INTRODUÇÃO: A nefrite lúpica é uma complicação frequente e de alta morbimortalidade do lúpus eritematoso sistêmico (LES). A evolução para insuficiência renal crônica terminal varia entre 8 e 15% dos casos, após um período de 5 anos. A fase inicial da nefrite se deve a uma atividade imunológica exacerbada que leva a sequelas renais, como a fibrose intersticial, sinéquias glomerulares, e glomeruloesclerose. Uma vez instalada, vários fatores aceleram a velocidade de progressão da insuficiência renal, como a presença de proteinúria residual, hipertensão arterial sistêmica e a etnia do paciente. Estudos recentes mostraram que a presença de polimorfismos do MYH9 são altamente prevalentes em pacientes com GESF (glomeruloesclerose focal e segmentar), nefropatia do HIV e em pacientes com doença renal crônica não diabética. Os polimorfismos do MYH9 mais relacionados com essas doenças são os do haplótipo E1, causados pelos polimorfismos rs4821480, rs2032487, rs4821481 e rs3752462, presentes principalmente na população negra e de hispano-americanos. No Brasil não há estudos sobre a prevalência desse gene. MÉTODOS: Nosso estudo analisou retrospectivamente 196 pacientes com nefrite lúpica, acompanhadas no ambulatório de glomerulopatias do Hospital das Clínicas da USP. Foram recuperados os dados clínicos e laboratoriais dos pacientes de janeiro de 1999 a dezembro de 2010. Foi feita análise dos polimorfismos do haplótipo E1 do gene do MYH9 (rs4821480, rs2032487, rs4821481 e rs3752462) e correlacionados com suas características clínicas e laboratoriais, apresentando como desfecho a duplicação da creatinina ou a evolução para doença renal crônica terminal. RESULTADOS: O tempo de seguimento médio dos pacientes foi de 6,1 anos, com a creatinina inicial média de 1,6 g/dL e proteinúria média de 3,9 g/dia. Dezenove pacientes não recuperaram função renal, mantendo-se em diálise. Dos 177 pacientes restantes 43 (24%) apresentaram o desfecho de duplicação (DC) da creatinina, ou necessidade de diálise (DRCT). Pacientes progressores eram tinham maior SLEDAI renal (10 vs 8,9 p=0,04), maior índice de cronicidade renal à biópsia (5 vs 2, p<0,001) e maior frequência de reativações da doença renal (flare renal) (82,9% x 53,8%, p=0,002), assim menores índices de remissão completa ou parcial (p<0,0001). Os 4 polimorfismos se segregam em conjunto, ou seja, como um haplótipo, pelo modelo de Hardy-Weinberg. Analisando separadamente cada polimorfismo, apenas o rs3752462, apresenta associação com o desfecho DC/DRCT, na análise por genótipo (CC/CT/TT, p=0,03) e quando feita análise TT/CT vs CC (p=0,02). Não houve relação dos polimorfismos com a etnia negra ou parda. Pacientes com haplótipo E1 eram progressores em 28% dos casos, conferindo um OR de 1,79 (IC 1,02 a 3,0) de DC/DRC. DISCUSSÃO: A presença do haplótipo E1 têm alta prevalência em pacientes portadores de nefrite lúpica no Brasil, sendo fator de risco para progressão da doença renal crônica / BACKGROUND: Lupus nephritis (LN) is a frequent complication with high morbidity and mortality of systemic lupus erythematosus (SLE). Chronic renal failure is observed in 8 to 15% of the patients after 5 years of follow up. LN is an inflammatory disease after a systemic autoimmune activation. Once inflammation is shutdown several renal and nonrenal factors, such as residual proteinuria, hypertension and ethnicity of the patient, may emerge and impose to the kidney a chronic phenotype (interstitial fibrosis, glomerular adhesions and glomerulosclerosis. Recently E1 haplotype (rs4821480, rs2032487, rs4821481 and rs3752462 polymorphisms) of the MYH9 gene was associated to progressive kidney diseases in patients with FSGS (focal segmental glomerulosclerosis), HIV nephropathy and non-diabetic chronic kidney disease, in african american and spanic american patients. In Brazil there is no data on this subject. METHODS: Retrospective analysis of 196 patients with LN followed in our outpatient glomerular disease ward were enrolled glomerulopathies. Patients clinical data from January 1999 to December 2010 were retrieved and MYH9 rs4821480, rs2032487, rs4821481 and rs3752462 polymorphisms were genotyped. Outcome was defined as doubling of serum creatinine, or end stage renal disease (ESRD). RESULTS: The mean follow-up of patients was 6.1 years, with an initial mean creatinine of 1.6 g/dL and mean proteinuria 3.9 g/day. On enrollment nineteen patients were on dialysis and did not recover renal function, they were withdraw from analyses of progressive kidney disease. On follow up, from 177 remaining patients, 43 (24%) showed the composite outcome: dialysis, or doubling creatinine. Progressors had higher renal SLEDAI (10 vs 8.9, p = 0.04), higher chronicity index at biopsy (5 vs 2, p <0.001) and more frequently renal flares (82, 9% vs. 53.8%, p=0.002), as well as lower rates of complete or partial remission (p <0.0001). The four polymorphisms segregate as a haplotype, according the Hardy-Weinberg model. Analysing each polymorphism, only TT/CT genotype from rs3752462 polymorphism was associated with the outcome of DC/ESRD (p = 0.02). E1 haplotype were associated with progression with an OR of 1.79 (CI 1.02 to 3.0). DISCUSSION: The presence of the E1 haplotype is associated with worse prognosis of chronic renal failure in lupus nephritis patients

Les Cellules Dendritiques sont un groupe hétérogène de cellules présentatrices d’antigènes, importantes pour le contrôle des réponses innées et adaptatives. Les Cellules Dendritiques Plasmacytoïdes (pCD) en représentent une population unique, aux caractéristiques phénotypiques et fonctionnelles particulières, notamment par leur capacité à produire de grande quantité d’Interféron de type I (IFN). Cette signature IFN marque la physiopathologie du Lupus Erythémateux Systémique (LES), maladie auto-immune systémique. Les mécanismes à l’origine de cette production excessive d’IFN par les pCD restent incomplètement élucidés. Nous montrons, dans notre étude, chez l’homme comme dans un modèle murin que les plaquettes, activées dans le LES, participent à la production d’IFN via le CD40L. Cette production en excès d’IFN, a pour conséquence une maturation et activation d’autres Cellules Dendritiques (CD) entrainant l’activation inappropriée des lymphocytes T. Chez le sujet sain, cette activation inappropriée du système immunitaire adaptatif doit être strictement contrôlée afin d’assurer l’homéostasie du système immunitaire. Il a été montré précédemment que de nombreux lymphocytes aux caractéristiques phénotypiques de type mémoire-effecteur (TEM) peuplent les tissus périphériques, notamment le tissu cutané. Ces TEM sont capables de s’activer et proliférer localement en réponse à un stimulus. Les Cellules de Langerhans (LC) sont des cellules dendritiques résidant au niveau cutané dans l’épiderme. Leur fonction est à ce jour l’objet d’une controverse entre une fonction immuno-stimulante (modèle humain) et une fonction immuno-régulatrice (modèle murin). Nous démontrons dans cette étude que les LC, à l’état basal, chez l’homme, induisent la prolifération de Lymphocytes T régulateurs (Treg) au niveau cutané, capables de bloquer la stimulation inappropriée des TEM cutanés. Cependant en présence d’un stimulus infectieux, les LC induisent préférentiellement la prolifération des TEM en limitant celle des Treg. Les LC semblent être à la fois immuno-régulatrices ou stimulantes en fonction du contexte biologique auquel elles sont confrontées. / Dendritic Cell (DC) are a heterogeneous group of antigen-presenting leukocytes that are important in activation of both the innate and adaptative arms of the immune system. Plasmacytoid Dendritic Cells (pDC) represent a unique population, characterized by their ability to produce large amounts of type I Interferon (IFN). This « IFN signature » is a prominent feature of Systemic Lupus disease (SLE). Mechanisms leading to the excessive production of type I IFN remain largely unknown. Here, in our present study, we demonstrate that platelets are activated in SLE patients by circulating immune complexes and represent a major reservoir of CD40L. Activated platelets potentiate the production of type I IFN by pCD through a CD40L/CD40 interaction. Excessive production of type I IFN by pCD leads to DC activation and maturation and inappropriate activation of auto-reactive T cells.Under steady state condition, inappropriate activation of the immune system must be tightly controlled. It has been previously shown that normal adult human skin contains a large number of resident T cells (TRM) expressing the phenotype of Effector Memory T cells (TEM). These TEMTRM are specific for antigens previously encountered through skin and can be activated and proliferate under specific stimulation. Langerhans Cells (LC) are a group of skin resident DC living in epidermis. There is currently substantial controversy regarding the physiologic role of LC with regard to immunoregulation versus immunostimulation. Here we show that under steady state condition, LC induce the proliferation of a small subset of TRM. These proliferating TRM express the phenotype of TREG and are functional. However this stimulation of TREG could be reversed in the presence of foreign antigen in a dose-dependant fashion, as the addition of a pathogen to LC and TRM led to diminished TREG proliferation and increased TEM proliferation. These findings establish a novel immunological role for LC in human skin, allowing for the constitutive maintenance of tolerance, while also permitting the stimulation of resident immune memory in response to infectious challenge

Le but de ce travail a été d’étudier l’implication de la réponse immunitaire innée au cours du lupus érythémateux systémique (LES) et de la polyarthrite rhumatoïde (PR). Dans le LES humain, nous montrons que les plaquettes sont activées par les complexes immuns circulants (CICs) et que le CD154 dérivé des plaquettes activées participe avec les CICs à la sécrétion aberrante par les cellules dendritiques d’interféron alpha, cytokine fondamentale dans la physiopathologie du LES. Dans deux modèles murins nous démontrons qu’inhiber l’activation plaquettaire améliore significativement les paramètres lupiques et la survie suggérant que l’activation plaquettaire pourrait constituer une nouvelle cible thérapeutique. Les cytokines inflammatoires tiennent un rôle central dans la physiopathologie de la PR mais les déterminants de leur production restent mal connus. Des polymorphismes du facteur de transcription interferon regulatory factor 5 (IRF5) sont associés au risque de développer une PR. Dans un modèle murin, nous montrons que l’inactivation du gène codant pour IRF5 améliore la maladie. IRF5 étant impliqué dans la signalisation des TLRs, nous mettons alors en évidence in vivo que l’inactivation des voies de signalisation des Toll-like récepteurs (TLRs) TLR3 et TLR7, récepteurs pour les ARN, améliore la maladie. Nous démontrons in vitro un effet synergique dépendant d’IRF5 de ces TLRs pour la production de cytokines inflammatoires. Enfin les souris invalidées pour la signalisation combinée des TLR3 et TLR7 sont spectaculairement épargnées par la maladie suggérant ainsi un rôle clé pour les ligands ARN endogènes dans la pathogénie de la PR. Ces données démontrent l’importance des mécanismes immunitaires innés dans la physiopathologie des maladies auto-immunes systémiques, apportant ainsi de nouvelles voies de recherche thérapeutiques. / The aim of the present sudy was to investigate the involvement of the innate immune system in the pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), two models of systemic autoimmune diseases. We show that in SLE patients, platelets are activated by circulating immune complexes. Activated platelet derived CD154 enhances interferon alpha production by immune complex-stimulated dendritic cells in a CD154/CD40 dependent manner. In lupus prone mice, we show that targeting platelet activation improves all measures of disease and overall survival suggesting that this process may provide a new therapeutic target. Proinflammatory cytokines play a central role in the pathogenesis of RA. Gain-of-function polymorphisms in the transcription factor interferon regulatory factor 5 (IRF5) are associated with an increased of developping RA. We now show that IRF5 deficiency substantially reduces disease in a mouse model of RA. As IRF5 participates in TLR signaling, we evaluated TLR requirements in this model and found a significant réduction in disease severity in mice deficient in signaling through the RNA-sensing TLR3 and TLR7. In vitro studies show that TLR3 and TLR7 synergize for proinflammatory cytokine production and that this synergy is markedly diminished in the absence of IRF5. Notably, mice with combined deficiency of TLR3 and TLR7 signaling developed minimal disease indicating an important role for endogenous RNA ligands in disease pathogenesis. Our results bring new elements in the comprehension of the role of innate immunity in the pathogenesis of systemic autoimmune diseases and provide new therapeutic targets.

Les exosomes apoptotiques (ApoExo) sont des vésicules extracellulaires (EVs) dérivées de lésions vasculaires et libérées par des cellules endothéliales (ECs) apoptotiques dont la taille, les protéines, le profil en ARN et l'activité enzymatique sont différents de ceux des corps apoptotiques classiques. Notre groupe a montré que les ApoExos accéléreraient le rejet vasculaire en association avec les anti-LG3 circulants, des auto-anticorps (auto-Ac) dirigés contre le LG3, le fragment 5' du perlécan. Nous avons également démontré le rôle de biomarqueur et le rôle effecteur des anti-LG3 dans les lésions vasculaires rénales, à la fois dans les reins natifs et transplantés. La néphrite lupique (NL) est une manifestation fréquente et grave du lupus érythémateux disséminé (LED). Il n'existe pas de biomarqueurs du dysfonctionnement rénal progressif dans la NL. Nous émettons l'hypothèse que les ApoExos stimulent des cellules B spécifiques qui existent dans le répertoire immunitaire normal et que les conditions pro-inflammatoires prévalant chez les patients atteints de LED, telles que l'activation accrue des récepteurs Toll-like (TLRs), amplifient cette réponse, conduisant à la production d'anti-LG3, un auto-Ac important dans l'établissement de la NL. Des cellules B productrices d’anti-LG3 ont été trouvées dans la cavité péritonéale de souris saines et ont produit des anti-LG3 suite à une stimulation in vitro avec des agonistes des TLR1/2, TLR4, TLR7 et TLR9. Il est intéressant de noter que ces cellules sont absentes de la cavité péritonéale de souris saines ayant reçu une injection d'ApoExos. En explorant l'importance fonctionnelle des TLRs dans le déclenchement d'une réponse auto-immune dans un modèle murin lupique, nous montrons que les agonistes de TLRs connus pour contribuer à la pathogenèse du LED (TLR2, 4, 7 et 9) déclenchent une production significativement plus élevée d'IgM anti-LG3, alors que la stimulation des TLRs qui ne sont pas associés à la pathogenèse du LED (TLR3 et 5) ne le fait pas. L’injection d'ApoExo a également déclenché l'axe auto-immun IL-23/IL-17 (mesuré par ELISA et essai cytokinique), augmenté les cellules B de centres germinatifs spléniques (mesuré par cytométrie de flux), augmenté les taux circulants d’IgG totaux, d’anti-LG3 et d’auto-Ac classiques du LED (mesuré par micropuce et ELISA) par rapport à l’injection de véhicule. Des niveaux élevés d'IgG anti-LG3 circulants sont observés chez les souris prédisposées au LED par rapport aux souris saines (mesurés par ELISA), ainsi qu'une proportion accrue de cellules B1 spléniques et de cavité péritonéale (mesurés par cytométrie de flux) augmentant avec l’établissement de la maladie. Ces observations suggèrent un rôle spécifique des ApoExos dans la modulation de la production d'auto-Ac qui, à son tour, déclenche l'involution microvasculaire importante dans les maladies auto-immunes et le rejet de greffe. Ces observations suggèrent également que les cellules B spécifiques de LG3 peuvent être modulées dans des conditions pro-inflammatoires telles que celles qui prévalent chez les patients atteints de LED, conduisant à la production d'auto-Ac. Une meilleure compréhension de l'impact de ces mécanismes permettra d'améliorer l'identification, la prédiction et la prise en charge de la NL. / Apoptotic exosomes (ApoExo) are vascular injury derived extracellular vesicles (EVs) released by apoptotic endothelial cells (ECs) with distinct size, protein, RNA profile and enzymatic activity from classical apoptotic bodies. Our group showed that ApoExo accelerated vascular rejection in association with circulating anti-LG3, autoantibodies (autoAb) against LG3, the 5’ fragment of the perlecan. We have also unravelled biomarkers and effector roles of anti-LG3 in kidney vascular damage in both native and transplanted kidneys. Lupus Nephritis (LN) is a common and serious manifestation of systemic lupus erythematosus (SLE). Biomarkers of progressive renal dysfunction in LN, are lacking. We hypothesize that ApoExo stimulate specific B cells that exist in the normal immune repertoire and that the pro-inflammatory conditions prevalent in SLE patients, such as increased Toll-like Receptors (TLRs) activation, amplify this response, leading to anti-LG3 production, autoAb of importance in LN development. B cells producing anti-LG3 were found in the peritoneal cavity of healthy mice and produced anti-LG3 AutoAb when stimulated in vitro with TLR 1/2, 4, 7 and 9 agonists. Interestingly, these cells disappeared from the peritoneal cavity of healthy mice infused with ApoExo. ApoExo infusion also triggered circulating IL-23/IL-17 autoimmune axis (measured by cytokines assay), increased splenic germinal centre B cells (measured by flow cytometry), increased total circulating IgG, anti-LG3 and classical autoAb (measured by microarray and ELISA) compared to vehicle infusion. Elevated circulating anti-LG3 IgG levels are found in SLE prone mice compared to healthy ones (measured by ELISA) as well as an increased proportion of splenic and peritoneal cavity B1 cells (measured by flow cytometry). Exploring the functional importance of TLRs in triggering such a response, we show that while TLR agonists known to contribute to SLE pathogenesis (TLR2, 4, 7 and 9) triggered significantly higher IgM anti-LG3 production, stimulation of TLR that are not associated with SLE pathogenesis (TLR3 and 5) did not. These observations suggest a specific role for ApoExo in modulating the production of autoAb which, in turn, trigger microvascular involution of importance in autoimmune diseases and transplant rejection. These observations also suggest that LG3-specific B cells may be modulated under pro-inflammatory conditions such as those prevalent in lupus patients, leading to production of autoAb. A better understanding of the impact of these mechanisms will lead to improved identification, prediction, and management of LN.

Lúpus eritematoso sistêmico juvenil é uma doença inflamatória autoimune multifatorial com elevação de citocinas inflamatórias e com gravidade variável. Estudos observaram associação entre exposição a poluição do ar e aumento do número de internações hospitalares devido à exacerbação das doenças reumáticas pediátricas e aumento no risco de crianças portadoras de lúpus eritematoso sistêmico juvenil apresentarem atividade de doença moderada /grave após exposição a material particulado e dióxido de nitrogênio. Exposição à poluição do ar pode acarretar agravo agudo em doenças reumatológicas pediátricas, incluindo lúpus eritematoso sistêmico juvenil. Objetivos: Avaliar o efeito da exposição real a poluentes atmosféricos sobre biomarcadores inflamatórios em condensado do ar exalado e sobre a fração de monóxido de nitrogênio em ar exalado em pacientes com lúpus eritematoso sistêmico juvenil. Avaliar ainda, a correlação entre os biomarcadores inflamatórios no ar exalado e atividade da doença. Métodos: Estudo longitudinal de painel de medidas repetidas realizado em 108 visitas consecutivas de pacientes com diagnóstico de lúpus eritematoso sistêmico juvenil sem doenças respiratórias. Por 4 semanas consecutivas, medidas diárias individuais de dióxido de nitrogênio, material particulado fino, temperatura ambiental e humidade relativa do ar foram obtidos. Este ciclo foi repetido a cada 2,5 meses ao longo de um ano, além disso, citocinas do condensado do ar exalado (interleucinas 6, 8, 17 e fator de necrose tumoral-alfa), fração exalada de monóxido de nitrogênio e parâmetros de atividade de doença foram coletados semanalmente. Modelos específicos de equação estimada generalizada foram usadas para avaliar o impacto destes poluentes no risco de Systemic Lupus Erythematosus Disease Activity Index 2000 >= 8, citocinas no condensado do ar exalado e fração exalada de monóxido de nitrogênio, considerando o efeito fixo para medidas repetidas. Os modelos foram ajustados para provas de fase aguda, índice de massa corpórea, infecções, medicações e variáveis meteorológicas. Resultados: Efeito positivo nas medidas de desfechos foi observado para um aumento do intervalo interquartil do material particulado fino (18.12 ?g/m3). Um aumento do intervalo interquartil na média móvel de 7 dias no material particulado fino (lag0 to lag6) foi associado a um aumento de 0.1pg/ml (95%IC:0.01;0.19) e 0.9pg/ml (95%IC:0.05;0,12) nos níveis de interleucina 17 e fator de necrose tumoral-? do condensado do ar exalado, respectivamente. Também foi observado um aumento no risco de Systemic Lupus Erythematosus Disease Activity Index 2000 >= 8 de 1.47(95%CI:1.10;1.84) associado ao aumento da média móvel de material particulado fino. Em adição, um efeito a curto prazo na fração exalada de monóxido de nitrogênio foi evidenciado, medida móvel de 3 dias do material particulado fino foi associada com um aumento de 0.75ppb (95%IC:0.38;1.29) na fração exalada de monóxido de nitrogênio. Conclusão: Exposição a partículas finas inaláveis pode aumentar a inflamação das vias aéreas e posterior inflamação sistêmica nos pacientes com lúpus eritematoso sistêmico juvenil / Systemic lupus erythematosus is an inflammatory multisystem, autoimmune disease with elevated inflammatory interleukins levels. Exposure to air pollution may trigger pulmonary inflammation/systemic inflammation. Objective: The objective of this study was to investigate the association between daily individual exposure to air pollutants and airway inflammation and disease activity in childhood-onset systemic lupus erythematosus patients. A longitudinal panel study was carried out in 108 consecutive appointments with childhood-onset systemic lupus erythematosus patients without respiratory diseases. Methods: Over four consecutive weeks, daily individual measures of nitrogen dioxide, fine particulate matter, ambient temperature, and humidity were obtained. This cycle was repeated every 2.5 months along 1 year, and cytokines of exhaled breath condensate (interleukins 6, 8, 17 and tumoral necrose factor-alpha), fractional exhaled nitrogen monoxide, and disease activity parameters were collected weekly. Specific generalized estimation equation models were used to assess the impact of these pollutants on the risk of Systemic Lupus Erythematous Disease Activity Index 2000 >= 8, exhaled breath condensate cytokines, and fractional exhaled nitrogen monoxide, considering the fixed effects for repetitive measurements. The models were adjusted for inflammatory indicators, body mass index, infections, medication, and weather variables. Results: An interquartile range increase in fine particulate matter 4-day moving average (18.12ug/m3) was associated with an increase of 0.05 pg/ml (95% CI 0.01; 0.09, p = 0.03) and 0.04 pg/ml (95% CI 0.02; 0.06, p = 0.01) in interleukin 17 and tumoral necrosis factor-alpha exhaled breath condensate levels, respectively. Additionally, a short-term effect on fractional exhaled nitrogen monoxide was observed: the fine particulate matter 3-day moving average was associated with a 0.75 ppb increase (95% CI 0.38; 1.29, p = 0.03) in fractional exhaled nitrogen monoxide. Also, an increase of 1.47 (95% CI 1.10; 1.84) in the risk of Systemic Lupus Erythematous Disease Activity Index 2000 >= 8 was associated with fine particulate matter 7-day moving average. Conclusion: Exposure to inhalable fine particles increases airway inflammation/pulmonary and then systemic inflammation in childhood-onset systemic lupus erythematosus patients

Made available in DSpace on 2014-07-29T15:25:17Z (GMT). No. of bitstreams: 1 Tese Denise Sisterolli Diniz.pdf: 6293055 bytes, checksum: d5c5b19907b7602ca5e99aa0414271f7 (MD5) Previous issue date: 2011-09-12 / Systemic Lupus Erythematosus - LES is an autoimmune disease involving multiple systems. In the Central nervous system-SNC is one of his more severe manifestations. One of the frames of Lupus neuropsychiatric - LES-NP, is a Demyelinating Syndrome - SD. SD in rare cases, may be the first manifestation of LES, resembling multiple sclerosis - MS in their symptoms. MS is autoimmune disease with involvement of SNC with components, degenerative and inflammatory, which can produce autoantibodies and general symptoms that make your final diagnosis. The disease occurs more in young women than in men, typically evolve with outbreaks and remissions. In order to contribute to the diagnosis of these two illnesses, we proposed a comparative analysis, considering clinical, laboratory, and image aspects. As there is a lack of definition of SD, aims to contribute to the definition and establishment of standards of compromise of the SNC by SD of LES-NP, comparing patients with LES-NP and MS (the prototype of CNS demyelinating disease), attended in rheumatology and neurology of the HC/FM/UFG, Goiania. GO, from 2009 to 2010, according to the demographic aspects (sex and age), clinical (time of illness, functional systems and EDSS), laboratory (Autoantibodies and liquid Cerebroespinhal - LCR), image (Conventional magnetic resonance - RM) and Neuropsychological - NPS (cognitive and emotional). It is one epidemiological, analytical and comparative cross-sectional study of LES-NP and MS, which was approved by the CEP/HC/UFG. Were used in the survey questionnaires: an analysis of demographic and personal and family background; other symptoms; Neurological exams and NPS; Analysis: image by RM, laboratory of autoantibodies and LCR; and symptoms of depression and anxiety by Beck inventory (BAI and BDI). The results demonstrated an increased frequency in patients of age; time of disease; Visual symptoms; changes of sensitivity; of coordination; of the March; retention and urinary incontinence; changes of speech and swallowing; depressive and psychotic symptoms in family and personal history; WMHs in parietal and frontal lobes, occipitais, corpus callosum, periventricular, justacortical, brain stem; the cerebellum; spinal cord and the Barkhof and Tintoré criteria - CBT. Other findings in the RM were: infarcts; Vasculitis; menigioma and thrombosis in patients with LES-NP. The involvement of SNC in LES was measured by clinical symptomatology, neurological examination, EDSS, image findings and neuropsychological tests. RM was predictive analytics to relate workload post-lesional adaptation in brain regions typically involved in in. The application of CBT in patients with SD of LES-NP, associated with the qualitative assessment of WMHs may define the differences of neurological involvement pattern of injury of the SD of LES-NP, those at. The dosage of autoantibodies was predictive analytics for the diagnosis of LES, especially the anti DNA, the anti-ENA and the LAC, which were unique in the LES-NP. The clinical examination and laboratory research, neurological autoantibodies antinuclear antibodies and image by RM offers great security in diagnosis of the two diseases.Show that there are aspects of the involvement of the SD in LES needing new assessments, using the markers of neuronal injury, the more specific and with studies of Immunogenetics compared with in, the prototype of CNS demyelinating disease. / Lúpus Eritematoso Sistêmico - LES é a doença auto-imune que envolve vários sistemas. No Sistema Nervoso Central - SNC é uma de suas manifestações mais graves. Um dos quadros neurológicos do Lúpus Neuropsiquiátrico - LES-NP, é a Síndrome Desmielinizante - SD. Em raros casos, SD pode ser a primeira manifestação do LES, se assemelhando a Esclerose Múltipla - EM em sua sintomatologia. EM é doença autoimune com envolvimento do SNC com componentes, inflamatório e degenerativo, que pode apresentar auto-anticorpos e sintomas gerais que dificultam seu diagnóstico final. As doenças ocorrerem mais em mulheres jovens que em homens, evoluem tipicamente com surtos e remissões. Com o intuito de contribuir para o diagnóstico dessas duas enfermidades, propusemos uma análise comparativa, considerando aspectos clínicos, laboratoriais e de imagem. Como há uma carência de definição da SD, pretende-se contribuir com a definição e o estabelecimento de padrões de comprometimento do SNC pela SD do LES-NP, comparando os pacientes com LES-NP e EM (o protótipo da doença desmielinizante), atendidos no serviço de reumatologia e neurologia do HC/FM/UFG, Goiânia/GO, de 2009 a 2010, segundo os aspectos demográficos (sexo e idade), clínicos (tempo de doença, sistemas funcionais e EDSS), laboratoriais (Autoanticorpos e Líquido Cerebroespinhal - LCR), de imagem (Ressonância Magnética Convencional - RM) e Neuropsicológicos - NPS (cognitivo e emocional). É um estudo epidemiológico, analítico, transversal, comparativo de LES-NP e EM, que foi aprovado pelo CEP/HC/UFG. Foram utilizados na pesquisa análise de questionários: um demográfico e de antecedentes pessoais e familiares; outro de sintomas; exames: neurológico e de NPS; avaliações: imagem por RM e laboratorial de auto-anticorpos e LCR; e sintomas de depressão e ansiedade pelo inventário de Beck (BAI e BDI). Os resultados demonstraram uma frequência aumentada nos pacientes de EM, em idade; tempo de doença; sintomas visuais; alterações de sensibilidade; da coordenação; da marcha; retenção e incontinência urinária; alterações da fala e deglutição; sintomas depressivos e psicóticos na história pessoal e familiar; alterações focais da substância branca (WMHs) nos lobos frontais, parietais e occipitais, corpo caloso, periventricular, justacortical, tronco encefálico; cerebelo; medula espinhal e os critérios de Barkhof e Tintoré - CBT. Outros achados na RM foram: infartos cerebrais; vasculites; meningioma e trombose nos pacientes com LES-NP. O envolvimento do SNC no LES foi mensurado pela sintomatologia clínica, exame neurológico, EDSS, achados de imagem e testes neuropsicológicos. RM foi preditiva em relacionar carga lesional em regiões cerebrais tipicamente envolvidas na EM. A aplicação dos CBT nos pacientes com SD do LES-NP, associado à avaliação qualitativa das WMHs pode definir as diferenças de padrão de envolvimento neurológico da lesão da SD do LES-NP, daquelas da EM. A dosagem de autoanticorpos foi preditiva para o diagnóstico de LES, especialmente o anti DNA, o anti-ENA e o LAC, os quais foram exclusivos no LES-NP. O exame clínico e neurológico, da investigação laboratorial de auto-anticorpos antinucleares e da imagem por RM oferece segurança no diagnóstico das duas enfermidades. Mostram que há aspectos do envolvimento da SD no LES que necessitam de novas avaliações, utilizando os marcadores de lesão neuronal, mais específicos e com estudos de imunogenética comparados com a EM, o protótipo da doença desmielinizante do SNC.

Lúpus eritematoso sistêmico juvenil é uma doença inflamatória autoimune multifatorial com elevação de citocinas inflamatórias e com gravidade variável. Estudos observaram associação entre exposição a poluição do ar e aumento do número de internações hospitalares devido à exacerbação das doenças reumáticas pediátricas e aumento no risco de crianças portadoras de lúpus eritematoso sistêmico juvenil apresentarem atividade de doença moderada /grave após exposição a material particulado e dióxido de nitrogênio. Exposição à poluição do ar pode acarretar agravo agudo em doenças reumatológicas pediátricas, incluindo lúpus eritematoso sistêmico juvenil. Objetivos: Avaliar o efeito da exposição real a poluentes atmosféricos sobre biomarcadores inflamatórios em condensado do ar exalado e sobre a fração de monóxido de nitrogênio em ar exalado em pacientes com lúpus eritematoso sistêmico juvenil. Avaliar ainda, a correlação entre os biomarcadores inflamatórios no ar exalado e atividade da doença. Métodos: Estudo longitudinal de painel de medidas repetidas realizado em 108 visitas consecutivas de pacientes com diagnóstico de lúpus eritematoso sistêmico juvenil sem doenças respiratórias. Por 4 semanas consecutivas, medidas diárias individuais de dióxido de nitrogênio, material particulado fino, temperatura ambiental e humidade relativa do ar foram obtidos. Este ciclo foi repetido a cada 2,5 meses ao longo de um ano, além disso, citocinas do condensado do ar exalado (interleucinas 6, 8, 17 e fator de necrose tumoral-alfa), fração exalada de monóxido de nitrogênio e parâmetros de atividade de doença foram coletados semanalmente. Modelos específicos de equação estimada generalizada foram usadas para avaliar o impacto destes poluentes no risco de Systemic Lupus Erythematosus Disease Activity Index 2000 >= 8, citocinas no condensado do ar exalado e fração exalada de monóxido de nitrogênio, considerando o efeito fixo para medidas repetidas. Os modelos foram ajustados para provas de fase aguda, índice de massa corpórea, infecções, medicações e variáveis meteorológicas. Resultados: Efeito positivo nas medidas de desfechos foi observado para um aumento do intervalo interquartil do material particulado fino (18.12 ?g/m3). Um aumento do intervalo interquartil na média móvel de 7 dias no material particulado fino (lag0 to lag6) foi associado a um aumento de 0.1pg/ml (95%IC:0.01;0.19) e 0.9pg/ml (95%IC:0.05;0,12) nos níveis de interleucina 17 e fator de necrose tumoral-? do condensado do ar exalado, respectivamente. Também foi observado um aumento no risco de Systemic Lupus Erythematosus Disease Activity Index 2000 >= 8 de 1.47(95%CI:1.10;1.84) associado ao aumento da média móvel de material particulado fino. Em adição, um efeito a curto prazo na fração exalada de monóxido de nitrogênio foi evidenciado, medida móvel de 3 dias do material particulado fino foi associada com um aumento de 0.75ppb (95%IC:0.38;1.29) na fração exalada de monóxido de nitrogênio. Conclusão: Exposição a partículas finas inaláveis pode aumentar a inflamação das vias aéreas e posterior inflamação sistêmica nos pacientes com lúpus eritematoso sistêmico juvenil / Systemic lupus erythematosus is an inflammatory multisystem, autoimmune disease with elevated inflammatory interleukins levels. Exposure to air pollution may trigger pulmonary inflammation/systemic inflammation. Objective: The objective of this study was to investigate the association between daily individual exposure to air pollutants and airway inflammation and disease activity in childhood-onset systemic lupus erythematosus patients. A longitudinal panel study was carried out in 108 consecutive appointments with childhood-onset systemic lupus erythematosus patients without respiratory diseases. Methods: Over four consecutive weeks, daily individual measures of nitrogen dioxide, fine particulate matter, ambient temperature, and humidity were obtained. This cycle was repeated every 2.5 months along 1 year, and cytokines of exhaled breath condensate (interleukins 6, 8, 17 and tumoral necrose factor-alpha), fractional exhaled nitrogen monoxide, and disease activity parameters were collected weekly. Specific generalized estimation equation models were used to assess the impact of these pollutants on the risk of Systemic Lupus Erythematous Disease Activity Index 2000 >= 8, exhaled breath condensate cytokines, and fractional exhaled nitrogen monoxide, considering the fixed effects for repetitive measurements. The models were adjusted for inflammatory indicators, body mass index, infections, medication, and weather variables. Results: An interquartile range increase in fine particulate matter 4-day moving average (18.12ug/m3) was associated with an increase of 0.05 pg/ml (95% CI 0.01; 0.09, p = 0.03) and 0.04 pg/ml (95% CI 0.02; 0.06, p = 0.01) in interleukin 17 and tumoral necrosis factor-alpha exhaled breath condensate levels, respectively. Additionally, a short-term effect on fractional exhaled nitrogen monoxide was observed: the fine particulate matter 3-day moving average was associated with a 0.75 ppb increase (95% CI 0.38; 1.29, p = 0.03) in fractional exhaled nitrogen monoxide. Also, an increase of 1.47 (95% CI 1.10; 1.84) in the risk of Systemic Lupus Erythematous Disease Activity Index 2000 >= 8 was associated with fine particulate matter 7-day moving average. Conclusion: Exposure to inhalable fine particles increases airway inflammation/pulmonary and then systemic inflammation in childhood-onset systemic lupus erythematosus patients

Para verificar a expressão das proteínas Fas e Bcl-2 em linfócitos e suas correlações com a atividade da doença foram avaliados 38 pacientes com lúpus eritematoso sistêmico de início na infância e 25 controles sem doença autoimune. Observou-se que as porcentagens de linfócitos T CD3+ e CD8+ e linfócitos B que expressavam a proteína Fas e a intensidade média de fluorescência da proteína Bcl-2 nos linfócitos T CD3+, CD4+ e CD8+ dos pacientes com lúpus foram significativamente maiores quando comparadas aos controles. Os pacientes com doença ativa apresentavam porcentagens de linfócitos B que expressavam a proteína Fas significativamente maiores que os pacientes com doença inativa e os controles e houve correlação direta entre estas porcentagens e o SLEDAI (p=0.02, r=0.38) / In order to verify the expression of Fas and Bcl-2 proteins on lymphocytes and their relationship with disease activity 38 patients with juvenile-onset systemic lupus erythematosus and 25 healthy controls were studied. The measurements showed that percentages of lymphocytes T CD3+ and CD8+ and B lymphocytes positively stained for Fas antigen and mean fluorescence intensity of Bcl-2 on CD3+, CD4+ and CD8+ T cells from lupus patients were significantly increased compared to healthy controls. Lupus patients with active disease presented percentages of lymphocytes B positive for Fas antigen significantly higher compared to patients with inactive disease and healthy controls and there was a statistically significant direct correlation between these percentages and SLEDAI score (p=0.02, r=0.38).

Para verificar a expressão das proteínas Fas e Bcl-2 em linfócitos e suas correlações com a atividade da doença foram avaliados 38 pacientes com lúpus eritematoso sistêmico de início na infância e 25 controles sem doença autoimune. Observou-se que as porcentagens de linfócitos T CD3+ e CD8+ e linfócitos B que expressavam a proteína Fas e a intensidade média de fluorescência da proteína Bcl-2 nos linfócitos T CD3+, CD4+ e CD8+ dos pacientes com lúpus foram significativamente maiores quando comparadas aos controles. Os pacientes com doença ativa apresentavam porcentagens de linfócitos B que expressavam a proteína Fas significativamente maiores que os pacientes com doença inativa e os controles e houve correlação direta entre estas porcentagens e o SLEDAI (p=0.02, r=0.38) / In order to verify the expression of Fas and Bcl-2 proteins on lymphocytes and their relationship with disease activity 38 patients with juvenile-onset systemic lupus erythematosus and 25 healthy controls were studied. The measurements showed that percentages of lymphocytes T CD3+ and CD8+ and B lymphocytes positively stained for Fas antigen and mean fluorescence intensity of Bcl-2 on CD3+, CD4+ and CD8+ T cells from lupus patients were significantly increased compared to healthy controls. Lupus patients with active disease presented percentages of lymphocytes B positive for Fas antigen significantly higher compared to patients with inactive disease and healthy controls and there was a statistically significant direct correlation between these percentages and SLEDAI score (p=0.02, r=0.38).

Les interférons de type I (IFN I) sont des cytokines antivirales aux propriétés puissantes. L’induction, la transmission et la résolution de la réponse immunitaire engendrée par les IFN I est minutieusement régulée. Le concept d’interféronopathie de type I, récemment individualisé par notre équipe, repose sur l’hypothèse que certaines pathologies seraient secondaires au déséquilibre de ces voies de signalisation complexes et à la sécrétion excessive et inappropriée d’IFN I. L’inhibition de celle-ci par des thérapeutiques ciblées permettrait de valider cette hypothèse, si les symptômes allégués s’amélioraient, voire disparaissaient. Ce travail de thèse s’est initialement concentré sur la caractérisation clinique et biologique des interféronopathies monogéniques et polygéniques, et secondairement sur l’identification moléculaire de nouvelles mutations du gène TMEM173 à l’origine de l’interféronopathie liée à STING, également appelée SAVI (STING associated vasculopathy with onset in infancy), syndrome auto-inflammatoire associant une atteinte sévère cutanée et pulmonaire. De nouvelles techniques ont permis la sélection de patients présentant une augmentation de l’IFN I en comparaison à des contrôles sains : la signature IFN I, qPCR de 6 gènes stimulés par l’IFN (IFN stimulated genes – ISGs) et le dosage d’IFN alpha sérique ou plasmatique par méthode du SIMOA (single molecule array) permettant la détection de molécules d’IFN de l’ordre du femtogramme (10-18g). Ces méthodes nous ont ainsi permis d’élargir le spectre clinique phénotypique des interféronopathies de type I, initialement considéré comme essentiellement neurologique. Les patients atteints du syndrome d’Aicardi-Goutières, première interféronopathie monogénique décrite, présentaient les signes suivants : dystonie, spasticité, décalage des acquisitions, calcifications intra-cérébrales et anomalies de la substance blanche. Cependant, l’utilisation systématique de nos méthodes de criblage associée à l’avènement des technologies de séquençage à haut débit (next generation sequencing – NGS) a permis de révéler un phénotype plus large, caractéristique des interféronopathies de type I : sur le plan cutané (engelures, vascularite nécrosante des extrémités, sclérodermie), pulmonaire (pneumopathie interstitielle isolée ou non), musculo-squelettique (arthralgies, arthrites, arthropathie de Jaccoud, myalgies et myosites), ophtalmologique (glaucome), néphrologique (néphropathies lupiques), gastro-entérologique (maladies inflammatoires chroniques intestinales précoces), associées à de l’auto-immunité ou un déficit immunitaire inconstants. Notre méthode de sélection nous a notamment permis d’identifier des patients présentant de manière variable des signes cardinaux de SAVI et une de trois nouvelles mutations activatrices dans une région spécifique du gène TMEM173 (codant pour STING). Ces mutations circonscrivent une région de la protéine à ce jour encore jamais impliquée dans le contrôle de la voie de l’IFN I. STING est une protéine du réticulum endoplasmique qui agit comme adaptateur cytosolique de senseurs intracellulaires d’ADN viral dans une voie de signalisation de l’IFN I. STING active TBK1 (TANK-binding kinase) et permet la transcription des IFN I par la phosphorylation d’IRF3. La Janus Kinase 1 (JAK1) et la tyrosine kinase 2 (TYK2) sont activées suite à la stimulation des récepteurs de l’IFN I et phosphorylent les facteurs de transcription STAT1 et STAT2, conduisant à l’expression de nombreux ISGs. Les analyses génétiques, de conformation tridimensionnelle, sur un modèle cellulaire in vitro (HEK293T) et ex vivo sur cellules mononuclées périphériques des patients nous ont ainsi permis de mettre en évidence pour ces mutations un caractère constitutionnellement activé, indépendant de la liaison au ligand cGAMP, mais transmettant ce signal à travers la voie d’aval par TBK1. (...) / Type I interferons (IFN I) are antiviral cytokines with potent properties. Hence, the induction, transmission and resolution of the immune response generated by IFN I is tightly regulated. The concept of the type I interferonopathies, recently formulated by our team, rests on the assumption that some diseases arise from a disturbance of this complex signalling pathway, leading to excessive and inappropriate IFN I secretion. On this basis, targeted therapeutics should improve or cure features of such type I interferonopathies, thereby providing a validation of the underlying hypothesis. This PhD project initially focused on the clinical and biological characterisation of monogenic and polygenic interferonopathies, and secondarily on the molecular identification of novel mutations in the gene TMEM173 causing the interferonopathy called STING associated vasculopathy with onset in infancy (SAVI), an auto-inflammatory syndrome with severe cutaneous and pulmonary features. Our selection of patients in comparison to healthy controls was made possible through the use of novel screening tools: IFN signature (qPCR of 6 IFN stimulated genes – ISGs), and measurement of IFN alpha protein levels in serum or plasma (SIMOA-single molecule array - enabling the detection of molecules of IFN in the femtogram [10-18g]) range. In this way, we have been able to expand the phenotypic spectrum of the interferonopathies, which was initially considered as primarily neurological. Patients with Aicardi-Goutières syndrome (AGS), the first described of the monogenic interferonopathies, exhibit dystonia, spasticity, developmental delay, intra-cranial calcifications and white matter abnormalities. However, the systematic use of our interferon screening assays, plus the advent of next-generation sequencing technology, has revealed a much broader set of features relevant to this novel disease grouping – involving the skin (chilblains, necrotising vasculitis, scleroderma), lungs (isolated lung interstitial disease or associated with other signs), musculoskeletal system (joint pain, arthritis, Jaccoud’s arthropathy, muscle pain and myositis), eyes (glaucoma), kidneys (lupus nephritis) and gastro-intestinal tract (early inflammatory bowel disease), as well features of autoimmunity and immunodeficiency. Using our screening assays enabled us to identify three patients variably exhibiting the core features of SAVI, all of whom were found to harbour distinct novel activating mutations in STING. These mutations highlight a protein domain not previously implicated in the control of IFN I signalling. STING is an endoplasmic reticulum protein, acting as a cytosolic adaptor of intracellular sensors of viral DNA in the type I IFN signalling pathway. STING activates TANK-binding kinase (TBK1), allowing transcription of IFN I through phosphorylation of IRF3. Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) are activated following stimulation of the IFN I receptor, leading to phosphorylation of the transcription factors STAT1 and STAT2 and the subsequent induction of a large number of ISGs. Genetic analysis, conformational studies, an in vitro cellular model (HEK293T) and ex vivo experimental data (using patient peripheral blood mononuclear cells - PBMCs) enabled us to confirm the constitutive activating nature of these variants, and show that this activation did not require binding with cGAMP, but was dependent on signalling through TBK1. Ruxolitinib, a JAK1/2 inhibitor, could antagonise this constitutive activation ex vivo. These results indicate a promising therapeutic approach in such patients, and more widely in the monogenic, and perhaps even, polygenic, interferonopathy context.