzidine (Liquid DAB+Substrate; Dako, Carpinteria, CA, USA), resulting in a brown reaction product. Finally, the tissuesectionswerecounterstainedwithMayer haematox-ylin and coverslipped. Sections of normal human tonsil wereusedaspositivecontrolforFoxP3.Asnegativecontrol, samples were treated as earlier, except that the primary antibody was replaced by a solution of bovine serum albumininPBS.
2.3. Immunostainingassessmentandstatisticalanalysis
After the immunohistochemicaltreatment, the tissue sec-tions were examined by two observers under a light microscope (Olympus CX31, Olympus Japan Co., Tokyo, Japan).Using100magnification,fivefieldswiththelargest number of immunostained cells were identified. In these fields,FoxP3+cellswerecountedat400magnificationand the totalnumber ofpositivecellsper casewascalculated, permittingthecalculationofthemeannumberofFoxP3+cells
perspecimen.
Theresultsobtainedweresubmittedtostatisticalanalysis. ComputationsweremadeusingtheStatisticalPackageforthe SocialSciences(version17.0;SPSSInc.,Chicago,IL,USA).For theintensityoftheinflammatoryinfiltrate,lesionsshowing inflammatoryinfiltrategradesIandIIwerecombinedintoone group and compared with those presenting inflammatory infiltrate grade III. The Kolmogorov–Smirnov test revealed absence ofanormaldistributionforthe number ofFoxP3+ cells. Therefore, to compare the number of FoxP3+ cells
according totype of lesion (PGs vs. RCs), thickness of the epithelial lining(atrophicvs.hyperplastic), andintensity of inflammatory infiltrate(grades I/IIvs. gradeIII),theMann– Whitney non-parametric test was performed. Spearman’s correlationtestwasperformedtoverifypossiblecorrelations betweenthenumberofFoxP3+cellsandtheintensityofthe
inflammatoryinfiltrate.Foralltests,significancelevelwasset at5%(P<0.05).
3.
Results
3.1. Morphologicalanalysis
Analysis of the inflammatory infiltrate in PGs revealed all cases(n=30;100%)withinflammatoryinfiltrategradeIII.In RCs,16cases(53.3%)showedinflammatoryinfiltrategradeIII, 13cases(43.3%)inflammatoryinfiltrategradeII,andonly1 case (3.3%) inflammatory infiltrate grade I. Morphological analysis of the epithelial thickness in RCs revealed the presenceofanatrophicepitheliumin25cases(83.3%)anda hyperplasticepitheliuminonly5cases(16.7%).
3.2. Immunohistochemicalanalysis
Analysis of the immunohistochemical expression of anti-FoxP3antibodyshowedthepresenceofFoxP3+cellsin28PGs
(93.3%)andin28RCs(93.3%).ThemediannumberofFoxP3+ cellswas2.40(0.00–9.60)inPGs(Fig.1A)and1.00(0.00–5.00)in RCs (Fig. 1B).The non-parametric Mann–Whitney test dis-closed a statistically significant higher median number of FoxP3+cellsinPGswhencomparedtoRCs(P=0.005)(Table1).
SincenocaseofPGwithinflammatoryinfiltrategradeIorII wasdetectedinthisstudy,analysisofthenumberofFoxP3+
cellsaccordingtointensityoftheinflammatoryinfiltratewas performedonlyinRCs.ThemediannumberofFoxP3+cells was 1.20 (0.00–5.00)in lesions with inflammatory infiltrate grade III (Fig. 2A) and 1.00 (0.20–4.40) in lesions with inflammatory infiltrate grade I/II (Fig. 2B). Comparison of themediannumberofFoxP3+cellsinrelationtotheintensity
oftheinflammatoryinfiltraterevealednostatistically signifi-cant difference between groups (P=0.465) (Table 1). In addition,Spearman’scorrelationtestdisclosednosignificant correlation between the number of FoxP3+ cells and the
intensityoftheinflammatoryinfiltrate(r= 0.090;P=0.635). Withrespecttothethicknessoftheepitheliallining,the mediannumberofFoxP3+cellswas1.40(0.20–2.80)inlesions with hyperplasticepithelium and1.00 (0.00–5.00)inlesions withatrophicepithelium.Thenon-parametricMann–Whitney test revealed no statistically significant differences in the mediannumber ofFoxP3+ cells betweenRCswithatrophic
Fig.1–(A)PresenceofFoxP3+cells(arrows)inPG(EnVision method,T400).(B)PhotomicrographshowingFoxP3+cells (arrows)inRC(EnVisionmethod,T400).
Fig.2–(A)PresenceofFoxP3+cells(arrows)inRCwith
inflammatoryinfiltrategradeIII(EnVisionmethod,T400). (B)PhotomicrographshowingFoxP3+cells(arrows)inRC withinflammatoryinfiltrategradeI(EnVisionmethod, T400).
epitheliumandRCswithhyperplastic epithelium(P=0.737) (Table1).
4.
Discussion
Periapicallesionsdevelopinresponsetochronicantigenic stimulationofperiapicaltissuecausedbybacterialinfection in root canal.13 As a consequence, innate and acquired
defencemechanismsareactivated,whichcomprisevascular andcellulareventsthatculminateinalveolarbone resorp-tionintheapexofthetooth.23Differentcellsareinvolvedin
thisprocess,includingneutrophils,macrophages,NKcells, Langerhanscells,andTandBlymphocytes,aswellasTreg cellswhichregulatetheimmuneresponse.3,24However,the
roleofTregcellsinthemodulationoftheimmuneresponse in PGs and RCs has not been fully established.9,13,19
According to Salomon et al.25 a combination of strong
antigen signalsand maximum costimulation isnecessary forthe development ofTregcells.This conditioniseasily found in periapical lesions due to the strong antigen stimulation arising from the root canals,1,5,9,13,19 a fact
explainingthepresenceofFoxP3+ cellsinmostPG(93.3%)
andRC(93.3%)specimensstudiedhere.
The present immunohistochemical study evaluated the numberofFoxP3+cellsinPGsandRCs.Theresultsshoweda significantlylargernumberofFoxP3+cellsinPGscomparedto RCs (P=0.005). Similarly, Fukada et al.9 investigating the
mRNAexpressionofFoxP3andofcytokinesinvolvedinTh1 (T-bet, interferon-g [IFN-g]) and Th2 (GATA-3, IL-4, IL-10) responsesbyrealtime-PCRinRCsandPGs,observedhigher expressionofFoxP3andIL-10inthelatter.Accordingtothese authors,Tregcellsplayanimportantroleinthemodulationof the inflammatory microenvironment of periapical lesions. The authors suggested a possible suppression of the Th1 responseinPGs.Ontheotherhand,Marc¸aletal.3evaluating
thenumberofFoxP3+cellsinRCsandPGs,foundnosignificant differencesbetweentheselesions.
Proinflammatory cytokines such as IFN-g and tumour necrosisfactoralpha(TNF-a)regulateTh1responses,whereas IL-4 is the key cytokine involved in Th2responses. These mediatorstriggerinflammatoryresponsesthatresultinthe progression of periapical lesions.3,23,26 These immune responses are modulated by FoxP3+ cells through direct
cellularmechanisms,mediatedbyCTLA-4,7,11,18,27orthrough
indirectmechanisms,mediatedbythesynthesisofTGF-band IL-10.4,7,9,13,27,28 In a comparative study of PGs and RCs,
Teixeira-Salum et al.4 detectedhigher expressionof TGF-b
inPGs,andofIL-4,IFN-gandTNF-ainRCs.Takentogether,the results reportedby theseauthors and the present findings suggestamoreeffectiveparticipation ofFoxP3+cellsinthe
modulationofinflammatoryresponsesinPGs,whichcanbe explained by the high expression of TGF-b, a molecule suppressingTh1and Th2responses,and bythe low levels ofproinflammatorymediatorsregulatingTh1(IFN-gand TNF-a)andTh2(IL-4)responsesintheselesions.Alsowithinthis context, the smaller number of FoxP3+ cells in RCs in
comparisonwithPGs,observedinthepresentstudy,together withthehighlevelsofinflammatorymediatorsandlowlevels ofTGF-bintheselesionsreportedinotherstudies,3,4maybe
relatedtotheexistenceofaminimalregulatoryenvironment inRCsthatmaycontributetothegreatergrowthpotentialof theselesions.2,29–31
FoxP3+ cells have been studied in diseases of different
aetiology,suchasrheumatoidarthritis,32atopicdermatitis,33
lichenplanus,34andintestinalinflammatorydisease.35Inan
animal modelof experimental periodontitis, Garlet et al.27
observedincreasedlevelsofTregcellsandhighexpressionof immunosuppressivecytokines(CTLA-4,TGF-b,andIL-10).The authorsalsofoundthatanimalstreatedwith anti-glucocorti-coid-inducible TNFreceptor(GITR),aninhibitorofTregcell function, exhibited a marked increase in bone loss and inflammatory cell migration, as well as a decrease in the expression ofimmunosuppressive cytokines. In agreement with these findings, Nakajima et al.8 suggested that in
gingivitisTregcellscontroltheimmunopathologicalprocess by preventing the destruction ofperiodontal bone. On the other hand, in established periodontitis Treg cells are recruited to suppress bone destruction through putative autoimmunemechanismsviaanegativefeedbacksystem.8
Ithasbeensuggestedthatthehighertheintensityofthe inflammatory infiltrate, the higher the antigen stimulation andconsequenttissuedamage.36Ontheotherhand,immune
surveillance by Treg cells would limit the tissue damage provoked by the host immune responseand by the direct actionofthemicroorganisms.27,37Despitethosereports,inthe
present study, no significant difference in the number of FoxP3+ cells was observedin terms ofthe intensity ofthe
inflammatory infiltrate of RCs (P=0.465). In addition, no significant correlation was found between the number of FoxP3+cellsandtheintensityoftheinflammatory infiltrate
(P=0.635).Thus,immunesurveillancebyTregcellsdoesnot seemtobemoreeffectiveinRCswithintenseinflammatory infiltrates.
Table1–MediannumberofFoxP3+cellsinperiapicalgranulomasandradicularcysts,andtheirdifferencesinrelationto
typeoflesion,gradeofinflammatoryinfiltrateandepithelialthickness.
Variable n Median Q25–Q75 Meanofranks Sumofranks U P Lesion Periapicalgranuloma 30 2.40 1.55–5.05 36.85 1105.50 259.50 0.005 Radicularcyst 30 1.00 0.55–2.80 24.15 724.50 Inflammatoryinfiltrate GradeI/II 14 1.00 0.60–3.05 16.75 234.50 94.50 0.465 GradeIII 16 1.20 0.25–2.35 14.41 230.50 Epithelialthickness Atrophic 25 1.00 0.60–2.90 15.74 393.50 56.50 0.737 Hyperplastic 5 1.40 0.20–2.60 14.30 71.50
The status ofthe lining epithelium (atrophic or hyper-plastic) has been described as a histological parameter indicating growth activity/inactivity in RCs.22 In quiescent
lesions (atrophicepithelium), no cystic growthis observed despitethepresenceofantigensandenzymesabletoinduce immuneresponses,epithelialproliferation,andbone resorp-tion.22Duringthisphase,thegrowthofRCsisprobablylimited byimmunosuppressiveresponses.22Nevertheless,no signifi-cantdifferenceinthenumberofFoxP3+ cellswasobserved
between RCs with hyperplastic and atrophic epithelium (P=0.735). In view of the small number of RCs with hyperplasticepithelium(n=5) inthe presentstudy,further investigationsareneededtoidentifypossibledifferencesin theimmuneresponsebetweenactive(hyperplastic epitheli-um)andquiescent(atrophicepithelium)RCs.
In conclusion, the present results demonstrated the presenceofFoxP3+ cellsininflammatory periapical lesions
andalargernumberofthesecellsinPGscomparedtoRCs, suggesting a greater participation of Treg cells in the modulationoftheinflammatoryresponseintheformer.In addition, the number of FoxP3+ cells was not intimately
correlatedwiththeintensityoftheinflammatoryinfiltrateor with the thickness of the lining epithelium in RCs. It is suggested that the presence of a less effective regulatory environmentinRCs,associatedwithhighlevelsof inflamma-torymediatorsasreportedintheliterature,contributetothe greatergrowthpotentialoftheselesions.
Funding
Nonedeclared.
Competing
interests
Nonedeclared.
Ethical
approval
TheethicalapprovalforourresearchwasgivenbytheEthics CommitteeoftheFederalUniversityofRioGrandedoNorte, Brazil(protocolnumber:056/10).
r
e
f
e
r
e
n
c
e
s
1. Garcı´aCC,SempereFV,DiagoMP,BowenEM.The post-endodonticperiapicallesion:histologicandetiopathogenic aspects.MedOralPatolOralCirBucal2007;12:e585–90. 2. NonakaCF,MaiaAP,NascimentoJG,deAlmeidaFreitasR,
BatistadeSouzaL,Galva˜oHC.Immunoexpressionofvascular endothelialgrowthfactorinperiapicalgranulomas,radicular cysts,andresidualradicularcysts.OralSurgOralMedOral PatholOralRadiolEndod2008;106:896–902.
3. Marc¸alJR,SamuelRO,FernandesD,deAraujoMS, NapimogaMH,PereiraSA,etal.T-helpercelltype17/ regulatoryT-cellimmunoregulatorybalanceinhuman radicularcystsandperiapicalgranulomas.JEndod 2010;36:995–9.
4. Teixeira-SalumTB,RodriguesDB,Gerva´sioAM,SouzaCJ, RodriguesJrV,LoyolaAM.DistinctTh1,Th2andTreg cytokinesbalanceinchronicperiapicalgranulomasand radicularcysts.JOralPatholMed2010;39:250–6.
5. MuglaliM,KomerikN,BulutE,YarimGF,CelebiN,SumerM. Cytokineandchemokinelevelsinradicularandresidual cysts.JOralPatholMed2008;37:185–9.
6. TengYT.Theroleofacquiredimmunityandperiodontal diseaseprogression.CritRevOralBiolMed2003;14:237–52. 7. GemmellE,SeymourGJ.ImmunoregulatorycontrolofTh1/
Th2cytokineprofilesinperiodontaldisease.Periodontol2000 2004;35:21–41.
8. NakajimaT,Ueki-MaruyamaK,OdaT,OhsawaY,ItoH, SeymourJG,etal.RegulatoryT-cellsinfiltrateperiodontal diseasetissues.JDentRes2005;84:639–43.
9. FukadaSY,SilvaTA,GarletGP,RosaAL,daSilvaJS,Cunha FQ.FactorsinvolvedintheThelpertype1andtype2cell commitmentandosteoclastregulationininflammatory apicaldiseases.OralMicrobiolImmunol2009;24:25–31. 10.Mittru¨ckerHW,KaufmannSH.Mini-review:regulatoryT
cellsandinfection:suppressionrevisited.EurJImmunol 2004;34:306–12.
11.CollisonLW,WorkmanCJ,KuoTT,BoydK,WangY,Vignali KM,etal.TheinhibitorycytokineIL-35contributesto regulatoryTcellfunction.Nature2007;450:566–9. 12.ShevachEM,TranDQ,DavidsonTS,AnderssonJ.The
criticalcontributionofTGF-betatotheinductionofFoxp3 expressionandregulatoryTcellfunction.EurJImmunol 2008;38:915–7.
13.Colic´ M,GazivodaD,Vucevic´ D,Majstorovic´ I,Vasilijic´ S, RudolfR,etal.RegulatoryT-cellsinperiapicallesions.JDent Res2009;88:997–1002.
14.YagiH,NomuraT,NakamuraK,YamazakiS,KitawakiT, HoriS,etal.CrucialroleofFOXP3inthedevelopmentand functionofhumanCD25+CD4+regulatoryTcells.Int Immunol2004;16:1643–56.
15.SchreiberTH.TheuseofFoxP3asabiomarkerand prognosticfactorformalignanthumantumors.Cancer EpidemiolBiomarkersPrev2007;16:1931–4.
16.SolomonGJ,MagroCM.Foxp3expressionincutaneous T-celllymphocyticinfiltrates.JCutanPathol2008;35:1032–9. 17.WadaDA,WilcoxRA,WeenigRH,GibsonLE.Paucityof
intraepidermalFoxP3-positiveTcellsincutaneousT-cell lymphomaincontrastwithspongioticandlichenoid dermatitis.JCutanPathol2010;37:535–41.
18.FontenotJD,RudenskyAY.Awelladaptedregulatory contrivance:regulatoryTcelldevelopmentandthe forkheadfamilytranscriptionfactorFoxp3.NatImmunol 2005;6:331–7.
19.AlshwaimiE,PurcellP,KawaiT,SasakiH,OukkaM, Campos-NetoA,etal.RegulatoryTcellsinmouseperiapical lesions.JEndod2009;35:1229–33.
20.WingK,SakaguchiS.RegulatoryTcellsexertchecksand balancesonselftoleranceandautoimmunity.NatImmunol 2007;11:7–13.
21.TsaiCH,WengSF,YangLC,HuangFM,ChenYJ,ChangYC. Immunohistochemicallocalizationoftissue-type
plasminogenactivatorandtypeIplasminogenactivator inhibitorinradicularcysts.JOralPatholMed2004;33:156–61. 22.MoreiraPR,SantosDF,MartinsRD,GomezRS.CD57+cellsin
radicularcyst.IntEndodJ2000;33:99–102.
23.RochaDAP,PereiraKMA,Gordo´n-Nu´n˜ezMA,CarvalhoRA, Galva˜oHC,CostaALL.Formacio´ndelosgranulomasy quistesradiculares:unarevisio´ndelosaspectos inmunopatolo´gicos.RevADM2007;64:91–6.
24.VignaliDA,CollisonLW,WorkmanCJ.HowregulatoryT cellswork.NatRevImmunol2008;8:523–32.
25.SalomonB,LenschowDJ,RheeL,AshourianN,SinghB, SharpeA,etal.B7/CD28costimulationisessentialforthe
homeostasisoftheCD4+CD25+immunoregulatoryTcells thatcontrolautoimmunediabetes.Immunity2000;12: 431–40.
26.JankovicD,LiuZ,GauseWC.Th1-andTh2-cell commitmentduringinfectiousdisease:asymmetryin divergentpathways.TrendsImmunol2001;22:450–7. 27.GarletGP,CardosoCR,MarianoFS,ClaudinoM,deAssisGF,
CampanelliAP,etal.RegulatoryTcellsattenuate experimentalperiodontitisprogressioninmice.JClin Periodontol2010;37:591–600.
28.WorkmanCJ,Szymczak-WorkmanAL,CollisonLW,Pillai MR,VignaliDA.Thedevelopmentandfunctionof regulatoryTcells.CellMolLifeSci2009;66:2603–22. 29. WhiteSC,SappJP,SetoBG,MankovichNJ.Absenceof
radiometricdifferentiationbetweenperiapicalcystsand granulomas.OralSurgOralMedOralPathol1994;78: 650–4.
30.SilvaTA,GarletGP,LaraVS,MartinsJrW,SilvaJS,Cunha FQ.Differentialexpressionofchemokinesandchemokine receptorsininflammatoryperiapicaldiseases.OralMicrobiol Immunol2005;20:310–6.
31.CarrilloC,PennarochaM,OrtegaB,Martı´E,Baga´nJV,VeraF. Correlationofradiographicsizeandthepresenceof radiopaquelaminawithhistologicalfindingsin70 periapicallesions.JOralMaxillofacSurg2008;66:1600–5.
32.SuzukiK,SetoyamaY,YoshimotoK,TsuzakaK,AbeT, TakeuchiT.DecreasedmRNAexpressionoftwoFOXP3 isoformsinperipheralbloodmononuclearcellsfrom patientswithrheumatoidarthritisandsystemiclupus erythematosus.IntJImmunopatholPharmacol2011;24:7–14. 33.HayashidaS,UchiH,MoroiY,FurueM.Decreasein
circulatingTh17cellscorrelateswithincreasedlevelsof CCL17,IgEandeosinophilsinatopicdermatitis.JDermatol Sci2011;61:180–6.
34.TaoXA,XiaJ,ChenXB,WangH,DaiYH,RhodusNL,etal. FOXP3Tregulatorycellsinlesionsoforallichenplanus correlatedwithdiseaseactivity.OralDis2010;16:76–82. 35.VeltkampC,AnstaettM,WahlK,Mo¨llerS,GanglS,
BachmannO,etal.ApoptosisofregulatoryTlymphocytesis increasedinchronicinflammatoryboweldiseaseand reversedbyanti-TNFatreatment.Gut2011;60:1345–53. 36.SantosLC,RamosEA,GurgelCA,deSantanaEJ,dosSantos
JN.ImmunohistochemicaldetectionofLangerhanscellsin dentalgranulomasandradicularcysts.JMolHistol 2007;38:201–5.
37.GuilliamsM,OldenhoveG,NoelW,He´rinM,BrysL,LoiP, etal.Africantrypanosomiasis:naturallyoccurring regulatoryTcellsfavortrypanotolerancebylimiting pathologyassociatedwithsustainedtype1inflammation.J Immunol2007;179:2748–57.
