Made available in DSpace on 2016-01-26T12:51:44Z (GMT). No. of bitstreams: 1 ricardoacayabadetoledo_dissert.pdf: 765730 bytes, checksum: bac7d4d4322f0a4d4a32d9be65a91344 (MD5) Previous issue date: 2013-03-20 / Introduction: Spondyloarthritis is a group of diseases with clinical, laboratory and image similar. Analysis of clinical manifestations of spondyloarthritis in patients with and without the HLA-B*27 were performed, but the results revealed great heterogeneity. Objectives: The aim of this study was to evaluate the clinical manifestations of spondyloarthritis according to the classification criteria of the European Study Group Spondyloarthropathy (ESSG) in the presence and absence of the HLA-B*27. Patients and methods: From a total of 156 patients with clinical suspicion referred for the investigation of gene HLA-B*27, 73 were diagnosed with spondyloarthritis according to the criteria of ESSG. The HLA-B*27 were identified using commercial kits (Dynal ReliTM SSO HLA-B Typing Kit, Invitrogen). Clinical data were collected from medical records of patients. The results were compared using the chi-square or Fisher exact test. The values of Odds Ratio (OR) and confidence interval of 95% were also calculated. The p-value equal to or less than 0.05 was considered significant. Results: Of the 73 selected patients, 53 (72.6%) were male and 20 (27.4%) were female. The mean age was 49.4 and did not differ between genders (p = 0337). The spondyloarthritis found among the 73 patients were: ankylosing spondylitis (n = 47; 64.4%), psoriatic spondyloarthritis (n = 9; 12.3%), undifferentiated spondyloarthritis (n = 9; 12.3%), spondyloarthritis enteropathica (n = 6; 8.2%) and reactive arthritis (n = 2; 2.7%). The average age of onset was equal to 39.1 (± 11.7) and did not differ between genders (p = 0.9057). Of the total, 35 (47.9%) patients were HLA-B*27 positive and 38 (52.1%) were negative. This gene was positively associated with ankylosing spondylitis (OR: 5.37, 95% CI: 1813-15905, p = 0.003) and negatively with spondyloarthritis enteropathica (OR: 0.07, 95% CI: 0003-1301, p = 0.025). The sacroiliitis was associated with the presence of the gene (OR: 10 552, 95% CI: 1260-88256, p = 0.014) and intestinal injury absence (OR: 0.195, 95% CI: 0038-0978, p = 0.048). Conclusions: The HLA-B * 27 was associated with ankylosing spondylitis, enteropathic but not to spondyloarthritis. The radiological signs of sacroiliitis prevailed in patients positive for HLA-B*27, while intestinal involvement was associated with the absence of this gene. especially in cases of dystrophic scoliosis. In both cases, studies with larger samples are needed to assess whether these trends are evident. / Introdução: Espondiloartrite é um grupo de doenças com características clínicas, laboratoriais e imagenológicas semelhantes. Análises das manifestações clínicas das espondiloartrites em pacientes com e sem o gene HLA-B*27 foram realizadas, mas os resultados revelaram grande heterogeneidade. Objetivos: O objetivo deste trabalho foi avaliar as manifestações clínicas das espondiloartrites utilizadas nos critérios de classificação do European Spondyloarthropathy Study Group (ESSG) na presença e na ausência do gene HLA-B*27. Casuística e método: De um total de 156 pacientes com suspeita clínica, encaminhados para investigação do gene HLA-B*27, 73 tiveram diagnóstico de espondiloartrites confirmado, de acordo com os critérios do ESSG. O gene HLA-B*27 foi identificado com o uso de kits comerciais (Dynal ReliTM SSO HLA-B Typing Kit, Invitrogen). Os dados clínicos foram colhidos dos prontuários médicos dos pacientes. Os resultados foram comparados com o uso dos testes Qui-quadrado ou o teste exato de Fisher. Os valores de Odds Ratio (OR) e intervalo de confiança a 95% também foram calculados. O valor p igual ou menor que 0,05 foi considerado significante. Resultados: Dos 73 pacientes selecionados, 53 (72,6%) eram do sexo masculino e 20 (27,4%), femininos. A média de idade foi igual a 49,4 e não diferiu entre os sexos (p=0.337). As espondiloartrites encontradas entre os 73 pacientes foram: espondilite anquilosante (n=47; 64,4%), espondiloartrite psoriásica (n=9; 12,3%), espondiloartrite indiferenciada (n=9; 12,3%), espondiloartrite enteropática (n=6; 8,2%) e artrite reativa (n=2; 2,7%). A média de idade de início dos sintomas foi igual a 39,1 (±11.7) e não diferiu entre os sexos (p=0,9057). Do total, 35 (47.9%) pacientes eram HLA-B*27 positivo e 38 (52.1%), negativos. Este gene foi associado positivamente à espondilite anquilosante (OR: 5.37; IC 95%: 1.813-15.905; p=0.003) e negativamente à espondiloartrite enteropática (OR: 0.07; IC 95%: 0.003-1.301; p=0.025). A sacroiliíte se associou à presença do gene (OR: 10.552; IC 95%: 1.260-88.256; p=0.014) e o comprometimento intestinal à ausência (OR: 0.195; IC 95%: 0.038-0.978; p=0.048). Conclusões: O gene HLA-B*27 foi associado à espondilite anquilosante, mas não à espondiloartrite enteropática. Os sinais radiológicos de sacroiliíte prevaleceram nos pacientes positivos para o gene HLA-B*27, enquanto o comprometimento intestinal foi associado à ausência deste gene.

Typning av hla-b*27:En jämförelsestudie mellan två analysmetoder för att påvisa HLA-B*27 molekylen i ankyloserande spondylitCarolina BermudezBermudez, C. Typning av HLA-B*27. En jämförelsestudie mellan två analysmetoder för att påvisa HLA-B*27 molekylen i Ankyloserande spondylit. Examensarbete i Biomedicinsk vetenskap, 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk vetenskap, 2018.Human leukocyt antigen (HLA) är vävnadsantigener, belägna på våra vita blodkroppar. HLA-B*27 allelen är starkt kopplat till Ankyloserande spondylit (AS). Det är en kronisk inflammatorisk ledsjukdom, som främst attackerar ryggraden, bäckenet och bröstkorgen. Det finns idag ingen enskild laborativ metod som med full säkerhet kan fastställa diagnos av denna sjukdom, innan de kliniska symtomen uppträder. Typning av HLA-B*27 ger endast information om närvaro eller frånvaro av antigenet, vid utredning av AS. Vidare är HLA-B*27 en polymorf och de olika alleltyperna varierar kraftigt, bland skilda etniska grupper samt mellan geografiska områden. Genetiska- och miljöfaktorer påverkar också. Sjukdomsutveckling i samband med närvaro av HLA-B*27 allelen, varierar därför från individ till individ. Därmed fungerar metoden endast som ett komplement-verktyg, för att ytterligare bekräfta diagnos. Syftet med denna studie var att med realtids-polymerase chain reaction (PCR), utföra typning av HLA-B*27 med Linkseq kit samt jämföra analysresultaten med uthämtade resultat från intern sjukhusdatabas, där typning av HLA-B*27 hade utförts med PCR-SSP (sekvens-specifika primers). Samtliga resultat stämde överens till 100%, vilket indikerar att metoden fungerar bra. Det finns studier som visat att HLA-B*27 molekylens fria tunga kedjor (HLA-B*272) har en starkare benägenhet än andra HLA-molekyler att binda in till killer immunoglobine-like receptorer (KIRs). Inbindning till KIRs med efterföljande ökad stimulering av interleukiner (IL) främst IL-17 och IL-23 bidrar till sjukdomsutvecklingen av AS. Dock finns ingen HLA-B*272 specifik antikropp som kan bevisa detta och det behövs därför ytterligare undersökning för att hitta en sådan. Därefter skulle en ny laborativ metod kunna utvecklas för att fastställa diagnos av AS i ett tidigt skede, innan de kliniska symtomen uppvisas. Nyckelord: Allelvarianter, Ankyloserande spondylit, HLA-B*27, KIR, PCR-SSP, Realtids-PCR. / typing of hla-b*27:a comparison study between two analysing methods for the detection of the HLA-B*27 molecule in ankylosing spondylitisCarolina BermudezBermudez, C. Typing of HLA-B*27. A comparison study between two analysing methods for the detection of the HLA-B*27 molecule in Ankylosing spondylitis. Degree project in Biomedical Laboratory Science, 15 credit points. Malmö University: Faculty of Health and Society, Department of Biomedical science, 2018.Human leukocyte antigen (HLA) are tissue antigens located on our white blood cells. The HLA-B*27 allele is strongly related to Ankylosing spondylitis (AS). It is a chronical inflammatory rheumatic disease that primarily affects the spine, the pelvis and the chest. At present, there is no single laboratory method that with all certainty may determine diagnosis of this disease, before the clinical symptoms appear. Typing of HLA-B*27 only gives information about the presence or absence of the antigen, upon the investigation of AS. Furthermore, HLA-B*27 is a polymorph and the different types of alleles, strongly vary among different ethnic groups and also between geographic regions. Genetic- and environmental factors also affect. Development of disease in conjunction with the presence of the HLA-B*27 allele, therefore varies from one individual to another. So, the method only functions as a complementary tool, to further confirm diagnosis. The aim of this study was to perform HLA-B*27 typing with realtime-polymerase chain reaction (PCR) using Linkseq kit and compare the analysed results with those results that were retrieved from the internal database of the hospital, where typing of HLA-B*27 had been performed with PCR-SSP (sequence specific primers). All results agreed with 100%, which indicates that the method functions well. There are studies that show that the heavy chains (HLA-B*272) of the HLA-B*27 molecule have a stronger affinity than other HLA-molecules of binding in to killer immunoglobulin-like receptors (KIRs). Increased stimulation of interleukins (IL) primarily IL17 and IL23, following binding to KIRs, contributes to the pathogenesis of ankylosing spondylitis. However, there is no HLA-B*272 specific antibody that may prove this and therefore more investigation is needed, in order to find one. A new laboratory method could then be developed to determine diagnosis of AS at an early stage, before the clinical symptoms emerge. Keyword: Allelvariants, Ankylosing spondylitis, HLA-B*27, KIR, PCR-SSP, Realtime-PCR.

Despite the introduction of antiretroviral therapy, the HIV/HIV epidemic remains an unsolved global health problem. Amongst all the host defence mechanisms, HLA class I molecules have shown the strongest genetic association with delayed disease progression, in particular HLA-B alleles. Numerous studies have shown that the HLAmediated CD8+ T cell responses play a central role in the immune control of HIV. Yet our understanding of HLA-mediated immune control of HIV remains incomplete, even when considering the best-defined epitopes restricted by the protective HLA alleles at a population level. The studies I have conducted and describe herein focus on two well-charaterised protective HLA-B molecules, HLA-B*81:01 and HLA-B*27:05; a third protective molecule, HLA-B*52:01, that has not been well-studied hitherto; and finally the most prevalent HLAB allele in many Asian populations such as Taiwan, HLA-B*40:01, which has an apparently neutral effect on viral replication. This thesis is centred on the Gag-specific immune response, since previous studies have shown the benefits of CD8+ T-cell responses targeting this conserved and immunogenic region of the HIV proteome, in particular the p24 capsid protein. I have investigated here HLA footprints driven by CD8+ T-cell pressure on HIV that are evident in the viral sequences of individuals expressing these HLA molecules. These footprints include novel escape and putative compensatory mutations. The impact of these variants on viral replicative capacity (VRC) and on HIV disease outcome clinical outcomes was examined via fitness assays. These studies identified several escape mutations that effectively cripple HIV. The distinct compensatory pathways available to the virus to mitigate the fitness cost of particular escape mutations were evaluated. In the course of these analyses I have demonstrated the critical influence of the viral backbone, including HIV clade, in combination with particular viral variants, on VRC. Computational modelling analysis has been applied to facilitate understanding of the mechanism by which certain mutants affect the stability of interactions between HLA and viral capsid protein. This thesis offers novel insights into immune control of the key HIV subtypes – B- and C-clade – and of the most severely affected populations – in Africa (South Africa) and Asia (India and Taiwan) – within the global epidemic. This work helps to better define the viral mutation landscape that is essential both for future vaccines designed to corner the virus, and for successful HIV cure strategies.