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
a,
Natália
Langenfeld
Fuoco
a,
Ana
Elise
Valencise
Quaglio
b,
Renata
Aparecida
de
Camargo
Bittencourt
c,
Bruna
Cambraia
Garms
d,
Thaís
Helena
da
Motta
Conceic¸ão
e,
Luiz
Claudio
Di
Stasi
b,
João
Tadeu
Ribeiro-Paes
e,∗aInstituteofBiomedicalSciences,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil
bDepartmentofPharmacology,BiosciencesInstitute,UniversidadeEstadualPaulista,Botucatu,SP,Brazil
cInstituteofHealthSciences,UniversidadePaulista,Assis,SP,Brazil
dSchoolofBiologicalandEnvironmentalSciences,UniversidadeFederaldaGrandeDourados,Dourados,MS,Brazil
eDepartmentofBiologicalSciences,UniversidadeEstadualPaulista,Assis,SP,Brazil
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t
i
c
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Articlehistory:
Received15January2014 Accepted5March2014 Availableonline2July2014
Keywords:
Inflammatoryboweldisease Stemcells
Adiposetissue Celltherapy
a
b
s
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t
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:jtrpaes@yahoo.com.br,joao.paes@pq.cnpq.br(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
r
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).1CDisatransmural
inflam-matoryconditionofmucosathatcanaffectanysegmentofthe gastrointestinaltract.2,3UnlikeCD,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.12Thus,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 wasfilteredthrougha70m-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-tionof1×105cells/cm2andincubatedat37◦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.,13withminor
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 inPBSataconcentrationof1×107cells/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.,14wasalsoperformed.
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±3.241a 0a
Control–PBS 7(5–9)b 3.70±0.741b 269.57±43.851b 83.33b
TreatedwithASC 6.5(4–9)b 3.51±0.415b 199.36±27.85b 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.8–12,18Inthiscontext,
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.11,13,22Thereisaclear
differenceinmeanW/Lratio,astheTable1shows.However, therewasnostatisticallysignificantdifferencebetween ani-malstreatedwithstemcellsversuscontrolanimals(Table1). This effect is possibly due to the severe injury caused by theadministrationofthealcoholicsolutionofTNBS,thereby complicatingtheregeneratingprocessoftheinjuredmucosa. Andoetal.11reportedthataninjectionofASCdirectlybeneath
thecolonsubmucosasignificantlydecreasestheweightofthe inflamed colon. Thisisanindicationthatthe location and routeofcelladministrationcaninfluencethefinaloutcome ofatreatment,aspreviouslyreportedbyotherauthors.23,24In
thisstudyweadoptedanIVinfusionrouteforASC,sothat thedivergenceamongtheresultsofthisstudyandofthose reportedbyAndoetal.11couldberelatedtothedifferent
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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