简介：Toinvestigatetheeffectsofantisenseoligonucleotidesontheexpressionofmacrophagemigrationinhibitoryfactor(MIF)onmacrophages,themousephosphorothioateoligonucleotidesweredesignedandsynthesizedwiththesequencesofantisense,5'-TACGGATACAAGTAGCAC-3'；Sense,5'-ATGC-CTATGTTCATCGTG-3'；Missense,5'-CTCTCAGACTCGATCTGT-3'.Thesephosphorothioateoligonucleotideswerethentransfectedintoculturedmacrophages(RAW264.7)byluciferasevector,andthetransfectedmacrophageswereincubatedwithLipopolysaccharide(LPS)(1ng/ml)forvariousperiodsoftimesandcollectedafterwards.ThecontentofMIFproteinintheculturalsupernatantswasdeterminedbyELISA,cellularRNAextractedandtheexpressionofMIFmRNAwasexaminedbyRT-PCRanalysis.TheexperimentalresultsshowedthatLPScouldinduceatime-dependentspecificexpressionofMIFonmacrophages,inwhichtheMIFmRNAincellsandtheMIFproteininculturalsupernatantsappearedafter3handreachedtheirhighestconcentrationat9-12hafterLPSstimulation.ThelevelsofmRNAandproteinsinthemacrophagestreatedwithantisenseolignucleotidesweredecreasedsignificantlyafterstimulationwithLPSincomparisonwiththatofstimulationwithLPSaloneorwiththatwithLPSplussenseormissenseoligonucleotides.TherewerenodifferencesamongthosewithoutLPSstimulation.ItisconcludedthatmacrophagesstimulatedwithLPSexpressMIF,andtheantisenseolignucleotidesofMIFinhibittheexpressionofMIFmRNAaswellasthesecretionofMIFproteinsinmacrophages.

简介：TheaimofthisstudyistoinvestigatetheeffectsofallitridinontheexpressionoftranscriptionfactorT-bet/GA-TA-3inmiceinfectedbymurinecytomegalovirus.BALB/cmicemodelsystemofmurinecytomegalovirus(MCMV)infectionwasestablished.Inwhich20modelmicewereallocatedrandomlyintoallitridintreatedgroup(n=10)andinfectedcontrolgroup(n=10).Allitridin(25mg·kg^-1·d^-1)wasusedintreatedgroupatthe24hbyintraperitonealroute(once/d×14d),andthesamevolumeofsalinesolutionwasinjectedcontrolmice.Normalcontrolmice(n=10),wereonlygivenwiththesamevolumeof0.89%sodiumchloride,withoutinfectionwithMCMV.TheexpressionlevelsoftranscriptionfactorT-bet/GATA-3mRNAweremeasuredbyRT-PCR,andtheexpressionlevelsofThelper1(Thl)cytokineIFN-γandTh2cytokineIL-10insupernatantofspleencellcultureweremeasuredbyELISA.ExperimentalresultsshowedMCMVinfectioncouldmarkedlydown-modulatetheexpressionofIFN-γandT-bet,andsignificantlyup-modulatetheexpressionofIL-10andGATA-3mRNA.AllitridincouldinduceincreasedexpressionoftranscriptionfactorT-betmRNAandThlcytokineIFN-γsignificantly(P<0.01),anddecreasedexpressionoftranscriptionfactorGATA-3mRNAandTh2cytokineIL-10markedly(P<0.01).ItisconcludedthatMCMVinfectionleadstodisequilibriumofThl/Th2cytokineexpression:thelevelofThlcytokineIFN-γdecreasessignificantlyandTh2cytokineIL-10overexpressesmarkedly.Allitridincanup-regulatetheexpressionofT-betandIFN-γ,andinhibittheexpressionofGATA-3mRNAandIL-10inMCMVinfectedmice,indicatingaTh1dominantstatewhichshouldenhancethespecificcellularimmunereactionsagainstCMVandbehelpfulforclearanceofthecytomegalovirusinhost.

简介：Toexploretheroleofnuclearfactor-κB(NF-κB)inthesignalpathwayofproteinkinaseC(PKC)regulatingtheproliferationandapoptosisofTlymphocytesinasthma.Tlymphocyteswereisolatedfromtheasthmaticmodelofguineapigsandtheasthmaticpatients.EithertheTcellsstimulatedwithPMAaloneorthosestimulatedwithPMAtogetherwithpyrrolidinedithiocarbamate(PDTC)wereincubatedfor1and24h.TheproliferationofandthepresenceofNF-κBinthecellsincubatedfor1hwereobservedbyMTTandimmunohistochemicalstaining,respectivelyAndthecellsincubatedfor24hwereobservedfortheapoptosisbyTUNEL.Alltheassayswereparalleledwithcontrols,andallthedatawereanalyzedstatisticallywiththesoftwareSAS.ThepercentageofcellsofnuclearpositivestainingofNF-scBandtheproliferationofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithPMAweresignificantlyhigherthanthoseofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithoutPMArespectively(P<0.01)andthoseofTlymphocytesfromnormalcontrolguineapigsandnormalcontrolpersonsstimulatedwithPMArespectively(P<0.01),andweresignificantlyreducedbyPDTC(P<0.01).TheapoptosisindexofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithPMAweresignificantlylowerthanthoseofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithoutPMArespectively(P<0.01)andthoseofTlymphocytesfromnormalcontrolguineapigsandnormalcontrolpersonsstimulatedwithPMArespectively(P<0.01),andweresignificantlyinducedbyPDTC(P<0.01).ThereweregoodpositivecorrelationbetweenthepercentageofcellsofnuclearstainingofNF-κBofTlymphocytesandtheproliferationofTlymphocytes(r=0.51-0.72,P<0.001),andalsogoodnegativecorrelationbetweenthepercentageofcellsofnuclearstainingofNF-scBandtheapoptosisindexofTlymphocytes(r=-0.55-0.71,P

简介：Toclonethegenecodingtheimmunodominantregioninthechlamydialprotease-likeactivityfactor(CPAF)fromChlamydophilapneumoniae,toanalyzeimmunocompetenceoftheexpressedprotein,andtoevaluateitsvalueinserodiagnosis,theCPAFimmunodominantregiongenewasamplified,ligatedintoapGEX6p-2vector,andthentheexpressedrecombinantproteinwaspurifiedwithglutathioneS-transferase(GST)agarosegelFFafterrenaturation,thenidentifiedbySDS-PAGEandWesternblot.AnewindirectELISAwasdevelopedwiththepurifiedproteinascoatingantigen.TheimmunogenicityoftherecombinantproteinwasevaluatedbyimmunizationtoNewZealandrabbits,anditsimmunoreactivitywasanalyzedbyreactingwithanti-C,pneumoniaeantibody.300clinicalserasampleswererespectivelyde-tectedbymicroimmunofluorescence(MIF)asreferencemethodandtheindirectELISA,andthediffer-encebetweenthetwomethodswasanalyzed.Cross-reactivityagainstChlamydiatrachomatiswasinvesti-gatedwiththeindirectELISAtodetectanti-C,trachomatispositiveantisera.Theresultsindicatedthata51.3kDarecombinantproteinwasobtained.Westernblotassayprovedthattherecombinantproteincouldmerelyspecificallyreactwithhumananti-C.pneumoniaeantisera.ThetitersofthespecificIgGan-tibodiesintheimmunizedNewZealandrabbitswereabove1:16000.Anti-C.pneumoniaeIgGpositiveandnegativereferencesereweredetectedwiththeindirectELISA,andtheconcordancerateofnegativeandpositiveresultswereboth100%(40/40).ThesensitivityandspecificityoftheindirectELISAincomparisonwithMIFwere93.8%(45/48)and100%(252/252)separatelybydetecting300clinicalserasamples,andtheconcordanceratebetweenthetwomethodswas99.0%.NocrossreactionagainstC.trachomatiswasfoundwiththeindirectELISAtodetectanti-C,trachomatispositiveantisera.Incon-clusion,thepreparedrecombinantproteinoftheCPAFimmunodominantregionshowsexcellentimmuno-competenceandcanbeusedtodevelopanewindirect

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