简介：EfficientactivationofTlymphocytesthroughTcellreceptor(TCR)dependsontwosignals．Thefirstsignal(signalone)isderivedfromTCRinteractingwiththeMHC／antigenicpeptidecomplex，whichconfersantigenicspecificitytotheimmuneresponse．Thesecondsignal(signaltwo)isprovidedbytheengagementofTcellsurfacereceptorswiththeirspecificligandsonantigenpresentingcell(APC)[1]．Asthereisgrowingevidenceforbidirectionalcommunicationsandsocalled“reversesignaling”fortraditionallydefinedligands，thedistinctionbetweenreceptorsandligandsbecomeslessclear．Thesepairsofmoleculesshouldbeviewedascosignalingmoleculesfunctioningincellswhichwouldexpresseitherthereceptorsortheligands.

简介：Toinvestigatewhetherestradiol(E2)playsaroleincell-contact-dependentregulatorymechanismofTcellactivation,westudiedtheroleofE2inregulatinggenetranscriptionofCTLA-4,ICOS,B7-1,B7-2andB7hinvitro.ThespleniccellsofnormalfemaleBALB/cmicewereactivatedbyConA.ThenthecellswereculturedwithE2(100pg/mlor50ng/ml)for24hor48h,respectively.ThecellproliferationwasmeasuredbyMTTassayandtheexpressionoftheco-stimulatorymoleculesmRNAwasexaminedbyRT-PCRanalysis.WefoundthatE2(100pg/ml,physiologicallevel)stimulatedtheactivatedspleencellsproliferation;inhibitedCTLA-4,ICOS,TGF-βandIL-10genetranscription;promotedB7-1andB7-2genetranscription.E2(50ng/ml,pregnantlevel)inhibitedtheproliferationoftheactivatedspleniccells;promotedCTLA-4,B7-1,IL-10butinhibitedB7-2andTGF-βgenetranscription.Therefore,weconcludethattheeffectsofE2onTcellactivationarepartiallythroughitsregulationontheco-stimulatorymolecules.Theco-stimulatorymoleculesarecrucialcomponentsofthecell-contactdependentregulatorymechanism,andE2mayregulateTcellactivationbythismechanism.

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简介：Analysisofthefrequencyofantigen-specificcytotoxicTlymphocytes(CTLs)exvivoislargelydependentontheuseofMHC/peptidetetramers.However,thelatterreagentshavenotbeenwidelyavailable,mostlikelybecauseoftheircostlyandtime-consumingproduction.InthisreportweutilizedaneconomicstrategytoconstructHLA/peptidetetramerswithrecombinantpeptide-linkedβ2microglobulin(β2m).TheHLA-A2-restricted,melanomaantigenMARTl-derivedpeptideMARTI27-35(AAGIGILTV)wasfusedtotheNterminusofhumanβ2mthrougha15-aminoacid(aa)-longlinkerbeforebeingrefoldedwiththerecombinantbiotinylatedHLA-A2heavychainectodomain.Theresulted2-component(2C)monomerwasthentetramerizedwithphycoerythin-labeledstreptavidin.Theexperimentalresultshowedthatthe2CHLA-A2/MARTI27-35monomerwasshowntobindtotheHLAclassⅠcomplex-specificmonoclonalantibodyW6/32andtheHLA-A2/MARTI27-35complex-specificsinglechainantibodyfragment(scFv)8.3,suggestingthecorrectnessofitsspecificity.Furthermore,the2CHLA-A2/MARTI27-35tetramerdetectedaspecificCD8^+TcellpopulationinHLA-A2-restrictedmelanomainfiltratinglymphocytesastheconventional3CHLA-A2/MARTI27-35tetramer.Theyieldof2CHLA-A2/MARTI27-35monomerwas2.5timesmorethanthatoftheconventional3Cmonomer.Takentogether,thesedataindicatethattheHLA-A2/MARTI27-35tetramercanbegeneratedconvenientlythroughtheuseofMARTI27-35peptide-β2mfusionproteins,whichcanfacilitatethemonitoringofHLA-A2-restricted,MARTl-specificCTLresponsesinpatientswithmelanoma.

简介：ToexploretheantiviraleffectandmechanismofpolysaccharidefromSpirulinaplatensis(PSP)onherpessimplexvimstype2(HSV-2),astandardstrainofHSV-2(333strain)wasusedtoinvestigatetheantiviraleffectofPSPinvitro.PSPinvariousconcentrationswasappliedtodifferentstagesofHSV-2replicationcycle.Finally,thevirusinfectivity(TCID50),cytopathiceffect(CPE),andMTTstainingmethodforviablecells(MTTassay)wereusedasmarkerstoevaluatetheeffectofPSPonHSV-2.ThequantityofHSV-DNAwasdetectedbyreal-timefluorescencequantitativePCR(FQ-PCR).TheHSV-2infectedVerocellultrastructureswereobservedbytransmissionelectronmicroscopy(TEM).TheresultsshowedthatPSPhadlittlecytotoxiceffectonVerocells,itcouldnotdirectlyinactivateHSV-2infectivity.PSPnotonlyinterferedinadsorptionofHSV-2toVerocellsbutalsoinllibitedHSV-2biosynthesisinthecells.FQ-PCRresultsshowedthattheinhibitoryrateonHSV-DNAalsoincreasedinadose-dependentandtime-dependentmanner.TEMalsoconfirmedthatPSPexhibitedpronouncedinhibitoryeffectonHSV-2.Inconclusion,theantiviraleffectofPSPonHSV-2maybeattributedtotheinhibitionofvimsadsorption,vimsreplicationandsynthesisincells.

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简介：Chlamydiatrachomatisoutermembraneprotein2(Ctomp2)isamajorimmunogeninchlamydialinfectionsandahighlygenus-conservedstructuralproteinofallChlamydiaspecies.Topurifytheproteinandtopreparemonoclonalantibodies(mAbs)againstit,therecombinantproteinwasinducedbyIPTG,whichwasconfirmedbySDS-PAGEandpurifiedbymeansofaNi2+-chargedresincolumn.ThedenaturedproteinwasrefoldedintheGSH-GSSHbuffergraduallyandidentifiedbyWesternblotting.ThentheBALB/cmicewereimmunizedwiththerecombinantproteintopreparethemAbagainstCtomp2.TheobtainedmAbswerecharacterized.GenitalspecimensweretestedwithindirectELISAmostlymadeofthemAbandcellculturein84patientswithgenitalsymptoms.Theresultsshowedthathigh-levelexpressionoftherecombinantproteinwasachieved,whichexistedasinclusionbodyandamountedto38%oftotalbacteriumprotein.AmAbagainstCtomp2wasobtained.ItbelongstoIgG2b.Thetiterswereashighas1:40000.TheWesternblottingshowedthatthemAbonlyreactedwiththerecombinantprotein.IthadnocrossingreactionsagainstE.coli,N.gonorhoea,M.hominis,U.urealyticumandM.penetrans.Ithadhighspecifity.Incomparisonwithgoldstandardtest-cellculture,thesensitivities,specificities,positivepredictivevaluesandnegativepredictivevaluesofindirectELISAwere95.24%,100%,100%and98.44%,respectively.Theabove-mentionedresearchworkcontributednotonlytothefurtherstudyofthestructureandfunctionofthisprotein,butalsototheestablishmentofthemethodforitsclinicalapplication,forithadnotbeenreportedbefore.

简介：TheaimofthisstudyistoinvestigatethefeasibilityandmechanismofhIL-2-preSDNAvaccineaspreventionandtherapeuticapproachagainstHepatitisB.EukaryonexpressionvectorinvolvinghIL-2andpreSgenewasconstructedwithrecombinanttechniqueandtransferredintonormalBALB/cmiceandHBVtransgenicmice(Tg-Mice)respectively.Tnenaseriesofdetectionwereperformed:detectionofanti-preS2,HBsantibodyandHBsAginBALB/cmiceandTg-micewithELISA,quantificationofHBVDNAcopiesinHBVTg-miceserumwithreal-timePCR,determinationofhepatitisdegreewithimmunopathologicalHEstaininganddetectionofliverfunction.Anti-preS1canbedetectedat4^th,6^thand10^thweekininoculatedBALB/cmice.Injectionwithgenegungainedanadvantageovermuscularandsubcutaneousinjectionsinceitacquiredjust1/10inoculationquantity(10μg/mouse).HighestexpressionofIgG2aat4^thweeksuggestedThl-mediatedimmuneresponse,whichfacilitatedHBVcleaning.OfallinoculatedHBVTg-mice,80%ofthemshowedanfi-preS2,HBsantibodypositiveandHBVDNAdecreased,and20%showednegativeforHBsAg.HEstainingtohepatictissueshowedobviousinfiltrationofinflammatorycells,swellingandgranulardegenerationofhepatocytes.Inourstudy,IL-2-preSDNAvaccinewhichcanprovokethehumoralandcellularimmuneresponseandbreaktheimmunetolerancesupportsthedesignationandconstructionofnewvaccineagainstHBVandspecificimmuneremedyforHBVcontinuousinfection.

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简介：Thepurposeofthestudyistoestablishafluorescencequantitativereversetranscriptionpoly-merasechainresponse(FQ-RT-PCR)methodforthequantitativedeterminationofIL-2mRNAandIL-4mRNAinThcells,withwhichtheThcellsstatusofthepatientswithgynaecologicaltumorsandchronicrenalfailure(CRF)canbeanalyzed.IL-2cDNAandIL-4cDNAwereprepared,andtheplasmidpMD18carryingIL-2cDNAorIL-4cDNAfragmentwasconstructedandclonedasthetemplateforquantitativedetermination.Theprimersandprobeslabelledwith6-carboxy-fluorescein(FAM)and6-carboxy-tetrarnethylrhodamine(TAMRA)wereprepared,andtheexperimentalconditionswereoptimizedtosetuptheFQ-RT-PCRmethodforquantitativedeterminationofIL-2mRNAandEL-4mRNA.Thcellsenrichedfromperipheralbloodmononuclearcells(PBMCs)of20healthyvolunteers(HVs),16gynaecologicalbenign(GB)cases,18gynaecologicalmalignant(GM)tumorcasesand16chronicrenalfailure(CRF)patientsweretestedforIL-2mRNAandIL-4mRNAbyFQ-RT-PCR.Thehouse-keepinggeneβ-actinwasusedastheinternalcontrolgeneoftheexperiment.Thestandardcurveforlogconcentrationofseriesofquantitativetemplatesvsthresholdcycle(CT)wasestablishedbylinearregression,andthelinearrangewas102-107copies/μl.TheimprecisiontestshowedtheCVofinter-assayandintra-assayofahighcontentsamplebyFQ-RT-PCRwere7.8%and12.5%,respectively.TheCVofinter-assayandintra-assayofalowcontentsamplewere10.8%and19.5%,respectively.TheIL-2mRNAexpressionsinThofthepatientswithgynaecologicalmalignanttumor(comparedwiththeHVsandthepatientswithgynaecologicalbenigndisease)andinThoftheCRFpatients(comparedwiththeHVs)weredeclinedsignificantlyandatthesametimetheIL-4mRNAexpressionincreasedsignificantly(P<0.001).Asimple,sensitiveandaccurateFQ-RT-PCRmethodforthequantitativedetectionofIL-2mRNAandIL-4mRNAhasbeenestablished.TheIL-2mRNAandIL-4m

简介：ToinvestigatetheeffectsofoverallalkaliofatraditionalChinesemedicine“Tongbiling”(brucineandstrychninealkaloidsinmain)onthecytokinesexpressioninTh1andTh2cellsinthesynovialfluidofpatientswithrheumatismarthritisandtheirsignalpathway,themononuclearcellsinthesynovialfluid(SFMC)ofpatientswereisolatedbyFicoll-Hypaquegradientcentrifugation,andtheCD3^+CD69^+andCD3^+HLA-DRantigenwereanalyzedbyflowcytometryincomparisonwiththoseoftheperipheralblood.TherestofcellswereculturedafterresuspensionwithRPMI1640culturemedium.Phorbol12,13-dibutyrate(PDB)andionomycinwereaddedsuccessivelyintotheculturewithvariousconcentrationofoverallalkaliTongbiling(TBL).After4hofcultivation,theexpressionofIFN-γandIL-4inCD3^+cellswereanalyzedbyflowcytometry.TheinfluenceofoverallalkaliTBL(100mg/I,)ontheintracellularcalciumwasinvestigatedafterFluo-3/AMlabelingandstimulationwithPDBandionomycinat1,2,4and10min,andtheinfluenceofTBLontheexpressionofCD3^+CD69^+cellsweredeterminedwithstimulationofPDBfor24hinthewholebloodlymphocytesculture.ItwasfoundthatthepercentageofTcellsbearingCD69wassignificantlyup-regulated(77%),whilethatofTcellsbearingHLA-DRwas44%inthesynovialmononucleatedcells.AfterPDBandionomycinstimulation,theexpressionofIFN-7inCD3~cellswereup-regulated,buttherewasnochangeontheexpressionofIL-4inCD3^+cells,indicatingthatratioofTh1/Th2wassignificantlyincreasedandThcellsdifferentiatetoThlcellsinmainly.FourconcentrationsofoverallalkaloidofTBI,(200mg/L,100mg/L,50mg/L,25mg/L)coulddown-regulatedtheexpressionofIFN-γinCD3^+cellsandtheTh1/Th2ratioobviously,butalltheconcentrationsoftheoverallalkaloidshadnoeffectontheexpressionofIL-4inCD3^+cells.100mg/Lconcentrationoftheoverallalkaloiddidnotdown-regulatetheintracellularcalciumlevel.Eachconc

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简介：ToconstructandexpressthefusionproteinStx2B-IntiminC300ofEHEC0157:H7,andtofurtherinvestigateitsimmunoprophyiacticpotential,thegeneofStx2B(stx2b)fromEHEC0157:H7chromosomewasclonedintopMD18-Tvector.Thereafter,theamplifiedgenewasclonedintoprokaryoticexpressionplasmidpET-28a(+)-eaeC300,whichwasconstructedpreviously.TherecombinantpasmidpET-28a(+)-stx2b-eaeC300wastransformedintoE.coliBL21(DE3).Afterinducement,theproteinStx2B-IntiminC300wassuccessfullyexpressedandanalyzedwithsodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDS-PAGE),WesternblottingandN-terminalaminoacidresidualsequencing.Toevaluateitsimmunoprophyiacticpotential,itwasprimarilypurifiedbyion-exchangechromatographyandinjectedinto30BALB/cmicewithAl(OH)3inthesubscapularregion.Tendaysafterthelastboostervaccination,20micewereattackedwithEHEC0157:H7lysateandtheprotectiveefficacywasobserved.Inthepresentstudy,thegeneofStx2B-IntiminC300wassuccessfullyclonedintopET-28a(+)vector.TheresultsofSDS-PAGEandWesternblottingassayshowedthatthefusionproteinwassuccessfullyexpressedintheinclusionbodyform,accountingfor25%oftotalexpressionproducts,anditsmolecularweightwasabout43kDa.TheresultoftheN-terminalaminoacidresidualsequencingshowedthatitwasidenticaltothatofthemoleculardesigned.Thepuritywasabout75%afterprimarypurification.AnimaltestsrevealedthatthefusionproteinStx2B-IntiminC300haselicitedhightiterofprotectiveantibodyrelatively.TheseresultsdemonstratethatthefusionproteinStx2B-IntiminC300issuccessfullyexpressedinprokaryoticexpressionsystemandshowscertainimmunoprophyiacticpotential.

简介：Wehaveconfirmedefficientanti-tumoractivitiesoftheperipherallymphocytestransducedwithap185HER2-specificchimericT-cellreceptorgenebothinmurineandinhumaninourpreviousstudies.TofurthertestthefeasibilityofchimericT-cellreceptorinabonemarrowtransplantationmodel,wefirst,madetwomurinetumorcelllines:MT901andMCA-205,toexpresshumanp185HER2byretroviralgenetransduction.MurinebonemarrowcellswereretrovirallytransducedtoexpressthechimericT-cellreceptorandgene-modifiedbonemarrowcellsweretransplantedintolethallyirradiatedmouse.Sixmonthsposttransplantation,p185HER2-positivetumorcells:MT-901/HER2orMCA-205/HER2wassubcutaneouslyorintravenouslyinjectedtomakemousemodelssimulatingprimarybreastcancerorpulmonarymetastasis.Theinvivoanti-tumoreffectsweremonitoredbythesizeofthesubcutaneoustumororcountingthetumornodulesinthelungsafterIndiainkstaining.ThesizeofthesubcutaneoustumorwassignificantlyinhibitedandthenumberofpulmonarynodulesweresignificantlydecreasedinmouserecipientstransplantedwithchimericT-cellreceptormodifiedbonemarrowcellscomparedwiththecontrolgroup.Ourresultssuggesttheefficientinvivoanti-tumoractivitiesofchimericT-cellreceptorgenemodifiedbonemarrowcells.

简介：WehavedevelopedandtestedchimericT-cellreceptors(TCR)specificforp185HER2.Intheseexperiments,retroviralvectorsexpressingtheN297orN29ξreceptorswereconstructedinpRET6.AmphotropicviralproducercellswereestablishedintheGALV-basedPG13packagingcellline.Ficollpurifiedhumanperipheralbloodlymphocytes(PBL)werevitallytransducedusinganoptimizedprotocolincorporatingactivationwithimmobilizedanti-CD3/anti-CD28monoclonalantibodies,followedbyviralinfectioninthepresenceoffibronectinfragmentCH296.Transducedcellswereco-culturedwithhumantumorcelllinesthatoverexpress(SK-OV-3)orunderexpress(MCF7)p185HER2toassayforantigenspecificimmuneresponses.BothCD4^+andCD8^+T-cellstransducedwiththeN297orN29ξchTCRdemonstratedHER2-specificantigenresponses,asdeterminedbyreleaseofTh1likecytokines,andcellularcytotoxicityassays.OurresultssupportthefeasibilityofadoptiveimmunothempywithgeneticallymodifiedT-cellsexpressingachTCRspecificforp185HER2.

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