Introdução: A Espondiloartrite axial (EpA-ax) é uma doença inflamatória e crônica do grupo das Espondiloartrites (EpA). Ela acomete o sistema músculo esquelético ocasionando inflamação das articulações axiais (especialmente da sacroilíaca). Quando essas manifestações são evidenciadas por exames de imagem, temos a caracterização da Espondilite Anquilosante (EA). Do contrário, chamamos de EpA-axial não radiográfica (EpA-ax-nr). A Espondilite Anquilosante (EA) se caracteriza pelo envolvimento inflamatório de articulações do esqueleto axial e apendicular. Seu diagnóstico é baseado em achados clínicos e radiológicos, além da elevação de marcadores inflamatórios e presença do HLA-B27. Várias são as metodologias que se propõem a identificar o HLA-B27 e a Polymerase Chain Reaction (PCR) é tida como referência. Sua ampla utilização esbarra, no entanto, em questões que levam em conta o custo, oferta do exame e o tempo até a obtenção do resultado. Neste contexto, a Citometria de Fluxo (CF) surge como uma alternativa capaz de ajudar o médico na identificação deste marcador genético que pode ser importante para o diagnóstico e prognóstico dessa doença. Objetivo: Avaliar a correlação entre as técnicas laboratoriais mais utilizadas na prática clínica (CF e PCR), comparando sensibilidade e especificidade para detecção do HLA-B27 em pacientes com diagnóstico estabelecido de EpA-ax. Métodos: Estudo transversal, comparativo, com pacientes maiores de 18 anos de idade selecionados por conveniência, coletados ao longo de 2015, com diagnóstico estabelecido de EpA-ax, segundo os do grupo internacional ASAS (working group - Assessment of SpondyloArthritis Intenational Society). Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. Para a análise estatística o coeficiente Kappa (P<0,005 foi adotado como parâmetro. O teste Exato de Fisher foi utilizado para comparar as variáveis categóricas, o teste t de Student, para variáveis quantitativas com distribuição simétrica de amostras independentes. E as variáveis com distribuição assimétrica foram comparadas pelo teste de Mann-Whitney. Resultados: O coeficiente Kappa obtido para a determinação da concordância entre os testes foi de 0,454. Foram incluídos no estudo 62 pacientes, desses, sessenta preencheram os critérios para diagnóstico de EA e 2 para EpA-ax não radiográfica (EpA-ax-nr), 64,5% dos pacientes eram do sexo masculino, 88,7% se autodeclararam brancos, a idade média (± desvio padrão) foi de 54,5±12 anos e o tempo mediano (percentis 25 e 75) de diagnóstico de 14 (9 e 24) anos. Dentre as características clínicas apresentadas pela população estudada houve diferença estatisticamente significativa para a artrite periférica, sendo mais frequente no grupo HLA-B27 negativo que no grupo HLA-B27 positivo (P=0,032). Na análise de correlação, 90,3% apresentaram tipagem HLA-B27 positiva por CF e 79,0% pela técnica de PCR. Tendo o PCR como padrão ouro, a CF apresentou uma sensibilidade de 98,0%, especificidade de 38,5% e uma acurácia de 85,5%. Conclusão: Apesar da baixa especificidade apresentada pela CF, nosso estudo demonstrou que a CF tem alta sensibilidade e boa acurácia o que a torna uma boa alternativa para ser utilizada como um teste de triagem na busca da caracterização da doença. Apesar da moderada concordância com a técnica de referência, a CF poderia ajudar o médico a excluir resultados falsamente negativos, racionalizando assim a investigação laboratorial para o diagnóstico da EA. / Introduction: Axial spondyloarthritis (SpA-ax) is an inflammatory and chronic disease of the Spondyloarthritis (SpA) group. It affects the skeletal muscle system causing inflammation of the axial joints (especially of the sacroiliac). When these manifestations are evidenced by imaging tests, a characterization of Ankylosing Spondylitis (AS). Otherwise, we call the non-radiographic SpA-axial (SpA-ax-nr). AS marked by the inflammatory involvement of the axial and appendicular skeletal joints. The diagnosis is based on clinical and radiological findings, as well as the on the elevation of inflammatory markers and presence of HLA-B27. There are several methodologies to identify the HLA-B27 gene and a Polymerase Chain Reaction (PCR) is considered the reference method. However, its use on a large scale does not progress because it takes into account the cost, offer of the exam and the running time to obtain the result. In this context, Flow Cytometry (FC) emerges as an alternative method, helping with the physician in the determination of this genetic marker, important for the diagnosis and prognosis of disease. Objective: To evaluate the correlation between FC and PCR, comparing sensitivity and specificity for detection of HLA-B27 in patients with established diagnosis of SpA-ax. Methods: A cross sectional study including 62 patients recruited during 2015 month was conducted in Hospital de Clínicas de Porto Alegre, an university public hospital. The sample, recruited by convenience in the SpA clinic, was composed of patients ≥ 18 years old, fulfilling the Assessment of Spondyloarthritis (ASAS) criteria SpA-ax. All participants underwent HLA-B27 typing through FC and PCR. The kappa statistic was used to calculate the concordance between the FC and PCR. Taking PCR as the gold standard, sensitivity and specificity of FC to detect HLA-B27 were calculated. Results: The Kappa coefficient obtained to determine the agreement between the tests was 0.454. Sixty two patients were included in the study, sixty met the criteria for diagnosis of AS and two for SpA-ax-nr, 64.5% of the patients were male, 88.7% were self-declared mean age (± standard deviation) was 54.5 ± 12 years and median time (25th and 75th percentiles) for diagnosis was 14 (9 and 24) years. Among the clinical characteristics presented by the population studied, there was a statistically significant difference for peripheral arthritis, wich was more frequent in the HLA-B27 negative group than in the HLA-B27 positive group (P = 0.032). In the correlation analysis, 90.3% presented HLA-B27 positive typing by FC and 79.0% by PCR technique. When PCR was considered the gold standard, CF had a sensitivity of 98.0%, specificity of 38.5% and an accuracy of 85.5%. Conclusion: Despite the low specificity presented by FC, our study demonstrated that FC has high sensitivity and good accuracy, which makes it a good alternative to be used as a screening test in the search for the characterization of the disease. Despite the moderate agreement with the reference technique, FC could help the physician to exclude falsely negative results, thus rationalizing laboratory investigation for the diagnosis of AS.

Background: Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease affecting primarily the spine and its adjacent structures. The disease is characterized not only by destructive joint changes but also by excessive osteoproduction, which can lead to gradual ankylosis of the spine and thus significantly reduce the mobility and quality of life. The pathogenesis of the disease is not yet fully understood, but a strong genetic background is suggested, along with dysregulation of tissue metabolism resulting from an imbalance of pro- and anti-inflammatory immune mechanisms. We are still lacking biomarker with sufficient sensitivity and specificity which could help to identify early diagnosis, to monitor subchondral damage, and to differentiate rapidly progressing patients. The aim of this work was to determine the levels of potential biomarkers of connective tissue metabolism, fat metabolism and new promising biomarkers for both disease subtypes, their relationship to disease activity and progressive structural changes. Results: We have shown increased serum/plasma levels of connective tissue metabolism biomarkers (especially matrix metalloproteinase mediated metabolites), which were able to differentiate patients with early and late forms of axSpA from healthy individuals (HC), were...

La SpA est un rhumatisme inflammatoire chronique fréquent, dont la prévalence est de 0,3% en France. Les mécanismes pathologiques qui en sont à l’origine demeurent largement incertains. Néanmoins, l’héritabilité de la maladie est élevée, impliquant de multiples facteurs génétiques, dont la région du complexe majeur d’histocompatibilité (CMH) et plus particulièrement l’allèle HLA-B27 qui y exerce un rôle prédominant. L’objectif de ce travail était d’identifier de nouvelles cibles moléculaires, en vue d’améliorer la compréhension de la physiopathologie de la SpA, par des approches génétiques et de génomiques fonctionnelles.La première partie de mon travail a consisté en l’identification de polymorphismes du CMH associés à la SpA et distinct de HLA-B27. Les études d’association portant sur les données génétiques de 3 cohortes indépendantes nous ont permis d’identifié 5 variants associés à la SpA indépendamment du HLA-B27. Les deux polymorphismes situés à proximité des gènes MICA et MAPK14 semblent particulièrement intéressants pour leur implication potentielle dans la pathogénèse de la SpA. En marge de cette étude, nous avons entrepris de déterminer la prévalence du HLA-B27 dans une cohorte française représentative de la population générale, qui était de 6,9% chez les témoins et de 74,2% chez les sujets atteints de SpA.Les études fonctionnelles conduites sur des cellules dendritiques dérivées de monocytes (MD-DCs) ont permis d’identifier un défaut de réponse proliférative des LT CD4+ stimulés par les MD-DCs de patients atteints de SpA, ainsi qu’une signature transcriptomique de 81 gènes caractéristique des MD-DCs de ces patients. Parmi les gènes validés, la surexpression d’ADAMTS15, de F13A1 et de SELL pourrait jouer un rôle dans l’inflammation liée à la pathologie, alors que la sous-régulation de CITED2 paraitrait corrélée à une dérégulation de la voie Wnt. Enfin, nos investigations sur les MD-DCS nous ont amené à identifier une corrélation entre l’haplotype d’ERAP1 prédisposant à la SpA, et un niveau accru d’expression de ce gène ainsi que de la protéine ERAP1. / Spondyloarthritis (SpA) is a frequent chronic inflammatory rheumatic disorder, with a prevalence of 0.3% in France. Pathological mechanisms leading to SpA remain largely uncertain. Nevertheless, the heritability of this disorder is high, likely involving multiple genetic factors, among which the major histocompatibility complex (MHC) region and particularly the HLA-B27 allele which plays a prominent role. The objective of this work was to achieve a better understanding of SpA physiopathology via genetic and transcriptomic approaches. The first part of my work consisted in identification of MHC polymorphisms associated with SpA, distinct of HLA-B27. Association studies based on the genetic data of 3 independent cohorts have allowed to identify 5 SNPs associated to SpA, independently of HLA-B27. Two polymorphisms localized next to MICA and MAPK14 genes seem particularly interesting for their implication in SpA pathogenesis. In parallel of this study, we characterized HLA-B27 prevalence in a French cohort corresponding to 6.9% in healthy controls and 74.2% in SpA patients. Functional studies on monocyte-derived dendritic cells (MD-DCs) revealed altered capacity to stimulate allogeneic CD4+ T cell responses by MD-DCs from SpA patients and a transcriptomic signature of 81 genes differentially expressed in those cells, as compared to those from healthy controls. Among validated genes, ADAMTS15, F13A1 and SELL could play a role in SpA inflammation, whereas CITED2 seemed to be correlated to Wnt pathway. Finally, a strong correlation between ERAP1 SpA-susceptibility haplotype and an increased expression of this gene and the ERAP1 protein has been identified.

No description available.

L’interface hôte/microbiote intestinal est un système d’interactions complexes dont le déséquilibre est associé au développement des maladies inflammatoires chroniques. Les traitements anti-TNFα sont très efficaces dans ces maladies, mais seulement chez certains patients. L’objectif de ce travail était de rechercher un lien entre composition du microbiote intestinal et réponse aux traitements anti-TNFα dans deux types de maladies inflammatoires chroniques, les spondyloarthrites et les maladies inflammatoires chroniques de l’intestin. Nous avons retrouvé des variations de la composition du microbiote intestinal après traitement par anti-TNFα chez des patients atteints de spondyloarthrite et avons identifié un nœud taxonomique prédictif de la réponse thérapeutique à trois mois. Ce nœud taxonomique, l’ordre des Burkholderiales, étant ainsi un biomarqueur potentiellement utilisable en pratique clinique, nous avons déposé une demande de brevet européen, qui est en cours d’instruction. Ce travail a été poursuivi par un nouveau protocole de recherche clinique incluant des patients atteints de spondyloarthrites mais aussi de maladies inflammatoires chroniques intestinales. Ce protocole est financé par le CHU de Bordeaux dans le cadre de l’Appel d’Offre Interne. Il permettra de valider les hypothèses de notre premier travail, en réalisant notamment des PCR quantitatives utilisant des amorces spécifiques de l’ordre des Burkholderiales. Nous avons de plus retrouvé in vitro pour la première fois à notre connaissance une modulation de la croissance bactérienne chez Bacteroides fragilis en réponse au TNFα humain. / The host/gut microbiota interface is a system of complex interactions whose imbalance is associated with the development of chronic inflammatory diseases. Anti-TNFα treatments are very effective in these diseases, but only in some patients. The purpose of this work was to find a link between the composition of the intestinal microbiota and clinical response to anti-TNFα treatments in two types of chronic inflammatory diseases, spondyloarthritis and inflammatory bowel disease. We found variations in the composition of the intestinal microbiota after treatment with anti-TNFα in patients with spondyloarthritis and identified a taxonomic node predictive of the therapeutic response at 3 months. This taxonomic node, the Burkholderiales order, being a biomarker potentially usable in clinical practice, we have filed a European patent application, which is currently under investigation. This work was continued by a new clinical research protocol including patients with spondyloarthritis but also with inflammatory bowel diseases. This protocol is funded by the Bordeaux University Hospital as part of the internal call for tenders. It will validate the hypotheses of our first work, notably by performing quantitative PCRs using specific primers targeting the order of Burkholderiales. In vitro, we have also found for the first time, to our knowledge, a modulation of bacterial growth in Bacteroides fragilis in response to human TNFα.

La spondyloarthrite (SpA) est une pathologie rhumatologique caractérisée notamment par une inflammation et par des ossifications excessives se formant au niveau des enthèses. Il s’agit de zones de fortes contraintes mécaniques où les tendons et ligaments sont ancrés dans l’os via une zone fibrocartilagineuse. La sphingosine 1-phosphate (S1P) est un lipide bioactif qui joue un rôle important à la fois dans le remodelage osseux et la réponse inflammatoire. Notre objectif était donc d’explorer le rôle de la S1P dans l’ossification excessive de la SpA. Nous avons observé que les taux sériques de S1P des patients atteints de SpA sont significativement supérieurs à ceux de donneurs contrôles. Nous avons utilisé comme modèle des cultures primaires murines d’ostéoblastes, de chondrocytes et de ténocytes et des cultures organotypiques d’enthèse de souris. Nous avons observé que les enzymes de synthèse de la S1P, les sphingosine kinases 1 et 2, contribuent à la minéralisation des ostéoblastes et des chondrocytes. L’effet pro-minéralisant de la S1P est partiellement médié par deux de ses récepteurs (S1P1 et S1P3). De plus, la production de S1P est stimulée suite à un étirement cyclique dans les ostéoblastes et les chondrocytes, et après un traitement avec les cytokines TNF-α et IL-17 dans les chondrocytes. Finalement, l’inhibition générale du métabolisme de la S1P par le Fingolimod conduit à une diminution de la minéralisation dans les ostéoblastes et encore davantage dans les chondrocytes. Ces résultats suggèrent que le métabolisme de la S1P participe à l’ossification excessive de la SpA. Des études in vivo sont maintenant nécessaires pour valider cette possibilité / Spondyloarthritis (SpA) is a rheumatic disease characterized in particular by enthesis ectopic ossification and inflammation. Enthesis is a zone of concentration of mechanical stresses where ligaments and tendons attach to bone through fibrocartilaginous connections. Sphingosine 1-phosphate (S1P) is a bioactive lipid that plays an important role in both bone remodelling and in inflammatory response. Our aim was to explore the role of S1P in SpA excessive ossification. We observed that serum S1P concentrations in SpA patients are significantly higher compared to control donors. We used primary mouse osteoblasts, chondrocytes and tenocytes as cellular models and organotypic cultures of mice enthesis. We observed that S1P synthetizing enzymes, sphingosine kinases 1 and 2, stimulate osteoblasts’ and chondrocytes’ mineralizing process. S1P pro-mineralizing effect was partially mediated by two of the S1P receptors (S1P1 and S1P3). Moreover, S1P production was enhanced by cyclic strain in osteoblasts and chondrocytes and by pro-inflammatory cytokines (TNF-α and IL-17) in chondrocytes. Finally, the inhibition of S1P metabolic pathway by Fingolimod reduced the mineralization in cultured osteoblasts and even more in chondrocytes. These results suggest that S1P metabolism participates in SpA excessive ossification. In vivo studies are now needed to validate this possibility

La spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent et invalidant. Plus d’une vingtaine de locus de susceptibilité à la maladie ont été identifiés à ce jour, dont HLA-B27 situé dans le complexe majeur d’histocompatibilité (CMH). L’objectif de ce travail était d’identifier de nouveaux facteurs génétiques de susceptibilité à la SpA grâce à une double approche d’études familiales et de génomique fonctionnelle. Dans la première partie, nous avons génotypé des familles multiplex de SpA. L’analyse de liaison non paramétrique a révélé la présence, en plus du CMH, d’un nouveau locus significativement lié à la SpA en 13q13. L’étude de ce locus nous a permis de restreindre la région d’intérêt à un intervalle de 1,3 Mb, dont le séquençage est en cours. Par ailleurs, l’étude d’association intra-familiale a identifié un SNP intronique de MAPK14 significativement associé à la SpA. Enfin, nous avons montré que l’un des SNPs du gène IL23R connu pour être associé à la spondylarthrite ankylosante était en fait associé à la présence d’une sacro-iliite radiologique dans la SpA. Parallèlement aux études familiales, nous avons comparé le transcriptome de cellules dendritiques de neuf patients atteints de SpA à celui de dix témoins sains. Nous avons ainsi identifié 81 gènes différentiellement exprimés. Nous avons aussi montré que l’expression génique d’ERAP1 (et à un moindre degré son expression protéique et son niveau d’activité enzymatique) étaient sous le contrôle de polymorphismes de ce gène associés à la SpA. / Spondyloarthritis (SpA) is a frequent and disabling chronic rheumatic disease. To date, more than 20 susceptibility loci have been identified, including HLA-B27 in the major histocompatibility complex (MHC). Most of the disease heritability remains to be elucidated. The aim of the study was to identify new genetic factors of susceptibility to SpA using an approach combining genetics and functional genomics. In the first part of this work, we genotyped SpA multiplex families with microarrays of 250,000 SNPs. Non parametric linkage analysis revealed a new locus significantly linked to SpA outside the MHC, in 13q13. Further studies on this locus allowed us to map the disease interval to a 1.3 Mb region, which will be soon sequenced. Moreover, family-based association study identified a significant association between one intronic SNP in MAPK14 and SpA. We also showed that one of the known ankylosing spondylitis-associated SNP in IL23R was indeed associated with sacroiliitis in SpA. We have also compared dendritic cells gene expression between nine SpA patients and ten controls and identified 81 genes differentially expressed. Moreover, we showed that ERAP1 gene expression (and at a less extent protein expression and enzymatic activity) is under the control of several polymorphisms in the gene which has previously been associated with SpA.