QUESTIONÁRIO CLÍNICO: - PSYCHOSOCIAL STRESS IN TYPE 1 BIPOLAR DISORDER

PSYCHOSOCIAL STRESS IN TYPE 1 BIPOLAR DISORDER

6. QUESTIONÁRIO CLÍNICO:

6.1. A Sra. possui diagnóstico de alguma das seguintes doenças?

Alzheimer ( ) Sim ( ) Não

Anemia hemolítica

( ) Sim ( ) Não

Artrite reumatoide

( ) Sim ( ) Não

115

Diabete tipo 1 (insulino‐dependente) ( ) Sim ( ) Não

Doença Cardiovascular ( ) Sim ( ) Não

Doença da tireóide

( ) Sim ( ) Não

Doença inflamatória do intestino

( ) Sim ( ) Não

Esclerodermia

( ) Sim ( ) Não

Gota ou outra artropatia induzida por cristais

( ) Sim ( ) Não

Hipertensão

( ) Sim ( ) Não

Lúpus eritematoso sistêmico

( ) Sim ( ) Não

Miastenia gravis

( ) Sim ( ) Não

Miosite

( ) Sim ( ) Não

Neoplasia maligna ( ) Sim ( ) Não

Parkinson

( ) Sim ( ) Não

Rinite alérgica ( ) Sim ( ) Não

Vasculite ( ) Sim ( ) Não 6.2. A Sra está tratando alguma doença infecciosa no momento? ( ) Sim ( ) Não 6.3. A Sra esteve tratando alguma doença infecciosa nas últimas duas semanas? ( ) Sim ( ) Não 6.4. A Sra possui alguma doença infecciosa crônica? ( ) Sim ( ) Não

116 6.5. A Sra faz uso de alguma das seguintes medicações? 6.6. Outros diagnósticos clínicos conhecidos: a) __________________________________________________________ b) __________________________________________________________ c) __________________________________________________________ 6.7. Outros medicamentos em uso pela paciente:

Hormônios (da tireóide, TRH) ( ) Sim ( ) Não

Anticoagulantes (Marcoumar ou Marevan) ( ) Sim ( ) Não Imunossupressores (Azatioprina, Metotrexato, Ciclosporina,

Etanercept, Leflunomida, outros) ( ) Sim ( ) Não

Glicocorticóides (Prednisona, Fludrocortisona, Prednisolona,

Dexametasona, Hidrocortisona, outros) ( ) Sim ( ) Não

Nome Farmacológico Iníc io mg / dia Suspensão

117 7. Exame físico: a. Dados antropomórficos: Peso Kg Altura cm IMC b. Sinais Vitais: FC bpm PA (1) mmHg PA (2) mmHg PA (média) mmHg 8. Exames Laboratoriais:

118 Análise de Urina (fita) Proteinúria ( ) sim ( ) não Glicosúria ( ) sim ( ) não ITU ( ) sim ( ) não Análise de sangue ‐ Laboratório Hemograma Hct % Hb g/dL VCM fL CHCM g/dL Leucócitos /µL TGO (AST) U/L TGP (ALT) U/L Glicemia mg/dL Creatinina mg/dL

119

120 10.

Anexo II – Demais produções do doutorado

1 0 . 1 . Artigo Científico

121

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Pleasecitethisarticleinpressas:doPrado,C.H.,etal.,ReducedregulatoryT cellsareassociatedwithhigherlevelsofTh1/TH17cytokines andactivatedMAPKintype1bipolardisorder.Psychoneuroendocrinology(2012),http://dx.doi.org/10.1016/j.psyneuen.2012.08.005

Reduced

regulatory

T

cells

are

associated

with

higher

levels

of

Th1/TH17

cytokines

and

activated

MAPK

in

type

1 bipolar

disorder

Carine

Hartmann

do

Prado

a,1

,

Lucas

Bortolotto

Rizzo

a,1

,

Andre´a

Wieck

,

Rodrigo

Pestana

Lopes

,

Antonio

L. Teixeira

,

Rodrigo

Grassi-Oliveira

,

Moise´s

Evandro

Bauer

a,e,

*

a_Laboratory_of_{Immunosenescence,}_Institute_of_Biomedical_Research,_Pontifical_Catholic_University

oftheRioGrandedoSul(PUCRS),PortoAlegre,Brazil

b_BD_Biosciences,_Sa

˜oPaulo,Brazil

c_Department_of_Internal_Medicine,_School_of_Medicine,_UFMG,_Belo_Horizonte,_Brazil

d_Faculty_of_Psychology,_PUCRS,_Porto_Alegre,_Brazil

e_Faculty_of_Biosciences,_PUCRS,_Porto_Alegre,_Brazil

Received17April2012;receivedinrevisedform16August2012;accepted16August2012

Psychoneuroendocrinology(2012)xxx,xxx—xxx KEYWORDS Bipolardisorder; Cytokines; Tcells; Lymphocytes; MAPK; RegulatoryTcells

Summary Bipolardisorder(BD)hasbeenassociatedwithanimmunologicimbalanceshownby

increasedperipheralinflammatorymarkers.Theunderlyingmechanismsofthisphenomenonmay

include changes in circulating cells and differential activation of mitogen-activated protein

kinases(MAPKs).Twenty-seven euthymicfemalesubjectswithBDtype I(allmedicated) and

24age-andsex-matchedcontrolswererecruitedinthisstudy.Lymphocyteswereisolatedand

stimulatedinvitrotoassessTh1/Th17/Th2cytokines(IL-2,IL-4,IL-6,IL-10,IL-17,IFN-gandTNF-

a)andMAPKphosphorylation.Theexpressionofphospho-MAPKs,alargepaneloflymphocyte

subsetsandcytokineswereassessed bymulti-colorflowcytometry. BDpatientshadreduced

proportionsofnaturalTregulatorycells(CD4+CD25+FoxP3+)(p<0.01)inparalleltohigher

cytokineproduction(allp<0.01)thanhealthycontrols.Inparticular,BDwasassociatedwitha

strongbiastoTh1ratherthanTh2profile.Therewasanexpansionofsenescence-associatedcells

(CD8+ CD28!) in BD (p<0.0001). Tcells of BD patients had an increased p-ERK signaling

(p<0.0001),indicatinglymphocyteactivation.Ourdatasuggestthatmultiplemolecularand

cellularmechanismsmaycontributetotheimmunologicimbalanceobservedinBD.Inaddition,

ourdataconcurtoanearlysenescenceprocessinthesepatients.

#2012ElsevierLtd.Allrightsreserved.

* Correspondingauthorat:InstitutodePesquisasBiome´dicas,HospitalSa˜o LucasdaPUCRS,Av.Ipiranga6690,28andar,P.O.Box1429,Porto Alegre,RS90.610-000,Brazil.

E-mailaddress:[email protected](M.E.Bauer).

1_Carine_Hartmann_do_Prado_and_Lucas_Bortolotto_Rizzo_contributed_equally_to_this_work.

Availableonlineatwww.sciencedirect.com

jo u rn al ho m e p age :w w w. el s ev i er. co m / lo c at e /p s y n eu en

0306-4530/$—seefrontmatter#2012ElsevierLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.psyneuen.2012.08.005

122 Introduction

Thereisgrowingevidencesuggestingthatthe immuneand inflammatorysystemsplayimportantrolesinthepathogen- esisof bipolardisorder (BD).Severalstudies have investi- gatedthepotentialroleofcytokinesinpsychiatricdisorders, basedontheirimportantactionsinmodulatingmetabolism ofcentralneurotransmitters,hypothalamic—pituitary—adre- nal(HPA)axisandneurotrophicsupport(Milleretal.,2009). Increasedplasmalevelsofpro-inflammatorycytokineswere observedduringBDmanicepisodes,includinghigherlevelsof IL-6,IFN-g,TNF-a,IL-2andserumsolubleIL-6receptor(sIL-

6R)(Kimet al.,2004,2010; O’Brienetal.,2006; Brietzke

etal.,2009;Barbosaetal.,2011).Wehaverecentlyobserved

that BD patients in mania had higher sTNFR1 levels than euthymicBDpatientsandcontrols(Barbosaetal.,2011).In addition, others have found elevated IL-4 levels (a Th2 cytokine) in patients with manic episodes (Kim et al.,

2004; Ortiz-Dominguez et al., 2007). Similar to mania,

increasedlevelsofIL-6andTNF-awerealsoobservedduring depressiveepisodes(Kimetal.,2004;O’Brienetal.,2006;

Ortiz-Dominguezetal.,2007).Itshouldbenotedthatpre-

vious studies assessed inflammatory markers in plasma/ serumsamples.Theanalysisofbiomarkersincellularsuper- natantsisadvantageoustoserum/plasmasamplingasitcan precise the cellular source of cytokines. The underlying mechanismsofthe immunologicimbalanceobserved inBD arelargelyunknown,andmayincludechangesincirculating lymphocytesandthedifferentialexpressionofintracellular signalingcascades.

Changesincirculatingleukocytesmaycontributetothe immunologic imbalance observed in BD. The analyses of lymphocytesubsets,particularlyT,BandNKcellsarevery scarceinBDthough.IncreasedpercentagesofactivatedT cells(i.e.CD3+HLADR+,CD3+CD25+andCD3+CD71+)and B cells (CD19+CD20+) were observed in BD compared to healthycontrols(Breunisetal.,2003).Thisactivationstate couldbetheoreticallyduetoalackofperipheralregulatory cells.Arecentstudydidnotobservechangesinregulatory Tcells (Tregs) in BD patients as compared to controls

(Drexhage et al.,2011).Tregs (CD4+CD25+ Foxp3+)play

key roles in suppressing excessive or misguided immune responses thatcan beharmful to the host. Inparticular, they are responsible for turning off immune responses againstself-antigensinautoimmune diseases,allergies or commensal microbes in certain inflammatory diseases

(Sakaguchietal.,2008). To date,theroles of CD8+reg-

ulatoryTcells(CD8+ CD28!and CD8+CD103+)in BD are largelyunknown.

TheimmunologicimbalanceobservedinBDcouldbealso explained by the differential expression of intracellular signaling cascades. Mitogen-activated protein kinases (MAPKs)areimportantintracellularsignaltransductionsys- temsandparticipateinaseriesofphysiologicalandpatho- logicalprocesses,includingcellgrowth,differentiationand apoptosis(Strniskovaetal.,2002;Sosaetal.,2011).Three majorMAPKcascadesareknown,includingtheextracellular signal-regulatedproteinkinase(ERK),c-junamino-terminal proteinkinase/stress-activatedproteinkinase(JNK/SAPK) andp-38. Studiesbasedinanimalmodelsfoundimpaired central MAPKssignaling associatedto behavioral changes andcognitivedeficitsrelatedtomooddisorders,suggesting

an important roleof thiscascadein psychiatricdisorders

(Mazzucchellietal.,2002;Einatetal.,2003a;Thomasand

Huganir, 2004;Yuanet al.,2010).Spiliotaki etal.(2006)

foundreducedlevelsofJNKinlymphocytesfromdepressed BDpatientswhencomparedtoeuthymicpatientsandcon- trolindividuals(Spiliotakietal.,2006).Yuanetal.(2010)

foundreducedERKlevelsinpre-frontalcortexofBD,major depression and schizophrenic patients compared to controls.TheERKpathwayregulatesgeneexpressionincluding proteinsassociatedtoapoptoticprocessesaswellasasso- ciatedwithcellproliferationanddifferentiation.Phosphor- ylationofERKcorrelateswithERKactivation(Yuanetal., 2010).Whilep38isassociatedtoapro-apoptoticpathway, ERKisactivatedbymitoticstimuli,beenassociatedtocell proliferation(Ramanetal.,2007;FurlerandUittenbogaart,

2010;Xieetal.,2010).Also,p38isoftenlinkedtoinflam-

mation and cellular non-responsiveness (anergy) (Raman

etal.,2007;FurlerandUittenbogaart,2010).Interestingly,

these two enzymes have reciprocal antagonistic actions

(Ramanetal.,2007).

Here, we assessed cellular and molecular mechanisms that may influence theinflammatorystateobserved inBD patients. Specifically, we determined (a) Th1/Th2/Th17 cytokinesinsupernatantsandaddressedthedistributionof (b)regulatoryT cellsandvariouslymphocytesubsets,aswell as(c)theintracellularexpressionofactivatedMAPKsp38(p- p38) andERK (p-ERK)in euthymictype 1BD patients and healthycontrols.

Methods

Subjects

Twenty-seveneuthymicfemalesubjectswithBDtypeIwere recruited by conveniencesampling at the Outpatient Psy- chiatric ClinicforWomenwithbipolardisorderofthePre- sidente Vargas Hospital,Porto Alegre, Brazil.Only women tookpart in thisstudy toavoid immunologicaldifferences associatedwithsexualdimorphism(Ghazeerietal.,2011). Age-andsex-matchedhealthycontrolsalsotookpartinthis study.Allsubjectsprovidedtheirwritteninformedconsent beforeinclusioninthestudyapprovedbytheEthicalCom- mitteeoftheinstitution.TheBDtype1diagnosiswasbased on clinical interview and confirmed with the Structured ClinicalInterviewforDSM-IV-AxisIDisorder(SCID-I)admi- nisteredbyanexpertandwell-trainedpsychiatrist.Severity of depressive and manic symptoms was assessed by the Hamilton Depression Rating Scale (HDRS) and the Young ManiaRating Scale (YMRS),respectively.All patientswere euthymic at the time ofblood collection. Therefore, this studywasdesignedtoexaminemoreenduringimmunological changesnotnecessarilyassociatedtomoodepisodes.Euthy- miawasdefinedbyYMRSandHDRSscores<8(Clarketal.,

2002). Exclusion criteria to both patients and controls included:(a) presence of major axisIpsychiatric disorder suchaspsychoticdisorder,mooddisorder(forcontrolgroup), anxietydisorderorsubstancerelateddisorderaccordingto SCID-I;(b)historyofaseveremedicalillness;(c)historyof brain injury; (d) presence of systemic diseases (including hypertension, inflammatory diseases, such as rheumatoid arthritis orinfection)or neurologicaldisorder,and(e)use

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of any substance that may induce immune or endocrine changes (exception of psychopharmacotherapy for BD patients).

Bloodcollectionandcellisolation

Twenty milliliters of peripheral blood were collected by venipuncturebetween1000hand1200handstoredinEDTA tubespriortoanalyses.Peripheralbloodmononuclearcells (PBMCs)wereisolatedbydensitygradientcentrifugationfor 30minat900!g.Cellswerecountedbymeansofmicro- scopy(100!)andviabilityalwaysexceeded95%,asjudged from their ability to exclude Trypan Blue (Sigma). PBMCs wereresuspendedincompleteculturemedium(RPMI-1640, supplemented with 0.5% gentamicine, 1% glutamine, 1% hepes, 0.1% fungizone, and 10% fetal calfserum, FCS;all fromSigma)andadjustedtoyieldafinal concentrationof 2!105cells/well.

Immunophenotyping

Alargepaneloflymphocytesubpopulationswasidentified by multi-color flow cytometry in freshly isolated PBMCs. Briefly,PBMCs werewashedinflowcytometrybuffer(PBS containing1%FCSand0.01%sodiumazide)andtreatedwith Fc Blocksolutionfor 20min.Inorderto evaluatespecific lymphocyte subsets, cells were stained for 30min with combinations of the following monoclonal antibodies: anti-CD3FITC,anti-CD3PECy5,anti-CD4PE,anti-CD4FITC, anti-CD8PE,anti-CD19PE,anti-CD56FITC,anti-CD28FITC, anti-CD45ROFITC,anti-CD69FITC,anti-FOXP3PECy5,anti- CD103FITC,anti-CCR7Cy7,anti-CD45RAFITC(allfromBD Biosciences,SanJose,CA,USA).Immediately afterstain- ing,cellswerewashed,resuspendedandanalyzedbyflow cytometry.Aminimumof20,000lymphocyteswereidenti- fiedbysize(FSC)andgranularity(SSC)andacquiredwitha FACSCantoIIflowcytometer(BDBiosciences).Theinstru- menthasbeencheckedforsensitivityand overallperfor- mance with Cytometer Setup and Tracking beads (BD Biosciences)priortodataacquisition.Datawereanalyzed using theFlowjo 7.2.5software(Tree StarInc., Ashland, OR,USA).

Quantificationofcytokines

To determine cytokine production, PBMCs were cultured (1.5_!105 _cells) _in _RPMI _medium _with _10% _FCS _(Sigma—

Aldrich)and1%phytohemagglutinin(PHA,fromInvitrogen, Carlsbad,CA,USA),for72hat37_8Candina5%CO2atmo-

sphere. The supernatants were collected and stored at "808Cforlateranalysis.Thesampleswerethawedinthe samedayandprocessedtogether.Multiplesolublecytokines (IL-2,IL-10,IL-4,IL-6,IFN-g,TNF-aandIL-17)weresimul- taneouslymeasuredbyflowcytometryusingtheCytometric Bead Array (CBA) Human Th1/Th2/Th17 Kit (BD Bios- ciences). Acquisition was performed with a FACSCanto II flowcytometer(BDBiosciences).Theinstrumenthasbeen checkedforsensitivityandoverallperformancewithCyt- ometerSetupandTrackingbeads(BDBiosciences)priorto dataacquisition.Quantitativeresultsweregeneratedusing FCAPArrayv1.0.1software(SoftFlowInc.,Pecs,Hungary).

The detectionlimits for these assays ranged from2.4 to 4.9pg/mL (IL-2, IL-10, IL-4, IL-6, IFN-g, and TNF-a) and 18.9pg/mLforIL-17.

AnalysisofintracellularactivatedMAPKsin lymphocytes

Activated MAPKs in lymphocytes were assessed by flow cytometric evaluation of intracellular phospho-p38 and phospho-ERKexpressioninCD3+CD4+andCD3+CD8+cells (Human T Cell Activation Kit, BD Biosciences). Isolated PBMCs were cultured in RPMI medium with 10% FCS and stimulated with 40nM phorbol 12-myristate 13-acetate (PMA)and1mMionomycin(IONO,allfromSigma—Aldrich) for15minat37_8Candina5%CO2atmosphere.Cellswere

harvested and immediately fixed and stored frozen ("808C)inCytofixsolution(BDBiosciences)forlaterana- lysis.Allsamples were thawed inthesame day and pro- cessed together to reduce variation. Cells were permeabilizedonicefor30minwithPhosflowPermBuffer III (BD Biosciences). Cells were washed (600!g, 6min), stainedfor60minatroomtemperature,washed(600!g, 6min) and resuspended in final concentration of 4.5!105cells/200mL, all in Pharmingen Staining Buffer (BD Biosciences). The4-color immunofluorescentstaining procedure wasperformed combiningthefollowingmono- clonal antibodies from BD Biosciences: anti-CD3 PerCP (clone SK7), anti-CD4 FITC (clone SK3) and anti-CD8 PE (cloneSK1)withanti-phospoERK1/2AlexaFluor647(clone 20A, whichrecognizesthephosphorylated threonine202, pT202,and tyrosine204, pY204 ofERK1 and thepT184/ pY186ofERK2)oranti-phosphop38AlexaFluor647(clone 36/p38, whichrecognizes theconserved dual phosphory- latedsitepT180/pY182ofp38a,b, gand d). Lyophilized humancontrolcells(BDBiosciences)wereusedaspositive andnegativecontrolsduetotheknownpresenceofmito- gen-activated (upregulation of phosphorylated MAPKs) or non-activated (basal levels of phospho-MAPKs) T cells in eachcontrol,respectively.Aminimumof20,000lympho- cyteswereidentifiedbysize(FSC)andgranularity(SSC)and acquired with a FACS Canto II flow cytometer (BD Bios- ciences).Theinstrumenthasbeencheckedforsensitivity andoverallperformancewithCytometerSetup&Tracking beads(BDBiosciences)priortodataacquisition.Datawere analyzed usingtheFlowjo 7.2.5software (TreeStarInc., Ashland,OR, USA).

Statisticalanalysis

Allvariablesweretestedforhomogeneityofvariancesand normalityofdistributionbymeansoftheLeveneandKol- mogorov—Smirnovtests,respectively.Forcontinuousvari- ables, differences between groups were analyzed by Student’st-testorMann—WhitneyUtestwhenappropriate. Statisticalinteractionsbetweencategoricalvariableswere comparedbymeansofthechi-square(x2)test.Statistical analyseswereperformedusing theStatisticalPackagefor Social Sciences, SPSS Statistics 17.0 software(SPSS Inc., Chicago, IL, USA). The significance level was set at a=0.05(two-tailed).

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124 Results

Characteristicsofthestudiedpopulations

Demographicandclinicalcharacteristicsofthesamplesare summarized in Table 1. Both groups were homogenous regarding age, gender, ethnicity, BMI and smoking habits. Mostpatientswereunderamultipledrugregimen(Table1). However,12patientswereunderlithiummonotherapy,one underantidepressantonly,onewithantipsychoticonlyand nonewithanticonvulsantonly.

Lymphocytesubsets

Wescreenedalargepanelofcirculatinglymphocytesubpo- pulationsbymulticolorflowcytometry,includingactivated, regulatory and immunosenescencemarkers (Table 2).Cells wereimmunophenotypedpriortocellcultures.Thestudied groupswerehomogenousregardingmostlymphocytemarkers. However,BDpatientshadalteredproportionsofregulatoryT cells(Fig.1).Inparticular,lowerpercentagesofnaturalTreg cells(CD4+CD25+FoxP3+)wereobservedinBDpatients,as showninFig.1CandD(U=100.00;p<0.01).Furthermore,BD patients hadareducedFoxP3 expression (!31.2%)intotal CD4+ cells as compared to controls (1576.08"363.46 vs. 2292"374.39,respectively),asestimatedbythemeanfluor- escenceintensity(U=75,p<0.05).Incontrast,BDpatients hadhigherfrequenciesofCD8+CD28!Tcellsascomparedto controls (U=165.00; p<0.0001). With respectto possible effectsofpharmacotherapy,nosignificantassociationswere foundwiththeimmunologicalmeasures(allp>0.05,assessed byMann—WhitneyTests).

Cytokineproduction

MultipleTh1/Th2/Th17cytokines(IL-2,IL-4,IL-6,IL-10,IL- 17,IFN-gandTNF-a)wereassessedinculturesupernatants byCBAs.Table3showsthecytokineprofilesfollowingpoly- clonal T-cell stimulation. All cytokineswere found signifi- cantlyincreasedinBDwhencomparedwithhealthycontrols (allp<0.01).Tofurtherinvestigatethecytokineprofileswe

comparedthecytokine ratiosbetween thetwogroups.BD patients showed higher IL-6/IL-4 (U=195.00;p<0.0001), TNF-a/IL-4 (U=91.00; p<0.0001), IFN-g/IL-4 (U=79.00; p<0.0001) and IFN-g/IL-10 (U=161.00; p=0.009) ratios comparedto controls (Fig.2),suggesting a strong biasto Th1ratherthanTh2profile.Therewerenostatisticaldiffer- encesregardingtheremainingcytokineratios(allp>0.05). No significant associations were found between pharma- cotherapy use and the immunological measures (all p>0.05,assessedbyMann—WhitneyTests).

Analysisofintracellularphospho-MAPKsin peripherallymphocytes

Wehavealsoanalyzedtheexpressionofphosphorylatedp-38 andp-ERKMAPKsinlymphocytesfollowingstimulationwith PMAandIONO.Fig.3showstheunstimulated(solidline)and activated(dottedline)profilesofarepresentativesample. The expression of p-ERK MAPK in T CD8+ (U=17.00; p=0.001)andCD4+cells(U=31.00;p=0.018),asestimated bythemeanfluorescenceintensity(MFI)wasfoundincreased inpatientsincomparisonwithcontrols(Fig.4C).Thiswasnot observedforthep-p38expression(Fig.4D).Therewereno significantdifferencesinthepercentagesofTcellsexpres- sing p-ERK or p-p38 between groups (Fig. 4A and B). No significantassociationswerefoundbetweenpharmacother- apy use and the immunological measures (all p>0.05, assessedbyMann—WhitneyTests).

Discussion

Datapresentedhere areinaccordance topreviousstudies suggesting an immune/inflammatory imbalance in BD. Briefly,patientshadlowerproportionsofnaturalregulatory Tcells (CD4+CD25+ FoxP3+)inparallel tohighercytokine productionwhencomparedtohealthysubjects(withstrong biastoTh1).Wealsoobservedanincreasedp-ERKsignalingin peripheralT-cellsubsetsofBDpatients,suggestingincreased lymphocyteactivation.

PBMCs of BD patients produced significantly higher amounts of IL-2, IL-4, IL-5, IL-10, IL-17, IFN-g and TNF-a

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Table1 Characteristicsofthestudiedpopulations.

BD HealthyControls p-Value

N 27 24 —

Age,years(mean"SD) 45.72"9.22 40.48"13.24 0.13

BMI(mean"SD) 27.92"4.20 26.48"3.26 0.23

Yearsofillness(meanandinterval) 10.44(1—46) — —

Ageatonset(mean"SD) 34.95"12.28 — —

HDRS(mean"SD) 4.96"2.21 — — YMRS(mean"SD) 1.70"2.09 — — Ethnicity(white/non-white) 21/6 23/1 0.09 Smoking 5 2 0.29 Lithium 18 — — Antidepressants 11 — — Antipsychotics 11 — — Anticonvulsants 3 — —

Datashownasmean(M)"standarddeviation(SD).Abbreviations:BMI,bodymassindex;BD,bipolardisorder;HDRS,HamiltonDepression RatingScale;andYMRS,YoungManiaRatingScale.DatawereanalyzedbyStudent’st-testorx2test.

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Table2 Immunophenotypingoflymphocytesubsets.

Markers Celltype BD(%) Healthycontrols(%) p-Value

CD3+CD4+ Th 48.51!6.83 48.19!6.98 0.50 CD3+CD8+ Tc 24.96!6.92 25.09!6.11 0.68 CD3"CD19+ B 8.59!4.61 7.40!2.30 0.30 CD3"CD56+ NK 11.18!11.47 13.02!8.51 0.22 CD3+CD56+ NKT 6.55!6.08 8.46!20.13 0.24 CD4+CD45RO+ Memory(Th) 27.86!9.73 27.40!5.10 0.94 CD8+CD45RO+ Memory(Tc) 10.52!6.60 9.99!3.01 0.94 CD4+CD25+ ActivatedTcell 1.34!0.72 1.97!1.66 0.50 CD3+CD69+ ActivatedTcell 2.32!1.33 2.27!1.23 0.34 CD8+CD28+ ActivatedTcell 14.24!7.96 15.68!5.08 0.25 CD8+CD28" RegulatoryTCell 17.92!6.76 12.55!5.08 0.002**

CD4+CD25+FOXP3+ RegulatoryTcell 2.33!2.46 7.18!10.14 0.014*

CD8+CD103+ RegulatoryTcell 0.695!0.31 1.05!0.68 0.12

CD4+CCR7+CD45RA" Centralmemory(Th) 44.51!11.22 45.40!9.97 0.97

CD4+CCR7"CD45RA" Effectormemory(Th) 16.42!5.68 14.42!5.21 0.29

CD4+CCR7+CD45RA+ Naı¨veTcell(Th) 34.47!13.75 34.86!11.56 0.94

CD8+CCR7+CD45RA" Centralmemory(Tc) 24.81!9.74 23.11!6.95 0.68

CD8+CCR7+CD45RA+ Naı¨veTcell(Tc) 40.85!12.15 44.68!9.65 0.38

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