Cell therapy in experimental model of inflammatory bowel disease

w w w . j c o l . o r g . b r

Journal

of

Coloproctology

Original

Article

Cell

therapy

in

experimental

model

of

inflammatory

bowel

disease

夽

Mônica

Yonashiro

Marcelino

_,

_Natália

_Langenfeld

_Fuoco

_,

Ana

Elise

Valencise

Quaglio

_,

_Renata

_Aparecida

_de

_Camargo

_Bittencourt

_,

Bruna

Cambraia

Garms

_,

_Thaís

_Helena

_da

_Motta

_Conceic¸ão

_,

_Luiz

_Claudio

_Di

_Stasi

_,

João

Tadeu

Ribeiro-Paes

a_{InstituteofBiomedicalSciences,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil}

b_{DepartmentofPharmacology,BiosciencesInstitute,UniversidadeEstadualPaulista,Botucatu,SP,Brazil}

c_{InstituteofHealthSciences,UniversidadePaulista,Assis,SP,Brazil}

d_{SchoolofBiologicalandEnvironmentalSciences,UniversidadeFederaldaGrandeDourados,Dourados,MS,Brazil}

e_{DepartmentofBiologicalSciences,UniversidadeEstadualPaulista,Assis,SP,Brazil}

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Articlehistory:

Received15January2014 Accepted5March2014 Availableonline2July2014

Keywords:

Inflammatoryboweldisease Stemcells

Adiposetissue Celltherapy

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Inflammatoryboweldisease,whichmainlyinvolvesCrohn’sdiseaseandulcerative recto-colitis,isaninflammatoryconditionofthemucosathatcanafflictanysegmentofthe gastrointestinaltract.Despitethefactthattheexistingtherapiesresultinimprovementin patient’ssymptomatologyandqualityoflife,thereisnocurativetreatment.Surgical treat-mentinvolvescomplexproceduresassociatedwithhighmorbidityandmortalityrates.In thiscontext,celltherapywithstemcellshasemergedasatreatmentwithbroad poten-tialapplicability.In thisstudy,weintended toverify the efficacyoftransplantationof adiposetissue-derivedstemcellsinratswithintestinalinflammationinducedby trini-trobenzenesulfonicacid.Thecellpopulationwasisolatedfromtheadiposetissueofinguinal regionofrats andprocessedforculturebymechanicaldissociation. Theanimalswere evaluatedwith respectto clinicalandbiochemicalaspects, aswellas bymacroscopic, microscopicandhistologicalanalyses.Intheexperimentalmodelofbowelinflammation by2,4,6-trinitrobenzenesulfonicacid,theinfusionofadiposetissuesignificantlyreduced thepresenceofadhesionsinthecolonandadjacentorgansanddecreasedtheactivityof myeloperoxidase,amarkerofneutrophilinfiltrationintheinjuredmucosa.Theresults sug-gestthatcelltherapywithadiposetissuecanpromoteand/oracceleratetheregenerationof damagedintestinalmucosa.Itisconcludedthatthepresenceofadhesionsandthe deter-minationofmyeloperoxidaseactivityprovideindicationsthatadiposetissuecanpromote and/oracceleratetheregenerationofinflammatorybowelmucosa.

©2014SociedadeBrasileiradeColoproctologia.PublishedbyElsevierEditoraLtda.All rightsreserved.

夽

StudyconductedatDepartmentofBiologicalSciences,UniversidadeEstadualPaulista(UNESP),AssisCampus,Assis,SP,Brazil.

∗ _{Correspondingauthor.}

E-mail:[email protected],[email protected](J.T.Ribeiro-Paes). http://dx.doi.org/10.1016/j.jcol.2014.06.004

Terapia

celular

em

modelo

experimental

de

doenc¸a

inflamatória

intestinal

Palavras-chave:

Doenc¸aInflamatóriaIntestinal Células-tronco

TecidoAdiposo TerapiaCelular

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e

s

u

m

o

ADoenc¸aInflamatóriaIntestinal(DII),consistindoprincipalmentedadoenc¸adeCrohne retocoliteulcerativa,éumacondic¸ãoinflamatóriadamucosaquepodeacometerqualquer segmentodotratogastrointestinal.Apesardasterapiasexistentesresultaremnamelhora dossintomasedaqualidadedevidadospacientes,nãohánenhumtratamentocurativo. Otratamentocirúrgicoenvolveprocedimentoscomplexosassociadosaaltastaxasde mor-bimortalidade.Nestecontexto,aterapiacelularcomcélulas-troncodespontacomoopc¸ão detratamentopotencialmentepromissora.Emfunc¸ãodestesaspectos,pretendeu-se,no presenteestudo,verificaraeficáciadotransplantedecélulas-troncoderivadasdotecido adiposo(ASC)emratoscominflamac¸ãointestinalinduzidaporácido trinitrobenzenosul-fonico(TNBS).AsASCsforamobtidaspordissociac¸ãomecânicadotecidoadiposodaregião inguinalderatoseprocessadasparacultivo.Osanimaisforamavaliados,considerando-se osaspectosclínicosebioquímicos,além deanálisesmacroscópica,microscópicae his-tológica.Nomodelodeinflamac¸ãointestinalinduzidaporTNBS,ainfusãodeASCsreduziu significativamenteapresenc¸adeaderênciasentreocóloneórgãosadjacentes,bemcomo diminuiuaatividadedamieloperoxidase(MPO),ummarcadordainfiltrac¸ãodeneutrófilos namucosalesada.OsresultadosobtidospermitemconcluirqueaterapiacelularcomASCs podepromovere/ouaceleraroprocessoderegenerac¸ãodamucosaintestinalinflamada.

©2014SociedadeBrasileiradeColoproctologia.PublicadoporElsevierEditoraLtda. Todososdireitosreservados.

Introduction

Inflammatoryboweldisease(IBD)comprisestwonosological entitiesofchronicandrecurrentnature:Crohn’sdisease(CD) andulcerativerectocolitis(URC).1_{CDisatransmural}

inflam-matoryconditionofmucosathatcanaffectanysegmentofthe gastrointestinaltract.2,3_{UnlikeCD,URCisadisease}

character-izedbydiffuseinflammationofcolonicmucosa.URCaffects therectum(95%ofcases)andproximalportionsofthecolon, inasymmetricandcontinuousmanner.4,5

Despite the introduction of new clinical therapeutic approachesandtheircontributiontoimprovethequalityof lifeofpatientswithIBDand/orinthepreventionofrecurrence, thereisnodefinitiveclinicalcurativetreatment.Thesurgical treatment,inturn,involvescomplexprocedures associated withhighratesofmorbidityandmortalityandpostoperative complications.6,7

Severalstudieshaveshownthatmesenchymalstemcells (MSC) may be involved in tissue regeneration, since they havetheabilitytomodulatetheimmuneresponsesof pro-inflammatorycells toinduce anenvironment witha more tolerant phenotype to inflammation.8–11 _{Furthermore, it is}

proposedthatthesecellsactinregulatingtheimmunesystem ininflammatorytissues,puttingintoactionproinflammatory cytokinesandthesecretionofchemokines.12_Thus,itcanbe

postulatedthatMSCcouldexertananti-inflammatoryaction inresponsetotheinductionofinjuryinexperimental mod-elsinvivoand,thus,actingasapotentialtherapeuticagentin inflammatorydiseasessuchasURCandCD.

Inlight ofthis, inthis study we intendedto verify the effectivenessoftransplantationofMSCderivedfromadipose

tissue(ASC)inratswithintestinalinflammationinducedby trinitrobenzenesulfonic acid (TNBS). In addition, with this approach we intend to shed light on pathophysiological aspectsofIBD,aswellastosuggestandevaluatethe effec-tivenessofnewalternativetherapies.

Method

Animals

Inthisstudy,Wistaralbinorats(Rattusnorvegicus),raisedinthe CentralBioterium,UNESP–campusofAssis,wereusedunder controlledtemperature(22◦_{C)andlighting(12hoflight/12hof} darkness)conditionsandfedwithasoliddietsupplemented withVitagold®_{(Tortuga,SaoPaulo,Brazil).}

Experimentalgroups

The animals were divided into the followingexperimental groups:blank(healthyanimals),control(animalswith TNBS-inducedbowelinflammationandtreatedwithphosphate–PBS buffer)andtreated(animalswithTNBS-inducedbowel inflam-mationandtreatedwithmesenchymalstemcells)groups.

Isolationbymechanicaldissociationandcultureof adiposetissue-derivedstemcells

The adipose tissue extracted from the groin of rats was mechanically dissociated with the help of two L-shaped syringe needles (BDTM_{, New Jersey, USA) in RPMI 1640}

antibiotic/antimycotic(Gibco®_{,NewYork,USA)forcell/tissue}

separation. Then, the solution containing dissociated cells wasfilteredthrougha70␮m-filter(CellStrainer–BDFalconTM,

NewJersey,USA)forseparatingcellulardebris.Thecollected materialwascentrifugedfor10minat900×g.Then,thecells wereseededinALPHA-MEMmedium(Gibco®_{,NewYork,USA)}

supplementedwith20%FetalCalfSerum–FCS(Gibco®_,New

York,USA)and1%antibiotic/antimycotictoafinal concentra-tionof1×105_cells/cm2_{andincubatedat37}◦_Cin5%CO

2.Cells

weremaintainedincultureand,whena60–80%confluencein relationtotheculturedishareawasreached,weredissociated withTrypleTM_(Gibco®_{,NewYork,USA).}

Invitrodifferentiationofadiposetissue-derivedstemcells

Inordertoinduceosteogenic,chondrogenicandadipogenic differentiation, specifickits (StemPro® _–Gibco®_{, New York,}

USA) were used, according to manufacturer’s instructions. Theosteogenicdifferentiationwasconfirmedbyanalysisof thematerialstainedwithAlizarinRedS(Sigma–Aldrich®_,St.

Louis,MO,USA),thechondrogenicdifferentiationwithAlcian Bluedye(Sigma–Aldrich®_{, St.Louis,MO,USA),andthe}

adi-pogenicdifferentiation withOilRedO (Sigma–Aldrich®_,St.

Louis,MO,USA).

ExperimentalmodelofIBD

Theinductionofthebowel inflammatoryprocess was per-formedbythemethoddescribedbyMorrisetal.,13_withminor

modifications. 2,4,6-Trinitrobenzenesulfonic acid – TNBS (Sigma–Aldrich®_{,St.Louis,MO,USA)wasfirstlyophilizedand}

subsequently preparedtoobtain asolutionof40mg/mLin 50%ethanol(v/v).Bowelinflammatoryprocessinductionwas performed by intracolonic administration of 0.25mL TNBS solutionatapoint8cmfromtherectum.Theanimalsinthe blankgroupunderwentthesameprocedure,butwith admin-istrationofPBSreplacingTNBS.Allanimalswere sacrificed sevendaysaftertheinductionofIBD.

Celltransplantation

Thecell infusionwas performedby intravenousroute (tail vein).Inthetreatedgroup,1mLofthecellsolutiondiluted inPBSataconcentrationof1×107_{cells/mLwasinfused.The}

controlgroupreceivedasimilartreatment;however,onlyPBS wasinfused.

Evaluationofanti-inflammatorybowelactivity

Theanimalswereevaluated,consideringfoodconsumption, bodyweightandonsetofdiarrhealstools.Attheendofthe experiment,thecolonswereextractedandanalyzedforbowel damage.

Macroscopicanalysis

Theweightand lengthofthe colonand presenceof adhe-sionsinthebowelandsurroundingorganswereevaluated.An

analysisofseverityandextentofbowelinjury,accordingtoa scalepreviouslydescribedbyBelletal.,14_{wasalsoperformed.}

Biochemicaldeterminations

Thedeterminationoftotalglutathione(GSH)contentwas per-formed according tothe method described by Anderson,15

andthedeterminationofmyeloperoxidase(MPO)activityin ratcolonfragmentswasperformedbythemethodofKrawisz etal.16

Histologicanalysis

Immediatelyafterthemacroscopicevaluationofthecolonic inflammatoryprocess,tissuesamples(0.5mm)adjacenttothe injuredareawerecollectedforhistologicalprocessing.These sampleswereplacedinhistologicalcassettes,fixedin4% for-malinandstainedwithhematoxylin–eosin(HE).

Statisticalanalysis

Resultswereexpressedasmean±standarddeviation. Differ-ences between means were tested byanalysis of variance (ANOVA), followed by tests of significance. Nonparametric data (scores) were expressed as median and analyzed by Kruscal–Wallistest.Discontinuousdatawereanalyzedby chi-squared(X2_{)test.Statisticalsignificancewasconsideredwhen}

p<0.05.

Ethicalaspects

ThisstudywasapprovedbytheEthicsCommitteeontheUse ofAnimals(CEUA),UniversidadeEstadualPaulista–UNESP,

campusofAssis(RegistrationNo.012/2011).

Results

Cultureandcharacterizationofadiposetissue-derived stemcells

AsshowninFig.1A,onday7ofcultureasignificantpopulation ofcellswithanelongated,fusiformappearancewasobserved. A70–80%confluencewasachievedaroundthe11thdayof cul-ture(Fig.1B).AfterthefirstdissociationprocesswithTryple®

(Gibco®_{, New York, USA), the cell growth became faster,}

becausethe70–80%confluencelevelwasreachedwithin3–4 days.Itwasalsoobservedthat,afterthethirdpassage,the culturehadassumedtheappearanceofauniformmonolayer ofspindlecells(Fig.1C), indicatingthatprobablytherewas no contaminationwithnon-adherent cells,suchasmature adipocytesanderythrocytes.

To verifywhether the cells isolated fromadipose tissue (ASC) met the requirements established by the Interna-tional Society of Cellular Therapy for validation of MSC,17

A

B

C

Fig.1–Behaviorofadiposetissue-derivedstemcellsatdifferentperiodsofculture.(A)Primaryculture(7days);(B)after11 days(70–80%confluence);and(C)homogeneousmonolayerofcellswithfibroblastoidaspect(thirdpassage).

Fig.2–DifferentiationofASCintochondrocytes,osteocytes andadipocytes.(A)CultureofASCintheabsenceofan inductionmedium(20×);(B)proteoglycanssynthesizedby chondrocytesandstainedwithAlcianBlue(40×);(C)bone matrixstainedwithAlizarinRedS(40×);and(D)lipid vacuolesstainedwithOilRedO(40×).

staining (Fig. 2C). Finally, the differentiation of ASC into adipocyteswasproven.AsseeninFig.2D,lipidvacuolesare stainedinred.

Evaluationofbowelanti-inflammatoryactivity

Thefood intake showed that the average consumption by healthy animals was higher, when compared with control

group animals. The consumption of blank group animals was statisticallydifferent,comparedtocontroland treated groups.However,therewasnostatisticallysignificant differ-encebetweencontrolandtreatedgroups.

Thedailybodyweightevaluationshowedthat,duetothe reductionoffoodconsumption,therewasadecreaseinbody weightofanimalssubjectedtotheadministrationofthe alco-holicsolutionofTNBS.Themeandeltabodyweightofblank groupshowedstatisticallysignificantdifference,when com-paredtocontrolandtreatedgroups.However,therewasno statisticallysignificantdifferencebetweentreatedandcontrol groups.

Thepresenceofdiarrhealstoolswasdailymonitoredin allgroupsofanimals.Withrespecttocontrolgroupanimals, anincidenceof100%wasobserved,whereasintreatedgroup animals the incidencewas 90%.An analysisofthe results showednostatisticallysignificantdifferencebetweencontrol andtreatedgroups.

Macroscopicevaluation

Colonweight/length(W/L)

There is a relationship for colonic injury resulting from inflammation,andthiscanbequantitativelyexpressedbyan increaseincolonicweight/lengthratio(W/Linmg/cm).With respecttotheresultspresentedinTable1,wenotedthatthere wasnostatisticallysignificantdifferenceinrelationtocolonic W/Lbetweencontrolandtreatedgroups.

Adhesions

Presenceofadhesionsintheintestineandsurroundingorgans isalsoconsideredacharacteristicfeatureofbowelmucosa inflammation.Table1shows 83.33%ofadhesionincontrol groupanimalsand20%inASC-treatedanimals.Astatistically

Table1–Macroscopicparametersfromtreatmentwithadiposetissue-derivedstemcells(ASC)inanexperimentalmodel ofinflammatoryboweldisease,byadministeringanalcoholicsolutionofTNBS.

Group Score(0–10)a _{Injuryextent(cm)}b _{Weight/lengthratio(mg/cm)}b _Adherence(%)c

Blank 0a ₀a _{106.77±3.241}a ₀a

Control–PBS 7(5–9)b _3.70±0.741b _{269.57±43.851}b _83.33b

TreatedwithASC 6.5(4–9)b _3.51±0.415b _{199.36±27.85}b ₂₀c

a _{Thescorevaluesareexpressedasmedian(range).}

b _{Injuryextentandcolonicweight/lengthratioareexpressedasmean±SEM(standarderrorofthemean).}

significantdifferencebetweencontrolandtreatedgroupswas found.

Severityandextentofbowelinjury

TheadministrationofTNBSincontrolgroupanimalstriggered aninflammatoryprocesscharacterizedbylesionsalongthe colon,between1.20and5.50cminlengthandascorebetween 5and9(Table1).Theresultsshownostatisticallysignificant differenceinrelationtothelesionlengthanditsscoreamong PBS–controlgroupanimalsversusASC-treatedanimals.

Biochemicalanalysis

The animals treated with the alcoholic solution of TNBS showed a statisticallysignificant difference in regard to a reduction in glutathione levels, when compared to blank groupanimals. AlthoughGSH levelswerehigher intreated animals,therewasnosignificantdifferencewhenconfronting theresultsamongPBS-treatedversusASC-treatedanimals.

Inthecaseofmyeloperoxidase(MPO)activity,itwasfound that the treatment with adipose tissue-derived stem cells resulted ina statisticallysignificant difference in terms of enzymeactivity,whencomparedtocontrolgroup,ascanbe seeninFig.3.

Histologicalanalysis

Fig.4Ashowsthenormalcoloniccytoarchitectureofahealthy animal, where the cells are intact, and mucosal, submu-cosalandmuscularismucosalayersandtheirdimensionsare

Blank PB S

Treatment

0 200 400 600 800 1000

***

*

MPO (U/g of tissue) a

b

c

Fig.3–Assessmentoftheactivityofmyeloperoxidase (MPO)inintestinalmucosaofaninflammatorybowel diseaseanimalmodel.Dataareexpressedasmean±SEM. Valueswithoutlettersincommondifferatp<0.05.

clearlyidentified,withoutanykindofmorphologicalchange. Fig. 4Bdepicts the colon ofan animal with TNBS-induced inflammatoryprocess,characterizedbymucosal disruption and the presence of abnormal cells as to form, size and number comparedtohealthy animals.Additionally, alarge submucosalcellinfiltrate,typicalofneutrophilmigrationdue tocolonicinflammation,isperceived.Fig.4Cdepictsthecolon

ofananimalundergoingtreatmentwithASC,wherealarge cellularinfiltrateduetoTNBS-inducedcolonicinflammation isnoted.However,thereisnomucosaldisruption–an occur-renceclearlyseeninPBS-treatedanimals.Themucosalcells arenotsignificantlyalteredinshape,andaclearrecoveryof coloniccytoarchitecturecanbeperceived.

Discussion

and

conclusion

Stemcellsderivedfromadiposetissue(ASC)havebeenused experimentally asa potentially promising alternative ther-apyinanumberofdiseases.Thetherapeuticeffectofstem cellsisattributabletoaprobableimmunomodulatoryaction, withsuppressionorinhibitionoftheinflammatoryprocess, aswellasbyangiogenesisstimulation.8–12,18_{Inthiscontext,}

IBDcanbeincludedinthegroupofdiseases thatcan ben-efitfromcelltherapywithstemcells.TheASCusedinthis studywasidentifiedasapopulationofelongated,fusiform cells,acharacteristicfeatureofMSCandshowingplastic sur-faceadherence.Furthermore,ASCpresented,wheninspecific conditionsofinduction,thefunctionalpropertyof differen-tiationin vitro inchondrogenic, osteogenicand adipogenic lineages,consistentwithvalidationcriteriareportedbyother authorsanddefinedbytheInternationalSocietyforCellular Therapy.11,17

Theenzymaticdigestionbyusingcollagenaseiscurrently the most widely used procedure for obtaining and isolat-ingASC.18,19 _{Inthisstudy,weusedaninnovativeandnovel}

techniqueofmechanicaldissociationforASCisolationand culture.Itshouldbeemphasizedthatthemechanical dissoci-ationmakesunnecessarytheuseofcollagenase,maintaining theefficiencyoftheprocessandbeingcheaper.

Itiswellestablishedthatoneofthesymptomsofpatients sufferingIBDisareductioninbodyweight,consideringthat thereisadecreaseinfoodintake,besidestheoccurrenceof gastrointestinal disturbances.20 _{Areductionin foodintake,}

withconsequentweightlossoftheanimalsafterthe induc-tionofIBD,wasseenincoliticgroups.However,itwasfound thatfoodintakeanddecreaseofbodyweightinASC-treated animals were similar to those for control group animals. According to Morris et al.,13 _{the IBD induction model by}

TNBS/ethanolproducesintestinalwallulcerationand thick-ening, without affectingsignificantly the long-term weight increaseofanimals.Thus,the needforalonger periodfor restorationofnormalfoodintakeand,thus,bodyweight,is inferred.

Commonly,diarrheaoccurswithaninflammationofthe colonandsmallintestine,asaresultfromtheactionof inflam-matorymediatorsinbowelepithelium.21 _{Theoccurrenceof}

thisobservationparameter,acharacteristicfeatureofIBD,was observedin90%ofASC-treatedgroupanimals,showingthat, after7daysoftreatment,thereisstillalargeamountof inflam-matorymediatorsinthecolon.Thesedataraisethepossibility thattheperiodforassessingtheeffectivenessofcelltherapy asanalternativetreatmentforIBDshouldbeextendedbeyond the7-dayperiodadoptedinthisstudy.

Otherindicatorsofseverityandextentofinjuryresulting fromIBDare:increaseinweightanddecreaseincoloniclength inanimalswithbowel inflammation.11,13,22_{Thereisaclear}

differenceinmeanW/Lratio,astheTable1shows.However, therewasnostatisticallysignificantdifferencebetween ani-malstreatedwithstemcellsversuscontrolanimals(Table1). This effect is possibly due to the severe injury caused by theadministrationofthealcoholicsolutionofTNBS,thereby complicatingtheregeneratingprocessoftheinjuredmucosa. Andoetal.11_{reportedthataninjectionofASCdirectlybeneath}

thecolonsubmucosasignificantlydecreasestheweightofthe inflamed colon. Thisisanindicationthatthe location and routeofcelladministrationcaninfluencethefinaloutcome ofatreatment,aspreviouslyreportedbyotherauthors.23,24_In

thisstudyweadoptedanIVinfusionrouteforASC,sothat thedivergenceamongtheresultsofthisstudyandofthose reportedbyAndoetal.11_{couldberelatedtothedifferent}

infu-sionrouteusedinthestudy.

ItisalsopossibletoevaluatetheintensityofIBDlesion severity by its length and scoring.25 _{Table 1 shows that}

thecolonicdamagecausedbytheinfusionofalcohol/TNBS wassevereandextensive,makingtherecoveryofintestinal mucosaunlikely after7daysofASCinfusion.In2009, Wei et al.26 _{compared theinfusionof hematopoieticstem cells}

(HSC),bonemarrowstemcells(MSC)andamixtureofbothcell typesinratswithTNBS-inducedcolitisafter7,14and21days. Theauthorsfoundthatadecreaseinthelesionscoreoccurred onlyonday21.Furthermore,this resultwas onlyobserved inthegrouptreatedwithaHSC/MSCcombination.Thus,we caninferthat,duetotheintensityandextentoftheinjury, itwouldbenecessarytoincreasethetimeperiodtoevaluate thestemcellperformancewithrespecttotheabilitytoreduce inflammationandpromoteregenerationoftheinjuredtissue. Anotherpointtobeconsideredreferstothepossibilityofa HSC/MSCco-infusion,consideringthatthesynergisticeffect ofthesedifferentcelltypescanactinabeneficialwayinthe treatmentofanumberofdiseases.27,28

The presence of adhesions among the colon and sur-rounding organs and/or tissues as a result of transmural inflammationisacommonfeatureofIBD,especiallyinCD.29

Table1showstheoccurrenceofasignificantreductioninthe incidenceofadhesionsincoliticanimalsandinASC-treated animals compared withcontrol group animals, suggesting that ASCacted asanagentprotecting and/orreducing the inflammatoryprocess.

Itcanbenotedthatinexperimentalmodelsofintestinal inflammationbyinstillationofTNBS,GSHdepletionresulting fromcolonicinflammationoccurred.22,25,30

ItcanbeseenthattheASCsuseddidnotpresentpotential topromoteincreasedlevelsofGSH.Itisnoteworthythatno studyonhumansoranimalmodelswithintestinal inflamma-tionrecordingdosagesoftotalglutathionelevelsafterinfusion ofstemcellshasbeenpublished.

Amongthebiochemicalanalyzes,myeloperoxidase(MPO) is an important tool for identifying the presence of neu-trophilic infiltrates and for revealing an inflammatory process as a result of the colonic inflammation. It is known that MPO levelreflects the quantityof neutrophils, and a decrease in its activity reflects a decrease in the injured tissue inflammation.16 _{In the present study, it}

anti-inflammatoryagent,diminishingthedamagecausedby TNBSadministration.11,30

The presence of adhesions and the determination of myeloperoxidaseactivityprovideanindicationthatASChave theabilitytopromoteand/oracceleratetheregeneration pro-cessininflamedintestinalmucosae.However,methodological adjustmentsshould beincluded infuture projects. Among thepropositions ofreadjustment,thereshouldbeincluded anevaluationoftheeffectivenessofcelltherapyforalonger periodoftime;cellinfusionbyotherroutesofadministration, inadditiontointravenousroute;andfeasibilityofthistherapy throughco-infusionofASCinassociationwithhematopoietic stemcells(HSC).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors thank the Mayor of Assis and its City Coun-cil (SP – Brazil) and the Consórcio Intermunicipal do Vale doParanapanema(CIVAP/Health)forfinancialsupport.The authorsMonicaYonashiroMarcelinoandNataliaLangenfeld FuocowerefundedbytheCoordenac¸ãodeAperfeic¸oamento dePessoaldeNívelSuperior(CAPES–Brazil).

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