Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry 英文参考文献.docVIP

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Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry 英文参考文献.doc

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Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry 英文参考文献

QuantificationofRegulatoryTCellsinSepticPatientsby Real-TimePCR–BasedMethylationAssayandFlow Cytometry RomanTatura1,MichaelZeschnigk2,MichaelAdamzik3,MichaelProbst-Kepper4,JanBuer1, JanKehrmann1* 1InstituteofMedicalMicrobiology,UniversityHospitalEssen,UniversityofDuisburg-Essen,Essen,Germany,2InstituteforHumanGenetics,UniversityHospitalEssen, Essen,Germany,3DepartmentforAnaesthesiologyandIntensiveCareMedicine,UniversityHospitalEssen,Essen,Germany,4InstituteforClinicalTransfusionMedicine, Sta¨dtisches KlinikumBraunschweiggGmbH,Braunschweig,Germany Abstract Duringsepsis,arelativeincreaseofregulatoryT(Treg)cellshasbeenreported.Itspersistenceisassociatedwithlymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expressionofmarkermoleculesisambiguous,asthesemoleculesareexpressedalsobyactivatednon-regulatoryTcells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activationstatus.Usingthisepigeneticmarker,weestablishedasingle-tubereal-timePCRbasedmethylationassay(QAMA) forrelativequantificationofTregcells.Validationwasperformedondefinedratiosofmethylatedandunmethylatedtarget + sequenceandonmixturesofTregandnon-regulatoryTcells.DNA-methylationwasmeasuredinCD4 Tcellsisolatedfrom bloodsamplesof30septicpatientsand30healthysubjectsandcomparedwithresultsofTregcellquantificationbyflow + cytometry based on CD4 CD25hiCD127low measurement. In septic patients both methods showed an increased ratio of TregcellstoallCD4 Tcells.Inhealthyindividuals,theresultsobtainedbybothmethodswereclearlypositivelycorrelated. However,thecorrelationbetweenbothmethodsinsepticpatientswasonlyweak.WeshowedthatquantificationofTreg + + cellsbyQAMAdetectsCD4 TcellswithunmethylatedFOXP3-TSDR,hiddenintheCD25med/low fractionofflowcytome