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1、Antigen selection in the lgE response and allergic disease. Yan WangYan WangSupervisor: Andrew Collins Supervisor: Andrew Collins CosupervisorCosupervisor: William Sewell: William SewellImmunoglobulinStructure of immunoglobulin Isotypes of immunoglobulinsImmunoglobulin DiversityGene recombination He
2、avy chain Heavy chain variable region (VH)variable region (VH)Somatic hypermutation Mutation leads Mutation leads to loss of to loss of specificity.specificity. Cell diesCell diesProgenyProgenyOriginalOriginalSelected cellSelected cell Mutation leads Mutation leads to improved to improved binding.bi
3、nding. Cell dividesCell dividesOther cellsOther cellsAntigen SelectionY YY YY YY YY YY YY YY YXY YY YY YY YY YY YY YY YY YY YY YY YX DivisionsDivisions SHMSHM DivisionsDivisions SHMSHMY YY YY YY YFunctionalFunctionalB B cell cellPlasma cellPlasma cellMemory B Memory B cellcellPre-mature Pre-mature B
4、 cellB cellIgE ResponsesParasite defencesAllergic diseaseHygiene Hypothesis of Allergic DiseasesGenetic susceptibilityGenetic susceptibilityThe hygiene hypothesisThe hygiene hypothesis states that a lack of early states that a lack of early childhood exposure to childhood exposure to infectious agen
5、ts increases infectious agents increases susceptibility to susceptibility to allergicallergic diseases. diseases. Hypothesis of IgE GenerationIgE sequences are not subjected to the antigen selection processes of the normal germinal center reaction IGHV gene polymorphismsAimAim: to carefully define t
6、he : to carefully define the Immunoglobulin Immunoglobulin heavy chain variable gene (heavy chain variable gene (IGHV) repertoireIGHV) repertoireIGHV genes are reported to be highly IGHV genes are reported to be highly polymorphicpolymorphicAre there any mistakes in the repertoire?Are there any mist
7、akes in the repertoire? Most alleles reported before 1995Most alleles reported before 1995 Lack of understanding of the organization of Lack of understanding of the organization of immunoglobulin gene locusimmunoglobulin gene locus The sequencing technology were not matureThe sequencing technology w
8、ere not matureMethodsCompilation of Database 226 IGHV previously reported alleles226 IGHV previously reported alleles 4718 rearranged VDJ gene sequences from EMBL 4718 rearranged VDJ gene sequences from EMBL Bioinformatic analysis The publications that reported each germline gene were The publicatio
9、ns that reported each germline gene were reviewedreviewed Multiple alignments of IGHV germline geneMultiple alignments of IGHV germline gene Aligned germline genes to rearranged sequences by Aligned germline genes to rearranged sequences by iHMMuneiHMMune- -align program align program Apparent mutat
10、ion numbers and patterns were Apparent mutation numbers and patterns were analysedanalysedResultsFive Level systems Five Level systems for classification of for classification of the IGHV germline the IGHV germline allelesallelesLevelDefinition1n = 44The reported sequence is a realallele. 2n = 38The
11、 reported sequence is a realallele. Similarities between thisallele and other known allelesmean that many alignments tothis allele are likely to be inerror.3n = 23The reported sequence may bea real allele. 4n = 17The reported sequence may bea real allele. Similarities betweenthis allele and other kn
12、ownalleles mean that many alignmentsto this allele are likely to be in error. 5n = 104The reported sequence is unlikelyto be a real allele. Most Level 5 alleles are never seen in the Most Level 5 alleles are never seen in the rearranged gene databaserearranged gene databaseMost Level 1 alleles are c
13、ommonly seen in the Most Level 1 alleles are commonly seen in the rearranged gene databaserearranged gene databaseResultsIdentifying new polymorphisms 12 12 putative alleles were identifiedputative alleles were identified Sequencing the putative alleles of IGHV3 and IGHV4 Sequencing the putative all
14、eles of IGHV3 and IGHV4 families in genomic DNA by PCRfamilies in genomic DNA by PCRIGHV3-49*p04, IGHV3-49*p05 and IGHV4-39*p07 IGHV3-49*p04, IGHV3-49*p05 and IGHV4-39*p07 were confirmed in the genomic sequencing were confirmed in the genomic sequencing Future workAustraliansPapua New Guineans Sampl
15、es investigationBlood samples from allergic and non-allergic individualsAt the same time, parasite infections will also be detectedTotal IgE analysis by ELISATotal IgE analysis by ELISAStool samplesStool samplesBlood samplesBlood samplesIgG and IgE SequencingTotal RNA will be extracted from peripher
16、al blood lymphocytesIsotype specific primerSemi-nested PCR in IgE sequencingBioinformatic analysisAligning of the IgE and IgG VH sequencesAligning of the IgE and IgG VH sequencesThe mutation frequencies and patternsThe mutation frequencies and patternsComparisonComparison IgG from Australians and Ne
17、w GuineansIgG from Australians and New Guineans IgE and IgG in IgE and IgG in parasitisedparasitised and allergic individuals and allergic individualsAcknowledgementsAndrew Collins, William SewellWendy Glenn, Margaret Cooley, Katherine Jackson, Jeanette Villanueva, James Lazenby Christine Whittall and Viveka SinghThank you!
