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OralToleranceStateofimmunologicalunresponsivenesstoantigeninducedbyfeeding.Itisafeatureofthecommonmucosalimmunesystem.ThemucosalimmunesystemConsistsofthegastro-intestinaltract,respiratorysystem,genito-urinarysystem,liver.CommonlymphoidcirculationEpithelialcellslinethemucosaLargestareaexposedtotheexternalenvironmentHeaviestantigenicloadFeaturesofmucosaltolerance?NormalimmunefunctionTolerancecanbelocalorsystemicItrequiresafunctionalimmunesystemSymbiosis-intheabsenceofcommensals,apoorimmuneresponsedevelopsandoraltolerancecannotbeinducedGeneralpropertiesofmucosaltolerance:Antigenspecific.Oftenpartial(eg.antibodiesinhibited,butTcellresponsesmayremain).Notcomplete(eg.maybeaquantitativereductioninantibodylevels).Waneswithtime.Generalpropertiesofmucosaltolerancecont’dEasiertoabrogatearesponsethanreduceandestablishedresponse.Goodimmunogensarebetteratinducingtolerance!(adjuvant…)Doseandroutedependent.BreakdownoforaltoleranceImmuneresponsestofoodleadstofoodintoleranceegcoeliacdiseaseImmuneresponsestocommensalbacterialeadstoinflammatoryboweldisease(IBD)egcrohn’sdisease,ulcerativecolitisBalanceRespond Don’trespond

fight anderadicate IgnorePATHOGENS SELF FOODMechanism?Centraltolerance

deletionofself-reactiveTcellsinthethymusPeripheraltolerance

anareaofveryactiveresearch!

1.deletion

2.immunedeviation

3.anergy

4.suppression/regulationRegulationofselftolerance?CentraltoleranceisincompleteTCRbindatlowaffinityandcanpotentiallyrecogniseanumberofMHC/peptideAuto-reactiveTcellsexistathighfrequencyintheperipheryAuto-immunity-isitaresultofdefectiveTcellregulation?1.DeletionMechanismof‘central’tolerance(negativeselectioninthethymus)ApoptosisofspecificTlymphocytes(egfas-fasL)Showntoplayarolein‘peripheral’toleranceinsitesofimmuneprivilege(egstromalcellsinthetestesexpressfasL)Peripheraldeletionofantigen-reactiveTcellsinoraltoleranceREF:Nature1995Jul13;376(6536):177-80ChenY,InobeJ,MarksR,GonnellaP,KuchrooVK,WeinerHLoralantigencandeleteantigen-reactiveTcellsinPeyer'spatches,inmicetransgenicfortheovalbumin-specificT-cellreceptorgenes.Thedeletionwasmediatedbyapoptosis,andwasdependentondosageandfrequencyoffeeding.Atlowerdosesdeletionwasnotobserved;insteadtherewasinductionofantigen-specificcellsthatproducedtransforminggrowthfactor(TGF)-betaandinterleukin(IL)-4andIL-10cytokines.Athigherdoses,bothTh1andTh2cellsweredeletedfollowingtheirinitialactivation,whereascellswhichsecreteTGF-betawereresistanttodeletion.Thesefindingsdemonstratethatorallyadministeredantigencaninducetolerancenotonlybyactivesuppressionandclonalanergybutbyextrathymicdeletionofantigen-reactiveTh1andTh2cellsDeletionsummaryGenerallyobservedathighdosesoffedantigen:Activationinducedcelldeath(AICD)mediatedbyfas/fasLinteractionsGrowthfactordeprival2.ImmuneDeviation

CD4+Tlymphocytesareactivatedbyantigenpresentingcells(APC)Th1cells-importantininflammatoryresponses(egdelayedtypehypersensitivity)Th2cells-importantinhelpingantibodyresponses.SuppressTh1cells(IL-4,IL-10).

ThereforeTh1immuneresponsesmaybeinhibitedifTh2cellsarestimulatedinstead.

Inhibitorycytokines

Transforminggrowthfactorbeta(TGF

)non-specificallyinhibitsthegrowthoflymphocytes(Th3)SpecificimmuneresponsescanbeinhibitedbyTh2cytokines(IL-4andIL-10)SomepopulationsofTlymphocytes(bothCD4andCD8)canconsumeIL-2,theTcellgrowthfactor.SurroundingcellsthereforefailtogrowOneexampleofmany

Feedingoralinsulintomicepreventsvirusinducedinsulin-dependentdiabetesinamousemodel.IL-4andIL-10weregeneratedwhichinhibitedaspecificimmuneresponse.REF:VonHerrathetal.,JClinInvest98,1324.1996BystandersuppressionAntigen-specificsuppressionisinducedbyfeedingSuppressionistriggeredbyre-encounterofantigenReleaseofinhibitorycytokineswillnon-specificallyinhibitothercells3.Anergy

Non-productiveantigenpresentation

Tcellsareactivatedbyantigenpresentingcells3signalsarerequiredtoactivateaTcellSpecificrecognition-TCR‘sees’therightMHC-peptidecomplex….signal1Costimulation-CD28bindsB7…signal2Cytokines-localmicro-environmentwillinstructthekindofTcellneeded…signal3Responsevsnon-response

Tlymphocyteactivationrequires2signalsSignal

Tcellproliferation+Signal

(IL-2&IL-2r)Signal

alone

NoproliferationSignal2absence/blockadeSomeepithelialcellsinthegutandlungnormallyexpressclassIIMHC,butnotcostimulatorymoleculesandthereforecannotprovidesignal2Reagents(egCTLA4Ig)havebeendevelopedtoblocktheinteractionofCD28withB7onAPCandthereforeblocksignal2AnergyResultsinaspecifichypresponsivenessAnergiccellsdonotrespondtospecificMHC+peptidepluscostimulationAnergiccellsmaythenblockAPC-andinhibitimmuneresponsesAnergiccellsmayconsumeIL-2Anergiccellsaremoresusceptibletoprogrammedcelldeath(apoptosis)3.RegulationTherehasbeenagreatdealofdiscussionof'suppressorcells’(especiallyinthe1980s)SuppressorcellshaveproveddifficulttocloneandphenotypeManycellsexertasuppressiveeffectArangeof‘regulatoryTcells(Treg)’havenowbeendescribedRegulatoryTcellsApopulationofCD4+TcellshasbeenimplicatedinthesuppressionofinflammatoryimmuneresponsesAntigenspecificTurnoffspecificinflammatoryimmuneresponsesMechanismunclear…TreginmurineinflammatoryboweldiseasePathogenic‘Tcells’(Tpath)-cantransferthediseaseto‘naïve’recipientsRegulatory‘Tcells’(Treg)-inhibitdisease&TpathTregareasubsetofhelperTcells(CD4)whichexpressCD25MajorareaofinvestigationinImmunologyResearchModelsoforaltoleranceEatsolubleantigenInjectantigenMeasureimmuneresponseTcellproliferationantibodyproductioncytokineprofileMultiplemodelsoforaltolerancehavebeenproposed(Weiner,1997)AnimalmodelsHumanmodelsClinicaltrialsAmurinemodel-GarsideMurinemodelinwhichOVA-specificTcellscouldbetrackedwithaspecificmonoclonalantibodyFedOVAWatchimmuneresponsebytrackingOVA-specificTcellsSmithKM,McAskillF,GarsideP.OrallytolerizedTcellsareonlyabletoenterBcellfolliclesfollowingchallengewithantigeninadjuvant,buttheyremainunabletoprovideBcellhelp.JImmunol2002May1;168(9):4318-25ResultsPRIMING-Ovainjectionresultedin:specificantibodyproductionproliferationofOVAspecificTcellsDTHresponseTOLERANCE-FeedingOvaabrogatedtheseresponsesdemonstratedthatprimingandtolerancecouldbeinducedinthismodel.Wheredidtheresponsestakeplace?PRIMINGd3peakofOVAspecificTcellsinperipherallymphnodeTOLERANCEd3peakofOVAspecificTcellsinperipherallymphnodeTcellproliferationPRIMINGTcelldivisioninperipherallymphnodes(pln),mesentericlymphnodes(mln)andpeyerspatches(pp)at2daysTOLERANCETcelldivisioninperipherallymphnodes(pln),mesentericlymphnodes(mln)andpeyerspatches(pp)at2daysTcellphenotypePRIMINGOvaspecificTcellsdevelopa‘memory’phenotype.Changesdetectedasearlyas6hafterfeeding.TOLERANCEOvaspecificTcellsdevelopa‘memory’phenotype.Changesdetectedasearlyas6hafterfeeding.Differences...EarlysystemicandlocalimmuneresponseinprimingandtolerancewasverysimilarHowever,laterimmuneresponseswereverydifferent(immunityvstolerance)TolerantTcellsdidnotmoveintoBcellareaandstimulatetheirexpansionPotentialCanoraltolerancebeusedtherapeutically?Doinbredanimalmodelsrelatetooutbredhumanpopulations?Canmechanismsofregulationbegeneratedexvivoorinvivoforclinicaltreatment?ClinicaltrialsAnumberofclinicaltrialsforauto-immunediseaseareinprogress:Disease Antigen MultipleSclerosis(MS) MyelinBasic Protein(MPB) RheumatoidArthritis(RA) TypeIIcollagen TypeIDiabetes Insulin Uveitis S-antigen TransplantRejection MHCmolecules HumanMStrial1ydoubleblindstudy6/15MSpatientsfedMBPhadattacks15/15controlMSpatientsfedplacebohadattacksThoseindividualsfedmyelinhadahigherfrequencyofTGF

producingcells(Fukuaraetal.,1996.JClinInvest98,70).HumanRAtrialsSeveralstudieshaveinvestigatedtheeffectoffeedingtypeIIcollagentoRApatientsStudyNoofcentresDose(mg) Time ResultsGermany 5 0,1,10 12weeksnd USA 6 0.02-2.5 12weeks 1+ InvestigatorsarefindingthatdoseandfrequencyareimportantfactorsDiabetesTypeIinsulindependentdiabetesmellitus(IDDM)Organspecificauto-immunediseaseTcellmediateddestructionofpancreaticbetacellsAnti-insulinantibodiesdevelopSusceptibilitycontrolledbyenvironmental&geneticfactors(particularlyMHCgenes)DiabetesPreventionTrial

(DPT-1)

Multi-centre,randomized,controlled,clinicaltrialdesignedtodetermineifitispossibletopreventordelaytheonsetoftypeIdiabetes(1994-2002)>50%riskinjectedinsulin25-50%risk

oralinsulinResultstodateInjectedinsulinfailedtopreventordelaytheonsetofdiabetes.SkylerJSetal.,(2002)Effectsofinsulininrelativesofpatientswithtype1diabetesmellitusNewEnglandJournalofMedicine346(22):1685-1691ResultstofollowTheOralInsulinTrialcompletedenrollmentonOctober31,2002.Studyinvestigatorsarenowcollectingfinaldatatodetermineiforalinsulincandelaytheonsetoftype1diabetes.TheyexpecttoannouncetrialresultsinJune2003.SummaryofhumanclinicaltrialsAgentsthatenhanceclinicaltoleranceareunderinvestigation,egothercytokines,adjuvants(cholera-toxin).HoweverclinicaltrialshavebeendisappointingPoorstudydesign/suboptimaldose/typeofantigen/route???/index.htmlDiffuseMALTLaminaproprialymphocytes(primarilyBcells)(LPmajorsiteofIgsynthesis)Laminapropria:thelayerofconnectivetissueunderlyingtheepitheliumofamucousmembraneDerivedfromO-MALTandrepresenteffectorandmemorycellsfromcellsstimulatedbyantigenIntraepitheliallymphocytes(IELs)PlasmacellsproducingdimericIgAAntigen-presentingcells(macrophagesanddendriticcells)ModesofAntigenSamplingStratified,non-keratinizedorparakaratinizedepithelia(oralcavity,pharynx,esophagus,urethra,vagina)AntigensamplingdependsonDendriticcellsLangerhanscells,phagocytic,antigen-presentingmotile“scouts”)Dendriticcellsmaythentransportantigentolocalandregionallymphoidfollicles.Simpleepithelia(bronchiole,intestine,bronchi)AntigensamplingdependsonMcellsandTransepithelialtransportDendriticcellsmayalsoparticipateinantigentransportDendriticcellsCaptureantigenintissuesTransporttosecondarylymphoidorgansProcessandpresenttoTcellsAnessentiallinkbetweeninnateandadaptiveimmunityMayalsorepresentthe“Achille’sHeel”ofthehost?(Cutleretal.2001)MaturationofDendriticCellsLossofendocyticandphagocyticreceptorsIncreasedexpressionofMHCUp-regulationofco-stimulatorymolecules(CD80andCD86)requiredforT-cellstimulationUp-regulationofCD40andadhesionmoleculesICAM-1andLFA-3Fcreceptors(endocytosis)decreaseThis"DC-precursor"foundinTDLlooksverymuchlikealymphocytewithseveralimportantexceptions.Mitochondriawerefarmorenumerousinthecytoplasm,andtheDCnucleuswasconvolutedwith

moredelicatelydistributedheterochromatinandlightereuchromatinthanisnormallyfoundinlymphocytes.(FromA.Anderson)AntigenSamplingacrossSimpleEpitheliaMucosalsurfacesgenerallylinedbyasinglelayerofepithelialcellsBarriersealedbytightjunctionsthatexcludepeptidesandmacromoleculesUptakeofantigenrequiresactivetransepithelialtransport(M-cellsorDendriticcells)Samplingisblockedbymechanismssuchaslocalsecretions,sIgA,mucins,etc.OrganizationofO-MALTM-CellFollicle-associatedepitheliumDomeregionGerminalCenterParafollicularregionLUMENLymphoidFollicleAntigenAdherencetoM-CellsAdherencefavorsendocytosisandtranscytosisAdherentmaterialstendtoevokestrongimmuneresponsesWidevarietyofpathogensadheretoM-cellsMechanismofadherenceisunclearManycommensalmicroorganismsavoidadherencetoM-cellsM-CellsMayServeasEntrysitesforPathogenicMicroorganismsBacteriaVibriocholeraeEscherichiacoliSalmonellatyphiSalmonellatyphimuriumShigellaflexneriYersiniaenterocoliticaYersiniapseudotuberculosisCampylobacterjejuniVirusesReoviruspoliovirusHIVAntigenRecognitionAntigentransportiseffectedbyM-CellswhichoccuroverOrganizedMucosa-AssociatedLymphoidTissue(O-MALT)Afterantigenstimulation,effectorB-lymphocytesleaveO-MALTandmigratetodistantmucosalorglandularsitesMigrationandHomingofLymphocytesDistributionofHomingSpecificitiesinMucosalTissues

Epithelialcellsliningpostcapillaryvenules(HEV’s)displayorgan-specificrecognitionsitescalled“vascularaddressins”Recognizedbycelladhesionmolecules“homingreceptors”HighEndothelialVenules(HEV)Containspecializedendothelialcellsliningpostcapillaryvenules.Displayorgan-specificrecognitionsitescalled“vascularaddressins”thatarerecognizedbyspecificcelladhesionmoleculesonlymphocytes.HEVcellsarecharacterizedby:ElongatedshapeandprominentglycocalyxonluminalsurfacePolarized,withadomedluminalsurfaceseparatedfromthebasolateralsurfacebyadherentjunctions,butnottightjunctionsCellsrestonabasallaminathatconstitutestherate-limitingbarriertomigratinglymphocytesHEV(continued)InO-MALT,HEV’sarepresentinT-cellareasbetweenBcellfolliclesInD-MALT,venuleshaveflatendothelialcellsthatsharemanyfeatureswithHEV’sHEV’sproducesulfatedglycolipidsandglycoproteinsintothevascularlumen(notknownwhethertheseproductsplayaroleinhomingorextravasation)AdhesionmoleculesclonedsofarbelongtofourmainproteinfamiliesIntegrinsSelectinsCAMs(celladhesionmolecules)Proteoglycan-link.coreproteinsModulationofHomingSpecificitiesNaivelymphocytespriortoantigenicstimulationdemonstratenomigrationpreferenceFollowingantigenicstimulation,lymphocytesacquirehomingspecificitiesLymphocytesinHEVLymphocytesadheringtoluminalsurfacesofHEVendothelialcells.Notemicrovillionsurfaceoflymphocytes.Cross-sectionofHEVTransepithelialTransportinMucosalImmunitySamplingSiteEnvironmentEffectorSiteDiffuseMALTOrganizedMALTMucosalorGlandularTissueTransepithelialTransportofIgAAntibodiesPolymericimmunoglobulinreceptoranditsintracellulartraffickingpoly-IgreceptorBindingofIgAtopolymericimmunoglobulinreceptorTransportandDistributionofIgAAntibodiesEffectorFunctionsofMucosalAntibodiesIgAantibodiesarenotgoodmediatorsofinflammatoryreactionscomplementactivationneutrophilchemotaxisphagocytosisImmuneExclusion/Serve“escort"functionBeneficialnottoinduceinflammationIntra-epithelialvirusneutralizationbyIgAExcretoryfunctionforIgARelationshipbetweenSystemicandMucosalImmunityOraltolerance(anergy)Oraladministrationofantigensuppressessystemicimmunity“MucosalInternet”Epithelialcell-ImmuneCellInteractionsMaybecriticalforinductionofadaptiveresponseDangertheoryEpithelialCellResponsetoPathogensRequirementsofProtectiveVaccinesBlockadherenceofmicroorganismtohostFacilitateclearancefromhostNeutralizetoxinMustrecognize“virulence”epitopesMustbeimmunogenicMustnotinduceautoimmunediseaseShouldinducelong-lastingimmunityMustinducethetypeofresponsethatiseffectivetoeliminatepathogen(eg.TH1orTH2)RationalStrategiesforMucosalImmunizationRequirementsSafetakenorallyLong-lastingduetocontinuedmaintenanceofmemorySurviveingastricandintestinalenvironmentsMustescapenormalclearancemechanismsMustcompeteforinclusionwithinM-CelltransportMustarriveintacttoantigen-processingcellsMustinducedimericsIgAreactivewithcellsurfaceRationalStrategiesforMucosalImmunization(continued)StrategiesforDeliveryofVaccineIntoO-MALTInertparticulatecarriersBiodegradablecopolymersImmune-stimulatingcomplexes(ISCOMs)HydroxyapatitecrystalsLivevaccinevectors(recombinant)Vacciniavirus

SalmonellaMycobacteriumbovisRationalStrategiesforMucosalImmunization(continued)StrategiesforEnhancingMucosalImmuneResponseCo-deliverywithcytokinesCo-immunogens(Choleratoxin)PeptidespresentedwithpotentT-cellepitopesOralVaccinesITHACA,N.Y.--TheBoyceThompsonInstituteforPlantResearchInc.(BTI),anaffiliateofCornellUniversity,announcedthatclinicaltrialswillbegintoday(July7)atRoswellParkCancerInstitute(RPCI)inBuffalo,N.Y.,totestthesafetyandimmunogenicityoftheworld'sfirstpotentialoralvaccineagainstthehepatitisBvirus.Thevaccinewillbedeliveredsimplybyeatingpotatoesgeneticallydesignedtocontainthevaccine.OralVaccineProtectsInfantsfromSevereRotavirusDiarrheaFirstSuccessinaDevelopingCountryAnoralvaccineagainstrotavirus--themostimportantcauseoflife-threateningdiarrheainchildrenunderage2--reducedseverediarrhealillnessby88percentinastudyofmorethan2,000infantsinVenezuela.Thisisthelargestandmostsuccessfultrialtodateofarotavirusvaccineamongchildreninadevelopingcountry.OralVaccines(cont’d)VaccineNowAvailableasanOralSeriesoraSingleDoseInjection

TyphoidfeverimmunizationisrecommendedforalltravelerstolessordevelopedcountriesespeciallythoseinCentralandSouthAmerica,Africa,SoutheastAsia,andTheIndianSubcontinent.ThehighestriskcountriesarePeru,India,Pakistan,andChile.However,abouthalfofallcasesoftyphoidfeverreportedinAmericantouristsareacquiredfromtraveltoMexicoeventhoughtheriskofdiseaseislowerthere.Typhoidfeverisgenerallyspreadpersontopersonespeciallybyfoodhandlerswhodonotwashtheirhandsadequatelyafterbowelmovements.Visitorswhostrayoffthebeatenpathandeatmealspreparedatfoodstandsorbystreetvendorsareathighestrisk.Carefullyselectingrestaurantswithcloseattentiontotheirsanitationstandardscanreducetherisk.Therenowisanoraltyphoidvaccineandanewsingledoseinjectablevaccinethatproducesfewersideeffectsthattheoldertwodoseinjectablevaccine.Bothvaccinesareequallyeffectiveandoffer65-75%protectionagainstthedisease.Alzheimer'svaccinelookspromising&BraindeteriorationslowedbynosedropsMedicalresearchershavesuccessfullytreatedAlzheimer’sdiseaseinmicebyputtingdropsofvaccineintheirnoses.Theythinkitwillultimatelybepossibletodothesamewithpeople."Weplantobeginhumantrialsnextyear,"saysHowardWeiner,aneurologistatHarvardMedicalSchoolwhohaspioneeredtheuseoforalandnasalvaccines.

OralVaccines(Cont’d)AVANTRECEIVESPATENTLICENSEONORALTYPHOIDFEVERVACCINENEEDHAM,MA(August22,2000):

AVANTImmunotherapeutics,Inc.(Nasdaq:AVAN)announcedtodaythesigningofacross-licensingagreementwithMeganHealthInc.forexclusiverightstoapatentportfoliosupportiveofAVANT’ssingle-dose,oralvaccinecandidateagainsttyphoidfever,calledTy800.

TheagreementallowsAVANTtofurtheritsclinicaldevelopmentofTy800inexpandedPhaseIIstudies,whileMeganHealthgainsnon-exclusiverightstouseAVANT'shigh-levelexpressionsystemforhumanandnon-humanvaccines.AgreementsSetInnovativeAIDSVaccineonFastTracktoDevelopingCountriesOralvaccinewould