Contents lists available atScienceDirect
Brain
Research
Bulletin
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s b u l l
Research
report
The
IFN-

1b
effect
on
Cu
Zn
superoxide
dismutase
(SOD1)
in
peripheral
mononuclear
blood
cells
of
relapsing-remitting
multiple
sclerosis
patients
and
in
neuroblastoma
SK-N-BE
cells
Simona
Damiano
a,
Anna
Sasso
a,
Bruna
De
Felice
b,
Giuseppe
Terrazzano
c,
Vincenzo
Bresciamorra
d,
Antonio
Carotenuto
d,
Nicola
S.
Orefice
d,
Giuseppe
Orefice
d,
Giovanni
Vacca
d,
Annamaria
Belfiore
a,
Mariarosaria
Santillo
a,
Paolo
Mondola
a,∗aDipartimentodiMedicinaClinicaeChirurgia,UnitàdiFisiologia,UniversitàdegliStudidiNapoliFedericoII,Italy
bDipartimentodiScienzedellaVita,SecondaUniversitàdiNapoli,Italy
cDipartimentodiScienze,UniversitàdellaBasilicata,Italy
dDipartimentodiNeuroscienzeeScienzeRiproduttiveedOdontostomatologiche,UniversitàdegliStudidiNapoliFedericoII,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received10April2015
Receivedinrevisedform16July2015
Accepted25August2015
Keywords: Interferon
Superoxidedismutase
Peripheralbloodmononuclearcells
Relapsing-remittingmultiplesclerosis
(RR-MS)
a
b
s
t
r
a
c
t
Multiplesclerosis(MS)isachronicinflammatorydemyelinatingdiseaseleadingtoaxonalinjury.Even iftheetiologyofMSisstillunknownthediseasebeginswithinflammationinvolvingautoreactiveT lymphocytesactivationingeneticallysusceptiblesubjects.Interferonbeta-1b(IFN1b)isoneofthe mostuseddrugintheMStherapy.
TheresultsobtainedinthisstudyshowthattheconcentrationofSOD1inCSFofrelapsing-remitting MS(RR-MS)patients,evaluatedbyenzyme-linkedimmunosorbentassay(ELISA),isdecreasedcompared topathologicalcontrols.Moreover,theWesternblottinganalysisdemonstratedthatSOD1inhuman peripheralbloodmononuclearcells(PBMC)inhealthycontrolswassignificantlyhighercomparedtoMS subjectsbeforestartingDMTtherapy.InadditionIFN1btherapycausesanincreaseofintracellular SOD1proteinaswellasmRNAlevelsinPBMC.Moreover,thetreatmentofneuroblastomaSK-N-BEcells withIFN1bincreasedSOD1proteinandmRNAlevels;thesedataalsosuggestthatneuroprotective effectofthisphysiologicalmoleculeis,atleastinpart,carriedoutthroughitseffectonSOD1.Thisstudy demonstratethatDMTtherapyisabletoincreaseSOD1expressioninPBMCofRR-MSpatients.Therefore, theeffectivenessofDMTtherapycanbeascribed,atleastinpart,toanincreasedlevelsofthisantioxidant enzymeasfurtherconfirmedbyinvitrostudiesinSK-N-BEcells.
©2015ElsevierInc.Allrightsreserved.
1. Introduction
Multiplesclerosis(MS)isachronicinflammatory demyelinat-ingdisorderoftheCentralNervousSystem(CNS)characterizedby theinfiltrationofCNSwhitematterbymyelin-reactivelymphocyte
Abbreviations: SOD1,superoxidedismutase;MS,multiplesclerosis;ELISA,
enzyme-linked immunosorbent assay; CSF, cerebrospinal fluid; CNS, central
nervous system;P Ctr, pathological controls; HD, healthydonator; SK-N-BE,
human neuroblastomacells; PBMC, peripheralbloodmononuclear cells;BFA,
brefeldinA;DMT,dimethyltryptamine;IFN,interferon;CDP,chronic
inflamma-torydemyelinatingpolyneuropathy;EAE,autoimmuneencephalomyelitis;RR-MS,
relapsing-remittingmultiplesclerosis.
∗ Correspondingauthor.Fax+390817463639.
E-mailaddress:[email protected](P.Mondola).
Tleadingtoprimaryorsecondaryaxonalinjury.Theetiologyof MS isnot yetknown; it hasbeen reportedthatgenetic factors couldplayimportantrolesinthepathogenesisofthisdisease(Ebers andSadovnick,1994;Oksenbergetal.,1993;Rebouletal.,2000; Kantarcietal.,2002;Tolide-Ieetal.,2014).Itisalsoknownthat environmentalfactors,suchassmoking(Russoetal.,2011),viral infectionsandvitaminDstatushavebeenlinkedtotheoccurrence ofMS(DeLorenzeetal.,2006;Marrie,2004).
AlthoughtheinitiatingeventofMSisamatterofdebate,the diseasebeginswithinflammationinvolvingautoreactiveT lym-phocytes(Dhib-Jalbutetal.,2002)thatcouldbetriggeredbyviral infectioningeneticallysusceptiblesubjects(Marrie,2004).In addi-tion, activated monocytes and macrophages can alsorelease a variety of neurotoxic molecules likenitric oxide, reactive oxy-genspecies(ROS)thatcontributetothedestructionofthemyelin http://dx.doi.org/10.1016/j.brainresbull.2015.08.009
EDSS,mean±SD 2.51±0.58
sheath(Dhib-Jalbutetal.,2002).Moreoverhistologicaldatafrom
thespinalcordofmiceaffectedbyautoimmuneencephalomyelitis (EAE),thatrepresentsagoodexperimentalanimalmodelofMS, confirma strongcorrelationof inflammationand axonal injury withperivascular lymphocytes infiltration that spread into the parenchyma(GiulianiandYong,2003).
Ithasbeenalsorecentlydemonstratedthatautoimmune pro-cessaffectsnotonlythewhitematterbutalsothegreymatterin thecourseofthedisease(Derakhshanetal.,2013).
ManyexperimentaldatasuggestasignificantroleofROSinthe pathogenesisofmyelindestructioninMSaswellasinother neu-rodegenerativediseases. Mitochondria constantly produce ROS, whichhaveimportantsignallingfunctions(Damianoetal.,2012, 2015).WhentherateofROSproductionexceedsthecellular antiox-idantcapacityitcancauseoxidativestressandextensivedamage toessentialcellcomponents(LinandBeal,2006;Witteetal.,2013). Howeversofaritisnotcompletelyclearwhetherthecorrelation betweenROSandMSisduetoadecreaseofenzymaticantioxidant defencemechanismsortoanoverproductionofROS.
Inpreviousresearchesweshowedthattheantioxidantenzyme Cu–Znsuperoxidedismutase(SOD1)is secretedbymany cellu-larlinesincludinghumanneuroblastomaSK-N-BEcells through amicrovesiclespathway(Mondolaetal.,2003)andincreases cal-ciumintracellularlevelsthroughanactivationofphospholipase C-proteinKinaseCpathway(Mondolaetal.,2004,1996).Ithasalso beenshownthatanimpairedsecretionofmutantsuperoxide dis-mutase1isassociatedwithneurotoxicityinfamilialamyotrophic lateralsclerosis(ALS)(Turneretal.,2005;TurnerandAtkin,2006); moreoveranendoplasmicreticulum(ER)stress,associatedwith anunfoldedproteinresponseandGolgiapparatusfragmentation inmutantSOD,ispresentinfamilialALS(Mondolaetal.,2004). RecentdatademonstratedthatSOD1isabletoactivate,through muscarinicM1receptor,atrasductionalpathwayinvolvingP-ERK andP-AKTinhumanneuroblastomaSK-N-BEcells(Damianoetal., 2013).
The aim of the present research was to evaluate theSOD1 amount in plasma and cerebrospinal fluid (CSF) of relapsing-remitting multiple sclerosis (RR-MS)subjects, pathological and healtycontrols;wealsoevaluatedhowinterferon1b(IFN)could modifytheexpressionofSOD1inneuroblastomaSK-N-BEcellsand inperipheralbloodmononuclearcells(PBMC)ofRR-MSpatients.
AfurthergoalofthisstudywastoinvestigatewhetherIFN beta-1bcanaffectexportofSOD1inneuroblastomaSK-N-BEcells.
2. Materialandmethods
2.1. Patients
Weenrolled23consecutiveoutpatientsoftheMSCentreof Fed-ericoIIUniversityHospital(NaplesItaly),fromMaytoNovember 2010withclinicallydefinitiveRR-MSaccordingtotheMcDonald criteria(Polmanetal.,2005),aged20–50yearswithoutrelapsenor corticosteroidstreatmentintheprevious3monthsnaïvetoany dis-easemodifyingtherapy.Patientsweretreatedwith250g/mlIFN beta-1b(8.000.000IU)onalternatedayswithsubcutaneous injec-tion.Visitswerescheduledwithmonthlyneurologicalevaluation andbloodsampleswerecollectedatbaselineandafter3monthsof
Fig.1. SOD1expressiondecreasesinCSFofMSpatients.
Enzyme-linkeddetection ofSOD1 incerebrospinalfluid ofcontrolversusMS
patients;thevaluesofSOD1areexpressedas%value.Errorbarsindicate±SE
(*P<0.001).Statisticalanalysishasbeenperformedbyusingt-teststudy.
therapy.Committeeapprovalwasobtainedandeachpatientgave theirsignedinformedconsenttoparticipateinthisstudy.
Wealsoenrolled20patientswithotherneurological inflam-matorydisease(ONID)suchasLES,SystemicSclerosis,Vasculitis andCIDP(ChronicInflammatoryDemyelinatingPolyneuropathy) usedaspathologicalcontrols(PCtr).Patientsandpathological con-trolswerematchedbyageandgender.CSFwascollectedfrom20 RR-MSpatientsandfrom23ONIDcontrols.Writteninformed con-sentwasobtainedfromeachpatientafteradetailedexplanation ofallprocedures.Thestudywasapprovedbythe“FedericoII” Uni-versityEthicalCommittee(No197/11/E1)andwasconductedin concordancewiththeDeclarationofHelsinki.
Inadditionweenrolled10healthydonators(HD)toevaluatethe amountofintracellularSOD1inPBMC.Demographicandclinical characteristicsaresummarizedinTable1.
2.2. Bloodcollection
Bloodsampleswereobtainedbetween07:00and09:00A.M. from23RR-MSpatientsand10healthydonators.Bloodtestingwas performedinthemorningaftera12-hfasteitherinpatientsorin healthydonators.Thesampleswerecentrifugedaftercollection at1500rpmfor10min.andcell-freesupernatantswerestoredat −80◦Cforfurtheranalysis.
2.3. PBMCisolation
PBMCcellswereisolatedfrom12mlofblooddrawnbyvein puncturefromHDandRR-MSpatients;diluted(1:1withPBS) sam-pleswerelayeredonthetopofFicollsolution(Ficoll-GEHealthcare) andspunat3000rpmfor25min.PBMCcellswerecollected,two timewashedinPBSandthenspunat3000rpmfor4min;thepellet fractionwasthendissolvedinRIPAbufferbeforepolyacrylamide gelelectrophoresis(PAGE)determination.
Fig.2. ProteinandmRNASOD1expressionincreaseinMSpatientsafterInterferontreatment.
PanelAshowstheWesternblottingevaluationofSOD1inPBMCinhealthyvoluntarysubjects(CTR)andinsubjectsaffectedbyMSbeforestartingtheinterferontreatment.
InpanelBthedifferentamountofSOD1inMSpatientsbeforeandafterthreemonthsofinterferontreatmentisshowed.PanelCindicatesthesemi-quantitativeRT-PCRof
SOD1beforeandafterthreemonthsofinterferontreatment.*P<0.001vsHDandvsTime0(PanelAandB).**P<0.005vsTime0(PanelC).
2.4. SOD1immunoblottingassayofPBMC
Western blotting was performed as previously described (Kantarcietal.,2002).BrieflyPBMCcells,washedtwicewithcold phosphate-bufferedsalinewerehomogenizedinbuffer contain-ing10mMTris–HCl,pH7.4,4mM2-mercaptoethanol,5mMEDTA addingamixofproteaseinhibitors.Nucleiandcelldebriswere eliminated by slight centrifugation at 3.000rpm for 5min; the supernatantwasthencentrifugedat100,000×gfor1hat4◦C.After centrifugationthepelletwasdiscardedand50goftotalproteins weresubjectedto10%sodiumdodecylsulfate-polyacrylamidegel electrophoresis(SDS-PAGE)underreducingconditions;thenthe proteinsweretransferred ontoa nitrocellulosefiltermembrane (GE-Healthcare,UK)withaTrans-BlotCell(Bio-RadLaboratories, UK)and transferbuffercontaining25mMTris, 192mMglycine, 20% methanol. Membranes were placed in 5% non-fat milk in phosphate-bufferedsaline,0.5%Tween20(PBST)at4◦Cfor2hto blockthenonspecificbindingsites.Filterswereincubatedwith specificantibodiesbeforebeingwashedthreetimesinPBSTand thenincubatedwithaperoxidase-conjugatedsecondaryantibody (GE-Healthcare,UK).AfterwashingwithPBST,peroxidaseactivity wasdetectedwiththeenhancedchemiluminescent(ECL)system (GE-Healthcare,UK).
SOD1wasdetectedbyarabbitpolyclonalantibodiespurchased bySantaCruzBiotechnology(USA)diluted1:1000in0.1%Tween 20PBS.
Thefilterswerealsoprobedwithananti␣-tubulinantibody (Sigma, USA). Protein bands were revealed by ECL and, when specified,quantifiedbydensitometryusingScionImagesoftware. Densitometricvalueswerenormalizedto␣-tubulin.
2.5. NeuroblastomaSK-N-BEcellsculture
HumanneuroblastomaSK-N-BEcells(AmericanTypeCulture Collection,ATCC,USA)andweregrowninmonolayerinRPMI1640 mediumsupplementedwith10%FBS,2mMl-glutamine,50g/ml streptomycinand50IU/mlpenicillin.Thecellswerekeptina5% CO2and95%airatmosphereat37◦C.SK-N-BEandgrownto
semi-confluencein60-mmdishes,thenwerewashedtwicewithPBSand incubatedfor18hinmediumcontaining0.2%FBSbeforethe exper-iments.Thecellswerethenincubatedfor3hwith100.000U/mlof IFN1ainpresenceof5g/mloftheinhibitorsofSOD1protein secretionBrefeldinA(BFA)andinpresenceandinabsenceof3lof 20MDPI.SK-N-BEandcelllysateswereobtainedinRIPAbuffer, containing50mM.
Tris–HCl,pH7.5,150mMNaCl,1%NP40, 0.5%deoxycholate, 0.1% SDS,2.5mM Na-pyrophosphate, 1mM -glycerophosphate, 1mMNaVO4,1mMNaF,0.5mMPMSFandacocktailofprotease
inhibitors(Roche,USA).Thecellswerekeptfor15minat4◦Cand disruptedbyrepeatedaspirationthrougha21-gaugeneedle.Cell lysateswerecentrifugedfor15minat13,000rpmandthepellets werediscarded.Fiftymicrogramsoftotalproteinswassubjected toSDS-PAGEunderreducingconditions.
After electrophoresis, the proteins were transferred onto a nitrocellulose filter membrane (GE-Healthcare, UK) with a Trans-Blot Cell (Bio-Rad Laboratories, UK) and transfer buffer containing25mMTris,192mMglycine,and20%methanol. Mem-branes were placed in 5% non-fat milk in tris-buffered saline, 0.1% Tween 20 (TBST) at 4◦C for 2h to block the nonspecific binding sites. Filters were incubated with specific antibodies before being washed three times in TBST and then incubated
Fig.3.SOD1proteinlevelsdeterminedbyELISAassayincreaseafterinterferontreatmentinSK-N-BEcells.
PanelAshowsthevalueofintracellularSOD1inneuroblastomaSK-N-BEcellsafterinterferontreatmentinabsenceandinpresenceofreactiveoxygenspeciesinhibitorDPI.
PanelBindicatesthevalueofextracellularSOD1afterthesametreatmentasindicatedinPanelA.InPanelCtheimpairmentofinterferon-inducedSOD1secretionafter
BrefeldinApretreatmentinabsenceandinpresenceofDPIisillustrated.*P<0.005vsDPI**P<0.001vscontrol.
withaperoxidase-conjugatedsecondaryantibody(GE-Healthcare, UK).
AfterwashingwithTBST,peroxidaseactivitywasdetectedwith theECLsystem(GE-Healthcare,UK).SOD1wasdetectedwith rab-bitpolyclonalantibodies;SOD1antibodieswerefromSantaCruz Biotechnology Inc. (USA), Protein bands were revealed by ECL and,whenspecified,quantifiedbydensitometryusing ScionIm-agesoftware.Densitometricvalueswerenormalizedto␣-tubulin antibodies.Eachexperimentwasthreetimesrepeated.
2.6. RNApreparation
TotalRNAsofcell lineswereextracted withHighPure RNA isolationkit(Roche),accordingtothemanufacturer’sinstructions, using2×106cells.TracesofcontaminatedDNAwereremovedwith
DNAseItreatment.
2.7. Semi-quantitativeRT-PCR
To improve the accuracy and sensitivity of the procedure, weperformedaone-stepquantitativereversetranscription poly-merasechainreaction,inwhichthereversetranscriptaseenzyme andaTaqDNApolymeraseblend,possessingaproofreading activ-itywerecombinedintheonetube,andasingle,non-interrupted thermalcyclingprogram.Quantificationwasachievedinasingle reactionby usingthehousekeeping beta-actingene as internal standard.100ngofRNAtemplatewasreverse-transcribedin50ll reactionmixcontaining200lMdNTPs,100mMDTT,0.25llRNase inhibitor,1.5mMMgCl2,and1llenzymemix(TitanOne-tube RT-PCRkit,Roche).Thesolutionwasincubatedfor30minat50◦Cinan automatedDNAthermalcycler(GeneAmp2400Perkin.Elmer).To performRT-PCRatoptimalconditionsandtostaywithinthe loga-rithmicallylinearproductformation,40cycleswerechosen(30sat 94◦C,30sat59◦C,60sat68◦C)andwerefollowedbyafinal exten-sionfor7minat68◦C.b-actin,SOD1,primerspairsweredesigned
toyieldPCRproductsofdifferentsizes,587bpforb-actin,290bp forSOD1.
The forward and reverse b-actin primers
were 5-CC AAGGCCAACCGCGAGAAGATGAC-30,
50-AGGGTACATGGTGGTGCCGCCAGAC-3, respectively; the forward andreverseSOD1primerswere5-CAGTGCAGGTCCTCACTTTA-3 and 5-CCTGTCTTTGTACTTTCTTC-3, respectively. To rule out genomic DNA contamination we performed a negative control whichcontainedRNAinsteadofcDNA.ThesignalintensitiesofPCR productswereseparatedona1.2%agarosegelandwerevisualized by ethidium bromide staining. The products’ signal intensities weredeterminedbycomputerizeddensitometricanalysisusing Fotoplot software. The expression of SOD1 was normalized to b-actinmRNAlevels.
2.8. DNAsequencing
Tocheckthespecificityoftheamplifiedproducts,DNAbands were eluted from the gel and purified; sequence analysis was determinedbytheBigDyeTerminatorCycleSequencingmethod (ABI-PRISMSequencer310PerkinElmer).
2.9. Enzyme-linkedimmunosorbentassay(ELISA)
The quantitative detection of CuZn superoxide dismutase (SOD1)inthecerebrospinalfluidwasdeterminedbytheBender Medsystemkit(BenderMedSystemDiagnostic,Vienna,Austria)as previouslydescribed(Mondolaetal.,2003).Theconcentrationof SOD1ontheliquorwasdeterminedinundilutedsamples calculat-ingtheaverageabsorbance(450nm)valuesforeachsampleand forstandardcurveintriplicate.Thereproducibilityofthemethod wasevaluatedinanindependentexperiment;thedetectionlimitof theassaywas0.07ng/mlandtheintra-assaycoefficientofvariation was4.8%.
2.10. Statisticalanalysis
Allresultsshownaremean±SEofatleastthreeseparate exper-iments.StatisticaldifferenceswereevaluatedusingaStudent’st test;forunpairedsamples.
3. Results
23RR-MSpatients,20ageandgendermatchedONIDpatients and10ageandgendermatchedHDwereenrolled.
3.1. SOD1detectioninCSF
SOD1determinationin cerebrospinalfluidin10 pathological controlsandin10 RR-MSpatientsisshown inFig.1.Ascanbe noticedinRR-MSsubjects,astatisticalsignificantdecreaseofSOD1 concentration(p<0.001)wasobservedrespecttopathological con-trolssuggestinganimpairedsecretionofthisantioxidantenzyme inRR-MSpatients.
3.2. DetectionofSOD1inPBMC
In Fig.2(Panel A)theWesternblotting analysisof SOD1in PBMCofvoluntaryhealthydonators(HD)andinRR-MSpatients wasshown.Ascanbenoticed,beforestartingDMTtherapy,the SOD1proteinamountwasstronglydecreasedcomparedtohealthy donators.EDSSanddiseasedurationwerenotrelatedtoSOD1level. IFNbeta-1btherapymodifiesSOD1proteinlevelsinPBMCof RR-MS;asshowninFig.2,afterthreemonthsofInterferon1b treatmentanincreaseofbothintracellularSOD1(PanelB)protein andmRNA(PanelC)levelsinPBMCwereobserved.
3.3. EffectofIFNbeta-1binSK-N-BEcells
InFig.3 (PanelA)theincrease ofintracellularSOD1protein amountafterincubationwith100ng/mlofIFNbeta1b3hisshown. TheeffectwasnotdependentonROSbecausethepreincubationof thecellsfor15minwith20MDPI,aninhibitorofROS,didnot impairinterferoneffectsonSOD1.Inaddition,inpresenceofBFA, thatimpairsSOD1secretion,theexportofSOD1stronglydecreases (panelC)suggestinganinvolvementofBFAonSOD1export.
The same increase (about 50%) of SOD1 mRNA levels was observed inneuroblastoma SK-N-BE cells after incubation with interferon1b(datanonshown).
4. Discussion
Multiplesclerosisinoneofthemostcommoncauseof disabil-ityinyoungadults,mainlyinfemalesubjects.Despiteanelevated numberofstudies,thepathogenesisofthisdiseasestillremains obscure.Manystudiesindicatethatthediseaseriskismore ele-vatedinfamilymembers withMSdiseasecompared togeneral populations(Sadovnicketal.,1997);inadditionseveral epidemi-ological data indicate that specific bacteria or virus infections, associatedtooxidative stresses,canbeinvolvedin thischronic demyelinating inflammation. The imbalance betweenROS pro-ductionand antioxidant defense mechanismsare associated to many neurodegenerativediseases, includedMS. Tothis respect theantioxidantsuperoxidedismutase(SOD)isoenzymescould car-riedoutakeyroleintheMSdisease.Wepreviouslyshowedfor thefirsttimethatCuZnsuperoxidedismutase(SOD1)isexported bymanycellularlinesincludinghumanneuroblastomaSK-N-BE cells(Mondolaetal.,2003,2004).Subsequently,observationsfrom severalauthorsalsoconfirmedthepresence ofSOD1inculture
mediumfromdifferentcelltypes(Turneretal.,2005;Urushitani etal.,2006;Gomesetal.,2007).
InthispreliminarystudyweevaluatedtheeffectofDMT ther-apyforashortperiodofthreemonthsonSOD1proteinamountand itsgeneexpressioninperipheralbloodmononuclearcells(PBMC) ofasymptomaticRR-MSpatients.WefoundthatafterDMT ther-apywithIFN1b,intracellularconcentrationofSOD1inPMBC ofRR-MSpatientsincreasedcomparedtothebasallevels(before treatment);inaddition,likewiseraisedthemRNASOD1levels.
Afurthergoalofourstudyhasbeeneithertoinvestigate,by invitroexperimentsonneuroblastomaSK-N-BEcells,theeffects ofIFN1bonintracellularcontentandexportofSOD1inorderto pointouttheroleofSOD1intheeffectivenessofIFN1btreatment. TheinvitrostudiesonneuroblastomaSK-N-BEcellsshowedthat IFN1b,increasesSOD1intracellularamountaswellasits secre-tionindicatingthatconstitutiveSOD1secretionismodulatedby thismolecule(Fig.3).
Thiseffectcannotbeascribedtoreactiveoxygenspecies(ROS) production by IFN  1b treatment because the ROS inhibitor diphenyleneiodonium-chloride (DPI) did not affected IFN  1b effects onintracellular and extracellularSOD1levels. Moreover IFN1btreatmentdidnotincreaseROSlevelsmeasuredby 5,6-carboxy-2‘,7-dichlorofluorescein-diacetate,DCHF-DA,(Molecular Probes,TheNetherlands)(datanotshown).
Theabsenceof ROSproductionin neuronalcells afterIFN  incubationforashortperiodwasalsoconfirmedbyothersauthors (Albonietal.,2013).
InCSFthepresenceofthethreeSODisoenzymeshasbeen pre-viouslydemonstrated;CuZn–SODaccountsfor∼75% oftheSOD activity,EC-SODfor 25%and Mn-SODfor <5%(Jacobsson etal., 2001).
TheconcentrationofCuZn–SODinCSFcouldbecorrelatedwith theamountofthisenzymeinthecytosolofcellsintheCNS.Inour study,thehigherlevelofSOD1detectedincerebrospinalfluidof controlsubjectscomparedtountreatedRR-MSpatientssuggests animpairedSOD1secretionthat,decreasingantioxidantdefences, could impair neuroimmunoregulatory responses and therefore, couldhavearoleinthepathogenesisofmultiplesclerosis.
Some studies demonstrated the suppression of the Lipopolysaccharide-induced in vitro production of two proin-flammatory cytokines, tumor necrosis factor ␣ (TNF␣) and interleukin  (IL-1) in subjects receiving spinal manipulative therapy (SMT) (Teodorczyk-Injeyanet al., 2006).It could beof interest to further explore the possible effects of SMT on the SOD1andothercellularantioxidantdefencesinmultiplesclerosis patients.
Therefore,thispreliminarystudyindicatesthatthe effective-nessofDMTtherapycanbeascribed,atlistinpart,toanincrease ofintracellularSOD1levelsinadditiontoaninductionofitsgene expressionthatcanbefinalizedtoimproveantioxidantdefence mechanisms. Finally ourdata on SK-N-BE cells pointed out on a newphysiologicalmechanism involvedin neuroprotectionby interferon1b.
Acknowledgements
TheauthorsaregratefultotheAcademicspinoffPRIUSs.r.l. SistemaIntegratoDiagnosieTerapiaforgivingtheopportunityto usescientificequipmentpresentinthelaboratory.WethankDr.I. Cerilloforclinicaldatacollecting.
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