Baganha, MF

Calado, G

Chieira, C

Loureiro, G

Machado, D

Martinho, A

Pais, M

Pereira, C

Santos, P

Tavares, B

Name not available

Genetic markers in chronic urticaria

CHUC Institutional Repository

Repositório Institucional dos Hospitais da Universidade de Coimbra

Oral Session 15Novel developments in immunogenetics and immunogeneomics85Quantitative proteomics: a strategy toidentify respiratory disease biomarkers inbronchial biopsiesO’Neil, S1; Ekerljung, L1; Sihlbom, C2; Carlsohn, E2;Krisiukeniene, A3; Stravinskaite, K3; Sitkauskiene, B3;Sakalauskas, R3; Lo¨tvall, J11Krefting Research Centre, Sahlgrenska Academy, Uni-versity of Gothenburg, Gothenburg, Sweden; 2Proteo-mics Core Facility, Sahlgrenska Academy, University ofGothenburg, Gothenburg, Sweden; 3Department of Pul-monology and Immunology, Kaunas University of Med-icine, Kaunas, LithuaniaBackground: Recent proteomic studies onrespiratory conditions investigate proteomechanges to identify protein biomarker can-didates for diagnosis, monitoring andtreatment. A major challenge in clinicalproteomics is the limited patient material.This is usually addressed by poolingpatients into disease groups. With sensitivequantitative proteomics methods, likeiTRAQ (isobaric tag for relative and abso-lute quantitation) technology, to identifyand quantify proteomic proﬁles with massspectrometry, it may be possible to deter-mine individual patient proteome differ-ences without the need for pooling. Thefeasibility of this strategy is examined withthe identiﬁcation of proteins in bronchialbiopsies of asthma and COPD patients, inan effort to identify disease and treatmentspeciﬁc protein biomarker candidates.Methods: Bronchial biopsies per patient,per timepoint, were taken from asthmatic(n = 14) and COPD (n = 16) subjects,treated with budesonide or placebo, beforeand after treatment. Healthy subjects(n = 4) and healthy smokers (n = 4) wereused as controls. Biopsies were randomisedover 20 sets of 4-plex experiments with theoperator being blinded to the disease sta-tus. Each 4-plex experiment contained acommon reference pool for normalisationpurposes. Proteins extracted from the biop-sies were digested, labelled with iTRAQreagents and fractionated using cationexchange chromatography, before each ofthe fractions was subjected to reversedphase nanoLC-MS/MS. The resulting spec-tra were analysed by Proteome Discovererand the Swissprot database was searchedfor protein identiﬁcations.Results: Bronchial biopsies yielded a small,but sufﬁcient amount of protein to conductproteomic experiments using iTRAQ tech-nology. Sixty-ﬁve micrograms of proteinwas digested, fractionated and analysed bynanoLC-MS/MS. Early results showed thatmore than 1000 proteins could be identiﬁedwith high statistical conﬁdence.Conclusion: Small amounts of patientbronchial biopsy material may be used toidentify and quantify proteins using theiTRAQ technology with a highly sensitiveMS system, without the need for poolingof samples from several patients. Thismethod may be used to identify airway dis-ease speciﬁc mucosal biomarkers.86Genetic markers in chronic urticariaMachado, D1; Pereira, C1; Martinho, A2; Loureiro, G1;Tavares, B1; Santos, P2; Calado, G1; Chieira, C1;Fontes Baganha, M3; Pais, M21Immunoallergology Department, Coimbra UniversityHospital, Coimbra, Portugal; 2Histocompatibility Centerof Coimbra, Coimbra, Portugal; 3Pulmonology Center,Coimbra University, Coimbra, PortugalBackground: The cellular role in chronicurticaria (CU) has been gradually recog-nised, however the genetic determinism isnot well identiﬁed. The aim of this workwas to evaluate the inﬂuence of HLAhaplotype and the association of geneticpolymorphism of some cytokines in CU.Methods: After signed informed consent,in a 15 patients population (10 female,mean age 45.7 ± 8.8 years and diseaseevolution time of 6.5 ± 5.9 years), withclinical criteria of CU and previous clinicaland laboratory evaluation, blood sampleswere collected to assess class I (locus Aand B) and class II (locus DR) HLAgenetic typing and evaluate cytokine singlenucleotide polymorphisms (SNP’s) in thepromoter region of IL-1 (a,b,R,RA), IL-2,IL-4, IL-6, IL-10, IL-12, IFN-c, TGF-band TNF-a. The results were comparedwith a group of 174 healthy individuals(group C).Results: It was observed a higher genotypefrequency in locus A for A*26 (NS) andA*68 (P = 0.048), while for A*01 (NS) itwas identiﬁed a lower frequency, comparingwith control group. In respect to locus B,the allele B*51 was the most frequent (NS);there was a higher frequency of B*38 in CUcomparing to group C (P = 0.047). Forclass II, the alleles DRB1*04, DRB1*07 andDRB1*15 were the most frequent, compar-ing to group C, but without statistical signif-icance. The most frequent haplotype wasthe A*01 B*08 DRB1*03, as in group C;however the second most frequent was A*02B*51 DRB1*11, corresponding to the ﬁfthmost frequent of the controls (4.7% versus1.4%, P 0.106). It was found a higher phe-notype frequency for the following cytokineSNP’s: IL10-592CC (P = 0.021), IL10-819CC (P = 0.021), TNFa-308GG (P =0.018), IL1a-889CC (P = 0.043), TGFb +869CT (P < 0.0001), TGFb+915CG (P =0.001) and IL2+166TT and GT (P = 0.014and 0.020). On the other hand, there werelower frequencies for IFNc+874TT(P = 0.001), TGFb+915GG (P = 0.023),IL2-166GG (P < 0.0001). In group CUthere were not ﬁnd heterozygous for TNFa-238AG (P < 0.0001) and homozygous forTNFa-308AA (P < 0.0001), TGFb +869TT and +915CC (P < 0.0001 and0.030), IL2-330TT (P = 0.002) and IL4-1098TT and -33TT (P = <0.0001 and0.014).Conclusion: The haplotype and cytokineSNP’s differences found suggest an immu-nogenetic contribution in CU pathogenesis.There were identiﬁed signiﬁcant changes incytokines of Th1 and Th2 response,explaining the immunologic and clinicalcomplexity of this syndrome.87Differential gene expression in thesensitisation and tolerance to olivepollen: microarray analysisAguerri, M1; Florido, F2; Mata, M3; Lo´pez-Cacho, J1;Gallardo, S1; Palomino, P1; Lahoz, C1; Ca´rdaba, B11Department of Immunology, Fundacio´n Jime´nezDı´az-Capio-Ciberes, Madrid, Spain; 2Department ofAllergy, Hospital Universitario San Cecilio, Granada,Spain; 3Genomic Department, Fundacio´n deInvestigacio´n, Hospital General Universitario-Ciberes,valencia, SpainBackground: Microarrays studies could bevery useful in the identiﬁcation of newroutes or pathways that can discriminatesensitization and natural or induced toler-ance against allergens.Methods: Subjects living in an area withextremely high load of olive pollen werecollected in two exposition moments, highpollen levels (pollen season) and low (out-side pollen season) distributed in 5 Groups: 2010 The Authors40 Journal Compilation  2010 Blackwell Publishing Ltd Allergy 65 (Suppl. 92): 1–77

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Genetic markers in chronic urticaria

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