Consumption of cranberry polyphenols enhances human γδ-T cell proliferation and reduces the number of symptoms associate...

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Title:
Consumption of cranberry polyphenols enhances human γδ-T cell proliferation and reduces the number of symptoms associated with colds and influenza: a randomized, placebo-controlled intervention study
Series Title:
Nutrition Journal
Physical Description:
Mixed Material
Creator:
Meri P Nantz
Cheryl A Rowe
Catherine Muller
Rebecca Creasy
James Colee
Christina Khoo
Susan S Percival
Publisher:
Nutrition Journal
Publication Date:

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Subjects / Keywords:
Cranberry
Proanthocyanidins
Immunity
γδ-T cell
NK cell

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Abstract:
Background: Our main objective was to evaluate the ability of cranberry phytochemicals to modify immunity, specifically γδ-T cell proliferation, after daily consumption of a cranberry beverage, and its effect on health outcomes related to cold and influenza symptoms. Methods: The study was a randomized, double-blind, placebo-controlled, parallel intervention. Subjects drank a low calorie cranberry beverage (450 ml) made with a juice-derived, powdered cranberry fraction (n = 22) or a placebo beverage (n = 23), daily, for 10 wk. PBMC were cultured for six days with autologous serum and PHA-L stimulation. Cold and influenza symptoms were self-reported. Results: The proliferation index of γδ-T cells in culture was almost five times higher after 10 wk of cranberry beverage consumption (p <0.001). In the cranberry beverage group, the incidence of illness was not reduced, however significantly fewer symptoms of illness were reported (p = 0.031). Conclusions: Consumption of the cranberry beverage modified the ex vivo proliferation of γδ-T cells. As these cells are located in the epithelium and serve as a first line of defense, improving their function may be related to reducing the number of symptoms associated with a cold and flu. Trial registration: ClinicalTrials.gov Identifier: NCT01398150.

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Source Institution:
University of Florida
Holding Location:
University of Florida
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All rights reserved by the source institution.
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AA00020087:00001

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RESEARCHOpenAccessConsumptionofcranberrypolyphenolsenhances human -Tcellproliferationandreducesthe numberofsymptomsassociatedwithcoldsand influenza:arandomized,placebo-controlled interventionstudyMeriPNantz1,CherylARowe1,CatherineMuller1,RebeccaCreasy1,JamesColee2,ChristinaKhoo3andSusanSPercival1*AbstractBackground: Ourmainobjectivewastoevaluatetheabilityofcranberryphytochemicalstomodifyimmunity, specifically -Tcellproliferation,afterdailyconsumptionofacranberrybeverage,anditseffectonhealthoutcomes relatedtocoldandinfluenzasymptoms. Methods: Thestudywasarandomized,double-blind,placebo-controlled,parallelintervention.Subjectsdrankalow caloriecranberrybeverage(450ml)madewithajuice-derived,powderedcranberryfraction( n =22)oraplacebo beverage( n =23),daily,for10wk.PBMCwereculturedforsixdayswithautologousserumandPHA-Lstimulation. Coldandinfluenzasymptomswereself-reported. Results: Theproliferationindexof -Tcellsinculturewasalmostfivetimeshigherafter10wkofcranberrybeverage consumption( p <0.001).Inthecranberrybeveragegroup,theincidenceofillnesswasnotreduced,however significantlyfewersymptomsofillnesswerereported( p =0.031). Conclusions: Consumptionofthecranberrybeveragemodifiedthe exvivo proliferationof -Tcells.Asthesecells arelocatedintheepitheliumandserveasafirstlineofdefense,improvingtheirfunctionmayberelatedtoreducing thenumberofsymptomsassociatedwithacoldandflu. Trialregistration: ClinicalTrials.govIdentifier:NCT01398150. Keywords: Cranberry,Proanthocyanidins,Immunity, -Tcell,NKcellIntroductionCranberriesandcranberryjuiceareassociatedwithpromotingurinarytracthealth[1,2]However,althoughthe responsibilityofimmunecellsistosurveytheirenvironmentandpreventbacterialandviralinfectionsfrom overwhelmingthebody,muchoftheliteratureregarding cranberryresearchhasfocusedonadherenceofbacteria [3-6]ratherthanmodificationofimmunefunction. Fourstudieshaveshowntheeffectsofcranberryon immunefunctionindiverseways:inarabbitmodelof infection-inducedoxidativerenaldamage,cranberryreducedinflammation[7];consumptionofacranberry beverageinahumaninterventionstudyresultedinareductioninpathogenin42%ofthesubjectswithoutalteringnormalvaginalmicrobiota[6];lowerlevelsof urinaryIL-6werefoundinpregnantwomenafterdrinkingcranberryjuiceforatleast3days[8];andanenhancedgenerationofanti-lymphomaantibodieswas detectedinanimmuno-competentmousemodelof lymphoma[9].Recentstudieshaveshowneffectiveness ofcranberryinreducingreoccurrenceofurinarytract *Correspondence: percival@ufl.edu1DepartmentofFoodScience&HumanNutrition,UniversityofFlorida,Box 110370,Gainesville,FL32611,USA Fulllistofauthorinformationisavailableattheendofthearticle 2013Nantzetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublicDomainDedication waiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwise stated.Nantz etal.NutritionJournal 2013, 12 :161 http://www.nutritionj.com/content/12/1/161

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infections[10-12].Recentclinicalinterventionswithcranberryhavefocusedonpyuriaandbacteriuria[13,14],but didnotexaminetheinfluenceonsystemicimmunity. Thesestudiessuggestthatsystemicimmunityismodified bythebioactivecompoundsincranberries,buthavenot directlyassessedit. Animmunecellparticularlysuitedtosurveillanceof thegenitourinarytractisthe -Tcell,whichisstrategicallylocatedintheepitheliumofboththeintestine andthereproductivetract.Wehaveshowninhuman consumptionstudieswithanencapsulateddriedfruit andvegetablejuicefraction[15],twocompoundsderivedfromtea[16],andConcordgrapejuice[17],that variousphytochemicalsmodify exvivo -Tcellproliferation.Phytochemicalsandproanthocyanidinsfromherbal preparationsalsointeractwith -Tcellsinvitro[18-20]. Cranberrypolyphenolsandproanthocyanidins,then, wouldseemtobepotentialcandidatesformodifying humanimmunity. Ourobjectivewastodetermineifapolyphenolcontainingfractionofcranberrywouldhaveimmunomodulatingactivitiesinhumans.Ourprimaryoutcome wasproliferationof -Tcells exvivo ;however,theproliferationofotherimmunecellswasalsoexamined.Our secondaryoutcomewastheevaluationofillnesssymptomsduringthe10-weekstudy.MaterialsandmethodsSubjectsAtotalof54healthysubjects(17menand37women), ranginginagefrom21to50years,withabodymass indexbetween18and30kg/m2,wererecruitedby postedadvertisementstoparticipateina10-wk,doubleblind,randomized,placebo-controlled,paralleltrial.The UniversityofFloridaInstitutionalReviewBoardapprovedthestudyprotocol,andinformedwrittenconsent wasobtainedfromeachsubject.Subjectswererequired tobegenerallyhealthy,andexclusioncriteriawere:takingimmunosuppressivedrugs,recentorchronicantibiotics,antioxidantsupplementsorprobiotics,orany flavonoid-containingsupplements;lactatingorbeing pregnantoronhormonetherapy;beingachronicuser ofnon-steroidalanti-inflammatorydrugs;havinganongoinginfectionorhypertensionthatrequiredmedication;consumingmorethan14alcoholicbeveragesper week,eatingmorethan7fruitsandvegetablesperday orfollowingavegetarianorstrictvegandiet.Participantswerenotallowedtobeginthestudyiftheywereill atthetimeofthefirstblooddraw.Participantswerein contactwiththeenrollingresearchassistantbye-mail andtelephone,weeklyormoreoften,throughoutthe study. Thepredefinedprimaryoutcomewasproliferationof -Tcellsin exvivo culture.Poweranalysis,( =0.05 andpower=0.80),basedondatawhereadifference wouldbesignificantiftheproliferationof -Tcellswas doublethatoftheplacebogroupwithanexpected standarddeviationof1.74,indicatedthat13subjects wouldbeneededpergrouptodetectastatisticaldifference.Apredefinedlevelofcompliancewasconsumption of80%oftheallottedjuice.StudydesignThestudywasconductedbetweenMarchandMayof 2009,tocoincidewithnormalcoldandinfluenzaseason intheSoutheast(CDC,http://www.cdc.gov/flu/;accessed October2011).TheCDCweeklyreportofinfluenzaactivityinthestateofFloridathatyearwasasfollows:March, widespreadtoregionalactivity;April,localtosporadicactivity;May,regionaltolocalactivity. SubjectsarrivedtotheFoodScienceandHumanNutritionbuilding,foraninitialbaselinefastingblooddraw (Day0)andwererandomlyassigned,bydrawingcards fromanopaqueenvelopethatwerenumberedeither246 or638,toreceivetheexperimentaltreatment[cranberry beverage(CB)]oraplacebobeverage(PB),bothprovided byOceanSprayCranberries,Inc.(Lakeville,MA).Both subjectsandinvestigatorswereblindedregardingthe treatmentgroups.TheCBcontainedcranberrycomponentsfromjuice,filteredwater,sugar,naturalflavors, citricacid,andsucralose.ThePBwasacolor-(Red40and Blue1),calorie-,andsweetener-matchedbeveragewithout cranberrycomponents.Theexperimentalbeverageisnot commerciallyavailable,butwasformulatedtocontaina levelofpolyphenolssimilartothatfoundincommercially availablecranberryjuicecocktails.Thefractionofcranberrycomponentsused,derivedfromthejuiceofcranberries,waspreparedandanalyzedbythemanufacturer (Table1).High-performanceliquidchromatographyanalysisofthefractionusedtopreparethebeveragewasconducted(manuscriptsubmitted). Subjectsweregivenbottlescontaining450ml(15oz) ofbeverageandinstructedtodrinkonebottlethroughouttheday,eachday,for70days(10wk).Participants weresuppliedwithmorebottlesthanwereneededto completethestudy,incaseschedulingconflictsdelayed thesecondblooddraw.Topreventbottlesfrombeing discarded,subjectswereinstructedtobringbackany remainingbottlesattheendofthestudy. Participantswerealsogivenadailyillnesslogtorecordanycoldandinfluenzasymptoms(listedbelow)they experiencedduringthe10-wkexperimentalperiod.Each daytheyhadtoanswerthisquestion:Didyouhaveany illnesssymptomstoday?Iftheyansweredyes,thenthey wereaskedtochecktheboxwitheachofthesymptoms theyhadthatday. Theprimaryoutcomeofthisstudywasdefined,prospectively,asphysiologicalchangesto -TcellproliferationinNantz etal.NutritionJournal 2013, 12 :161 Page2of9 http://www.nutritionj.com/content/12/1/161

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exvivo culture.Illnesssymptomswereconsideredassecondaryoutcomesbecauseofthesmallnumberofsubjects, andbecausetheywereself-reported.Theseoutcomes weredefinedasincidence(numberofpeoplereportingan illnesspergroupandthenumberofillnessespergroup), duration(totalnumberofdayswithatleasttwosymptoms andaveragenumberofdayspergroup),andtotalnumber ofsymptomspergroup.Symptomslistedinthedailydiary were:runnyorcongestednose,cough,sneezing,fever and/orchills,sorethroat,headache,wheezing,andintestinaldistress(nausea,vomiting,diarrhea,and/orabdominalcramps).Allergysymptomswerenotincludedinthe analysisofsymptoms.Subjectswereinstructedastothe differentmanifestationsofcolds,influenzaandallergies: Colds – symptomsoccuroneatatime,generallylastfor 5-7days,yellow/greenishnasaldischarge,mayormaynot beaccompaniedbyafever,withslightbodyachesand pains; Influenza – symptomsoccurrapidly,lastupto 14days,nonasaldischarge,oftenahighfeverandsometimeschills,withseverebodyachesandpains,including headache; Allergy – symptomsoccurrapidlyandallat once,lastaslongastheallergy-causingagentispresent, clearandwaterynasaldischarge,notassociatedwitha fever,andnobodyachesorpains.Therecordofthemedicationstakenduringanillnessalsohelpedtodistinguish allergyfromcoldsandinfluenza.Subjectswereaskedto reportiftheymissedclassorwork,whethertheysought medicaltreatment,iftheywereprescribedanymedicationsasaresultofseekingtreatment,whichover-thecountermedicationstheytook,andwhethertheyhada significantdecreaseinnormalactivitiesduetoillness symptoms. At10wk,studyparticipantsreturnedforafinalblood drawandtocompleteanexitquestionnaire.Theexit questionnaireincludedquestionstodetermineifsubjectsexperiencedanysideeffectsfromthebeverage, theirestimateofcomplianceandiftheygenerallyadheredtotheinclusion/exclusioncriteria.Todetermine efficacyofblinding,subjectsreportedwhetherthey thoughttheyhadreceivedtheCBorthePBandwere askedwhytheybelievedthat.Studycompliancewas assessedbycomparingthenumberofbottlesofbeverage returnedattheendofthestudy(primaryassessmentof compliance)withthenumbertheyshouldhavereturned.Bloodcollectionandperipheralbloodmononuclearcell (PBMC)isolationBloodwasobtainedfromfastingsubjectsonDay0 (baseline)andat10wk.Fastingrequiredeatingnofood aftermidnightofthepreviousday.Bloodwascollected intoone10mlsodiumheparintubeforPBMCisolation, andone10mlSST ™ tube(Vacutainer,BectonDickinson, FranklinLakes,NJ)toobtainserum.Alltubeswere processedwithin2hofbloodcollection.SerumwasremovedfromSST ™ tubesaftercentrifugation(2,000g, 10min,4C)andusedasautologousseruminculture media.Aliquotsofserumwerefrozenat 80Cforantioxidantanalysis.Wholebloodwasdiluted1:1with0.9% NaClandlayeredonagradient(LympholyteHCellSeparationMedia,CedarlaneLaboratoriesLtd.,Burlington, NC)toseparatePBMCbycentrifugation(800g,20min, 20C).Themononuclearcelllayerwasremovedand washedtwicewithRPMI1640(Cellgro,Mediatech,Inc., Manassass,VA)completemedium(100,000U/Lpenicillin;100mg/Lstreptomycin;0.25mg/Lfungizone;50mg/ Lgentamicin;2mmol/LL-glutamine;25mmol/LHEPES buffer).CellpelletswereresuspendedinRPMI1640 completemediumandcountedonahemocytometer.CultureofPBMCforproliferationandcytokineproductionOnDay0,1.0106PBMCinRPMI1640complete mediumcontaining50 M2-MEand10%autologous serumwereseededintotwowellsofduplicate24-welltissuecultureplates(Costar,CorningIncorporated,Corning, NY).Phytohemagglutinin(PHA-Lfrom Phaseolusvulgaris )atafinalconcentrationof10 g/ml,wasaddedto onesetofwellsoneachplate,whiletheothersetwas broughttovolumewithRPMI1640completemedium. Theplateswereincubatedat37Cinahumidified5% CO2atmosphere.After24h,supernatantfluidsfromone platewereharvestedandfrozenat 80Cforfuturecytokineanalysis.OnDay3,humanrecombinantIL-2(BD Biosciences,SanJose,CA),ataconcentrationof1ng/ml, wasaddedtoallwellsofthesecondplate,whichwasincubateduntilDay6whencellswereharvestedandprocessedforflowcytometry. Table1Chemicalcharacterizationaofthecranberry treatmentandplacebobeveragesCranberrybeveragePlacebobeverage Proanthocyanidins,%dwbb65-77%0-1% Sugars,%dwb0.77-1.12%0% Anthocyanins,%dwb6.8-11.3%Notdetected Organicacids,%dwb0.5-0.9%0.1-0.2 Phenolicacids,%dwb7.1-7.5%Nottested Flavonols,%dwb6.8-10.0%Nottested TotalSolids,%dwb87.0-107.8%0.1-1.2% Sucralose( g/mL)152149 VitaminCNotdetectedNotdetected ORACc( MAA/g)62.1Notdetected Colorant(Red40/Blue1)None1% Brixbyrefractometry(o)0.250.23aCharacterizationofthebeverageswasbystandardmethodologyand performedbythemanufacturerofthebeverages.Valueswereassessedinthe labofSSPbystandardmethodologyandwerecomputedagainstanascorbic acid(AA)standardcurve.bdwb,dryweightbasiscORAC,oxygenradical absorbancecapacity.Nantz etal.NutritionJournal 2013, 12 :161 Page3of9 http://www.nutritionj.com/content/12/1/161

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FlowcytometryPBMCwereanalyzedbyflowcytometryonDays0and6 ofculture,usingcellsurfacemarkersforidentification. Specificcellpopulationnumberswereexpressedasa percentageoftotalcells[Bcells,monocytesandnatural killer(NK)cells]orasapercentageoftheCD3+population( -Tcellsand -Tcells).Todetermineproliferationchangesofthevariouscelltypesatbothblood draws,thefoldchangeofindividualsubjectswascalculatedastheratioofnumbersofculturedcellstounculturedcells(Day6/Day0).Ifnoproliferationoccurred duringthattime,theratiowouldequal1.0.Then,the foldchangebeforebeverageconsumption(baseline)was subtractedfromthefoldchangeafterconsumption (10wk).AntibodieswereobtainedfromeBioscience,Inc. (SanDiego,CA)andusedtodetectspecificcelltypes,as follows: -Tcells(PE-CD3andFITC-TCR); -T cells(PE-CD3andFITC-TCR);NKcells(PE-CD314 andFITC-CD56);Bcells(PE-CD19)andMonocytes (FITC-CD14).Afterstaining,cellswerewashedandfixed with1%paraformaldehyde.DatawasacquiredonaBD BiosciencesFACSortflowcytometerwithCellQuestPro software(BDBiosciences,SanJose,CA)within48h. FlowJoAnalysisSoftware,version7.5(TreeStar,Inc., Ashland,OR)wasusedfordataanalysis.Cytokineleveldeterminationincellculturesupernatant fluidsLevelsofcytokines(IFN,IL-1 ,IL-1 ,IL-13,MIP-1 andTNF)secretedbyPBMCinculturefor24h,were quantifiedusingaHumanCytokineMultiplexImmunoassaykit,accordingtothemanufacturer ’ sdirections (MilliporeCorp.,Billerica,MA).Standardsandcontrols wereprovidedwiththekit.Thebeadswereanalyzedon aLuminex200instrument(LuminexCorporation, Austin,Texas)withxPONENT3.1software.Inaddition, levelsofIL-17weredeterminedbyELISAaccordingto manufacturer ’ sdirections(eBioscience,SanDiego,CA) withkitstandardsandcontrols.Thefluidswerethawed oniceandusedundilutedintheassay.Finalabsorbance wasmeasuredat450nmonaSPECTRAmax340PC platereaderanddataanalyzedusingSOFTmaxPro5.2 (MolecularDevices,Sunnyvale,CA).Allvaluesareexpressedaspg/ml.Serumoxygenradicalabsorbancecapacity(ORAC)TheORACassayconsistedofmonitoringtheinhibitionof decayoffluoresceininthepresenceoftheperoxylradical generator2,2 azobis2-amidinopropanedihydrochloride (AAPH).Therateoffluorescencedecaywasmonitored overtimebycalculatingtheareaunderthefluorescent decaycurveandquantifiedusingastandardcurveof Trolox(0.312-2.5 M,FlukaChemical/Sigma).Antioxidantactivityinprotein-freeserawasdeterminedusingthe ORACassay,aspreviouslydescribed[21,22]fora96-well microplatereader,withsomemodifications.Briefly,atcollectionserawerediluted1:5in6%meta-phosphoricacid andfrozenat-80C.Afterthawing,theywerecentrifuged toprecipitateprotein(12,000g,5min,4C)anddiluted 1:10inphosphatebuffer.Then,50 lofeachsamplewere pipettedintoduplicatewells.Serumsamplesforquality controlwerepreparedinourlaboratoryandusedineach assayplatetodeterminevariability.Platetoplatevariabilitywas7.1%.Fluoresceinsolution(20nM)wasaddedto allwells.Theplatewasmixedfor3minutes,andthen equilibratedfor7minutesto37CinaSPECTRAmax GeminiXPSfluorescentplatereader(MolecularDevices, LLC,Sunnyvale,CA).FreshlypreparedAAPHfree-radical solution(140mMinphosphatebuffer)wasadded(50 l), andtheFITCfluorescencedecaywasmonitoredat32-sec intervalsfor40min(excitation,485nm;emission,538nm; cutoff,530nm).ORACvalueswerecalculatedasthearea underthecurveusingSOFTmaxPro5.2Software (MolecularDevices,LLC,Sunnyvale,CA),andthedata expressedin molTroloxequivalents.StatisticalanalysisForthecytokineandimmunecellmeasures,anonparametricWilcoxon/Kruskal-WallisRanksumtestwas performedonthedifferencesinlevelsbeforeandafter supplementation.Illnessandsymptomdatawasanalyzed bylogisticalregressionwithageneralizedlinearmodel andanexponentialdistribution.Anover-dispersionparameterwasused,duetolarger-than-expectedvariance (JMP,version8,Cary,NC).Anominalalphalevelof0.05 wasusedtodeclarestatisticalsignificance.Asub-group analysisofallprimaryindiceswasperformedtodetermine andtoaddressblindingissues,comparingthosewho guessedcorrectly(n=5)withthosewhodidnot.ResultsOf68peopleassessedforeligibility,54individualsenrolledinthestudy(Figure1).Duringthestudy,9people withdrew:sixbecausetheywereunabletoreturnforthe finalblooddraw,twobecauseofthetasteofthebeverage, andonewithnoexplanation.Ofthe54subjectsenrolled, 45(83%)completedthestudy.Overallcompliance,determinedbyreturnedbottlecount,was96.1%3.7%bottles ofbeverage(Table2).Allparticipantsconsumedatleast 80%oftheirallocatedbeverage;therefore,noonewasexcludedfromdataanalysisbasedonnoncompliance. Regardingblinding,allsubjects(100%)intheplacebo groupthoughttheyweredrinkingthePB,whileonlyfive ofthe22peopleintheexperimentaltreatmentgroup (23%)guessedtheyweredrinkingtheCB(Table2).As therewasasignificantdifferencebetweentheproportion ofpeopleguessingcorrectlyversustheproportionof peopleguessingincorrectly( p <0.0001,FisherexactNantz etal.NutritionJournal 2013, 12 :161 Page4of9 http://www.nutritionj.com/content/12/1/161

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test),withnearlyeveryoneinthestudybelievingthey wereontheplacebo,biastowardtreatmentwasnotanticipatedtoinfluenceoutcome.Asubgroupanalysisindicatedthattherewerenosignificantdifferencesinany oftheparameters,butone,whencomparingwhoguessed correctlywithwhoguessedincorrectly.Thoseinthe groupthatguessedcorrectlyhadslightlyhigherMIP1 levelsatbaselinecomparedto10week,orcomparedtoeithertimepointofthosewhodidnotguesscorrectly.Interactionsbetweenguessingcorrectlyandtimewerenot significant.Sincethiswasobservedinthebaselinevalue, beforeanyonestartedthestudy,thebiologicalsignificance ofthisstatisticaldifferenceisunknown. Thetotalincidenceofcoldsandinfluenzawerenotstatisticallydifferentbetweenthetwogroups(Table2).However,theproportionofthetotalnumberofsymptomswas statisticallylowerintheCBdrinkinggroup( p =0.031). Sinceonly11.1%ofthesubjectsthoughttheywereconsumingtheactivetreatment,abiasbywhattheythought theyweredrinkingwasnotsuspected.Thereportofintestinaldistressasasymptomwasstatisticallygreater ( p =0.021)inthePBgroup,ascomparedtotheCB group.Noothersymptomshowedanystatisticaldifference.However,becausesubjectsintheplacebogroupreportedafewmoresymptomsineachcategory,thetotal numberofsymptomswasproportionallygreaterinthat groupcomparedtotheCBgroup,andtotalsymptoms werestatisticallydifferentbetweenthegroups. ThepercentageofspecificallylabeledPE+-FITC+cells inthelymphocytepopulation(FSC/SSC)wasdeterminedattheblooddrawsbeforeandafterconsumption, inbothfreshlyisolatedPBMCandinPBMCthatwere culturedforsixdays.Proliferationof -TcellswassignificantlyimprovedafterCBconsumption(Table3) comparedtotheplacebo.NKcellproliferationdidnot achievesignificancewhentreatmentswerecompared. TherewasnoeffectofCBon -Tcells,Bcellsor monocytes.Theantioxidantactivityoftheserumwas determinedinde-proteinatedsamples,butthegroups werenotstatisticallydifferent(datanotshown). Cytokinelevelsinthesupernatantfluidsof24h PBMCculturedwithPHA-Lweredetermined,resulting Assessed for eligibility (n=68) Excluded (n=14) Not meeting inclusion/exclusion criteria (n=7) Would not consume red dye or sucralose in beverage (n=2) (n=5) Analyzed group #638 (n=22) Excluded from analysis (n=0) Allocated to #638 (n=27) Received allocated intervention (n=27) Discontinued intervention (n=5) All unable to return for final blood draw Allocated to #246 (n=27) Received allocated intervention (n=27) Discontinued intervention (n=4) 1 unable to return for final blood draw 2 did not like sucralose taste of juice 1 never responded Analyzed group #246 (n=23) Excluded from analysis (n=0) Allocation Analysis Enrollment Randomized into treatment groups #638 or #246 Unable to be present for blood draws (n=54) Figure1 Flowdiagram:Studyparticipanteligibilityassessment,enrollment,groupallocationandanalysis. Peoplerecruitedforthestudy wereassessedandthosedeemedeligiblewereenrolled.Subjectswererandomizedintooneoftwobeveragegroups.Bottlesofthetwo beverages,cranberryandplacebo,werereceivedfromOceanSprayCranberries,Inc.andlabeledeither#638or#246.Subjectsandinvestigators wereblindedregardingthetreatmentgroups.Statisticalanalysiswasperformedondatafromallsubjectscompletingthestudy.Investigators wereunblinded(cranberrybeverage:#638;placebobeverage:#246)followingcompletionofthedataanalysis. Nantz etal.NutritionJournal 2013, 12 :161 Page5of9 http://www.nutritionj.com/content/12/1/161

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inanobservationofvariabilityamongsubjects.Therefore,thechangebetweencytokinesecretionat10wk andsecretionatbaselinewascomparedbetweentheplaceboandtreatmentgroups(Table4).Theabilityof PBMCtosecreteinterferon(IFN)wassignificantly increasedafterCBconsumption( p =0.041).Othermeasuredcytokineswerenotstatisticallydifferent.DiscussionTheprimarypredefinedoutcomemeasurewasachange intheabilityof -Tcellsderivedfromperipheralblood immunecellstoproliferatein exvivo culture.The -T cellsshowedanimprovedabilitytoproliferateinculture withPHA-L,aftertheCBwasconsumed.Thecranberry fractionpowderwaspreparedfromthejuiceofcranberriesandoneservingofthelowcalorieCB(450ml) containedpolyphenollevelscomparabletoaservingof cranberryjuicecocktail(250ml).Theproanthocyanidin fractioncontainedbothA-andB-typelinkages.Cranberriesareknowntohavehighantioxidantactivity,yet serumantioxidantactivitywasnotdifferentbetweenthe groupsorfrombaselineto10wk(datanotshown). Pharmacokineticstudiesthatmeasureimmediateaccumulationofpolyphenolsinbloodshowthattheturnover israpid;about2-4hours[23-25].Thus,alackofchange inserumantioxidantactivityisnotsurprisingwhen Table2DemographicsandcoldandinfluenzasymptomsCharacteristicPlacebobeverageCranberrybeverageP Demographic n 2322 Age(y)SDa24.03.324.95.8 Male59 Female1813 BMIb,initial21.52.723.24.00.103 ChangeinBMI(kg/m2)SD 0.0950.52 0.0630.370.810cNo.blinded/guessedcorrectly(%)23/23(100)5/22(23)<0.001 Complianced(%)SD96.34.695.93.7NSDeColdandInfluenzaSymptoms nf20150.624 Totalincidenceg31210.282hTotalcoldandinfluenzasymptoms3542970.031hIncidenceofintestinaldistress35150.021hTotaldaysmissedwork/school29200.329hTotaltimesreportedadecreaseinactivity28160.154haSD,standarddeviationbBMI,bodymassindexcp valuederivedfrom t -testdBasedonbottlecounteNSD,notsignificantlydifferentfNumberofpeoplewho reportedsymptomsgNumberofcoldsandcasesofinfluenzareported.Someindividualshadmorethanoneillnessduringthe10wkperiodhPvaluederivedfrom z -testofproportions. Table3Immunecellproliferationafoldchangebafter cranberrybeverageconsumptionCelltypePlacebobeverageCranberrybeverageP -Tcellc1.200.26f3.860.50<0.001 NKdcelle0.150.120.330.190.068 -Tcellc 0.160.09 0.160.080.602 Monocytee 0.110.29 0.300.450.883 Bcellse0.810.161.320.400.235aAfterisolation(Day0),cellswereplacedintocultureinRPMI-1640with autologousserumandPHA-L,IL-2andIL-15for6daysbAratioofculturedcell numbers(Day6/Day0)wasusedtocalculateproliferationfoldchanges. Baselinefoldchangevaluesweresubtractedfromvaluesobtainedat10wkcPercentageoftheCD3+populationdeterminedby2-colorstainingdNK, naturalkillerePercentageoftotalcellsfWilcoxon/Kruskal-Wallisranksumstest wasusedtodeterminesignificantdifferences.Valueswerecalculatedforeach subjectandthemeanSEMdetermined. Table4CytokinessecretedbyPBMCaduring24hculturebCytokinePlacebobeverageCranberrybeveragePcIL-1 519.7115.5d 187.8107.10.088 IFN24.833.4148.180.20.041 TNF189.7155.9367.2140.20.452 IL-1716.811.326.216.70.973 IL-1 36.520.0 27.929.70.496 MIP-1 968.3533.11120.0470.30.395 IL-13 3.17.1 11.510.60.156aPBMC,peripheralbloodmononuclearcellsbOnDay0,PBMCwereculturedin RPMI-1640withautologousserumandPHA-L,IL-2andIL-15cPvaluesaretwotailedbasedontheWilcoxon/Kruskai-WallisRanksumtestdValuesreported arethemeanchangesSEMofthe10-wkdataminusthebaselinedata.Nantz etal.NutritionJournal 2013, 12 :161 Page6of9 http://www.nutritionj.com/content/12/1/161

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measuredintheserumoffastingsubjects,aswedidin thisstudy. Weproposethat -Tcellproliferation exvivo isa surrogatemarkerforimmunefunction.Wehaveshown inpreviousstudiesthattheirproliferationcanbemodifiedbydiet[15-17].Theissueofbioavailabilitymustbe addressedsincemanystudiesshowthatpolyphenolsdo notaccumulateintheblood,butarerapidlymetabolized.Intestinalimmunecellsareabletointeractwith thecontentsofthelumenbecauseoftheirlocationin thePeyer ’ spatchesandtheintra-epithelium.Migration ofintestinalimmunecellsiswelldocumented[26,27] andmigrationpatternshavebeenshowntobeinfluencedbydiet[28,29].Althoughdataspecificallyregarding -Tcellsislacking,wehypothesizethatwemeasure changesinbloodimmunecellsbecausetheymigrate aftertheyhaveinteractedwithluminalbioactivecompoundsintheintestine. Inaddition,largermolecules,attimes,areableto translocateacrosstheintestineandfindtheirwayto cellsinthelaminapropriaandthemesentericlymph nodes.Bacterialtoxinsarefoundintactintheblood orlymph[30,31].Proteinsareabsorbedintact[32,33] andprocyanidindimersaretransferredtotheserosal sideofenterocytesintheisolatedsmallintestine[34] orinaCaco-2cellmodel[35].Jutila ’ sgrouphas shownthattheproanthocyanidincontentofherbsinteractswithreceptorsonthe -Tcellandprimes thatcell[36].Afterpriming,thecellisabletorespondfaster,andtoagreaterextent,thanifitwere notprimed.Directinteractionofdietarycomponents inthelumen,ortranslocationofdietarycomponents intothelaminapropria,couldresultinprimingof immunecells.Finally,changesdetectedinsystemic bloodcellsmayalsooccurbecauseofthefermentationofunabsorbedpolyphenolsinthelargeintestine, resultinginbioactivecompoundsthataresubsequentlyabsorbed.Thestudywasnotdesignedtodistinguishamongthesedifferentmechanisms. Changesinimmunefunctionmayberesponsiblefora reducednumberofcoldandinfluenzasymptoms.Althoughtheincidenceofcoldsandinfluenzaweresimilar betweenthetwogroups,thetotalnumberofsymptoms waslowerafterconsumingtheCB.Combining  missed work ’ and  lowerabilitytoperformanormaldailyroutine ’ resultedina p valueof0.056.Thisstudywasnot poweredoncoldandinfluenzasymptoms;itdoesnot achievestatisticalsignificance,sotheideathatcranberry consumptionhasaneffectonahealthoutcomeisonlya suggestion.Furthermore,thedataoncoldandinfluenza symptomswereself-reportedandphysiciansdidnot confirmthepresenceofcoldorinfluenzapathogens. However,MacintyreandPritcharddemonstratedthat self-reportedsymptoms,aswellassymptomseverity, washighlycorrelatedwithassessmentsmadebyphysicians[37],thereforewefeelconfidentthatthesymptoms reportedbythesubjectsaccuratelyreflectthesymptoms theyhad. Weexaminedcytokinesbasedonprevious invitro data(manuscriptsubmitted)inwhichPBMCfromhumandonorswereincubatedwithvariouscranberryfractions.Forevaluationinthisstudy,wechosethose cytokinesthatweresecretedinresponsetotheaddition ofthecranberryfractions.Theresultsfromparticipants inthisstudyshowedtheresponseofthecellsto exvivo stimulationwasextremelyvariable.Drawingfirmconclusionsabouttherolecranberryplaysincytokineproductionispremature,yetthedatasuggestthatcranberry contributestoananti-inflammatoryeffect.Cranberry proanthocyanidinshavebeensuggestedasbeingantiinflammatoryin invitro studies[38,39]andinrabbits [7].Onehumaninterventionstudyshowedthatthelevel ofurinaryIL-6wasreducedinpregnantwomenwho consumedcranberryjuicecomparedtoaplacebo[8],yet othercytokinesmeasuredinthatstudydidnotachieve statisticalsignificance,perhapsduetothesmall n of eachgroup.ConclusionsInsummary,dailyconsumptionofacranberrybeverage containingcranberrypolyphenolsandproanthocyanidins atlevelssimilartocommerciallyavailablejuicefor10 wk,waseffectiveinincreasingtheproliferationresponse of -TcellsandperhapsNKcells.Overall,theimprovedproliferationresponsewascoupledwithlower productionofaninflammatorycytokine.Improvement offunctionalimmunity,particularlyofacelllocatedin theepitheliumandresponsibleforbarrierprotection, mightbeanothermechanismbywhichcranberryisable tomaintainurinarytracthealth.Thesephysiological changescouldbe,inpart,responsibleforourbeneficial healthoutcome,areductionofthenumberofillness symptoms.Abbreviations AAPH: 2,2 azobis2-amidinopropanedihydrochloride;CB:Cranberrybeverage; ELISA:Enzyme-linkedimmuosorbentassay;FITC:Fluoresceinisothiocyanate; NC:Notcalculated;NK:Naturalkiller;NSD:Notsignificantlydifferent; ORAC:Oxygenradicalabsorbancecapacity;OSC:OceanSprayCranberries, Inc.;PB:Placebobeverage;PBMC:Peripheralbloodmononuclearcell;PHA-L: Phytohemagglutinin;PE:Phycoerythrin;TCR:T-cellreceptor. Competinginterests CKisanemployeeofOSC.SSPreceivesnoothercompensationfromOSC outsideofthefundingforthisresearch.Theotherauthorshavenoconflicts ofinterest. Authors ’ contributions ThestudywasconceivedbySSPandCK,andSSPwasresponsibleforthe studydesign.MPN,CAR,CM,andRCcontributedtothedesignofthestudy andwereresponsibleforcoordinatingtheintervention,performingtheNantz etal.NutritionJournal 2013, 12 :161 Page7of9 http://www.nutritionj.com/content/12/1/161

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assaysandwritingsomeofthepaper.JChelpedwiththestatisticalanalysis. CKwasresponsibleforthepreparation,analysisandrandomizationofthe beverages.SSPinterpretedthedata,wrotethepaper,andisresponsiblefor finalcontentofthemanuscript.Allauthorsreadandapprovedthefinal manuscript. Acknowledgements FundingforthestudywasprovidedbyOceanSprayCranberries,Inc.(OSC). Weacknowledgethetalentsofourundergraduatestudentvolunteers:Jillian Lozada,ChelseaChapkin,andSophiaPasqualini.WealsothankNealBenson andstaffattheFlowCytometryCoreFacility,InterdisciplinaryCenterfor BiotechnologyResearch,UniversityofFlorida. Authordetails1DepartmentofFoodScience&HumanNutrition,UniversityofFlorida,Box 110370,Gainesville,FL32611,USA.2DepartmentofStatistics,Universityof Florida,Gainesville,FL32611,USA.3OceanSprayCranberries,Inc.,Lakeville, MA02349,USA. Received:25September2013Accepted:9December2013 Published:13December2013 References1.JepsonR: Cranberriesforthepreventionofurinarytractinfections. 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37.MacintyreS,PritchardC: Comparisonsbetweentheself-assessedand observer-assessedpresenceandseverityofcolds. SocSciMed 1989, 29: 1243 – 1248. 38.BodetC,ChandadF,GrenierD: Cranberrycomponentsinhibitinterleukin-6, interleukin-8,andprostaglandinEproductionbylipopolysaccharideactivatedgingivalfibroblasts. EurJOralSci 2007, 115: 64 – 70. 39.Madrigal-CarballoS,RodriguezG,SibajaM,ReedJD,VilaAO,MolinaF: Chitosomesloadedwithcranberryproanthocyanidinsattenuatethe bacteriallipopolysaccharide-inducedexpressionofiNOSandCOX-2in raw264.7macrophages. JLiposomeRes 2009, 19: 189 – 196.doi:10.1186/1475-2891-12-161 Citethisarticleas: Nantz etal. : Consumptionofcranberrypolyphenols enhanceshuman -Tcellproliferationandreducesthenumberof symptomsassociatedwithcoldsandinfluenza:arandomized,placebocontrolledinterventionstudy. NutritionJournal 2013 12 :161. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Nantz etal.NutritionJournal 2013, 12 :161 Page9of9 http://www.nutritionj.com/content/12/1/161