INTRODUÇÃO: O diabetes tipo 1 autoimune (DM1A) está associado a alterações na imunidade inata e adaptativa. A agressão autoimune, órgão específica, determina a destruição das células beta do pâncreas e a deficiência da produção de insulina. O infiltrado inflamatório do tipo linfomononuclear, configurando a insulite, e a escassez ou a ausência das células ?, definem o quadro histológico do DM1A. Os autoanticorpos contra antígenos das células beta, que geralmente se desenvolvem na fase pré-clínica, conferem predisposição para DM1A. No entanto, é difícil definir quando e quais indivíduos progredirão para o diabetes manifesto, justificando a busca de outros biomarcadores que auxiliem nas indicações de tratamentos preventivos. Nesse contexto, sabe-se que os microRNAs (miRNAs), pequenos RNAs que atuam pós transcrição, desempenham papel crucial na regulação de genes, integrando fatores genéticos e ambientais e influenciando o funcionamento de órgãos e tecidos de maneira pontual ou sistêmica. OBJETIVOS: avaliar o envolvimento biológico e a relevância da expressão de miRNAs na resposta imunológica e na função das células ? na patogênese do DM1A. MÉTODOS: analisamos o perfil dos miRNAs séricos em 4 grupos, a saber: pacientes portadores de DM1A, até 6 meses do diagnóstico (DM1A recente), (n=30); pacientes portadores de DM1A com duração de 2-5 anos (DM1A 2-5)(n=26) e indivíduos com autoanticorpos pancreáticos positivos sem diabetes (AcP) (n=25), os quais foram comparados aos indivíduos controles saudáveis(n= 29). A expressão dos microRNAs foi obtida com ensaios individuais TaqMan® MicroRNA Assays 5x primers e TaqMan MicroRNA Human Array Card A, (Applied Biosystems- Forster City CA, USA) constituído por 377 alvos e 4 endógenos. Os dados de expressão foram analisados no Software Cloud, (Thermo Fisher Scientific) e no programa Limma (Linear Models for Microarray and RNA-Seq Data). RESULTADOS: Não houve diferença nas características demográficas, como idade, cor auto referida e sexo entre os grupos (p > 0,05). Pacientes portadores de DM1A (recente e com duração de 2-5 anos), diferiram do grupo controle pelos valores elevados de glicose, hemoglobina glicada, títulos de autoanticorpos pancreáticos, e menores de peptídeo C (p < 0,05) e foram semelhantes entre si. Os portadores de autoanticorpos (AcP) tinham características intermediárias entre os grupos: menores valores de HbA1c e de anticorpo anti-tirosina-fosfatase (anti-IA2) e maiores de peptídeo C em relação aos dois grupos com diabetes. Diferiram dos controles apenas pelos maiores títulos de anticorpo anti-insulina (IAA) e anti-descarboxilase do ácido glutâmico 65 (anti-GAD65). A frequência dos alelos HLA de risco para diabetes (-DR3 ou -DR4 e -DQ2 ou DQ-8) decresceu dos grupos DM1A recente e DM 2-5 para AcP e controles. Foram avaliados 135 miRNAs que estavam expressos em 20% ou mais das amostras dos quatro grupos analisados. Maior expressão foi observada em 13, 4 e 33 miRNAs dos grupos AcP, DM1A recente e DM1A 2-5 respectivamente e menor em 11, 7 e 31 miRNAs destes grupos. Destes, 4 miRNAs foram diferencialmente expressos nos grupos AcP, DM1A recente e DM1A 2-5 em relação ao grupo controle. Os miRNAs: miR -16, miR-195 e miR-454, relacionados com regeneração endócrina do pâncreas, efeito anti-inflamatório e resposta à injúria da célula ? estavam diminuídos nestes 3 grupos. O miR-200a, implicado em apoptose das células beta, estava aumentado nos grupos AcP e DM1A recente e diminuído nos pacientes com maior duração do diabetes (DM1A 2-5), possivelmente devido à escassez destas células. Outros 8 miRNAs apresentaram expressão diferente da do grupo controle em dois dos grupos avaliados, e tendência semelhante no terceiro grupo, sendo 4 deles elevados (miR-193a-5p, miR- 323-3p, miR-423-5p, e miR-92a) e 4, diminuídos (miR-191, miR-19a, miR- 376a, miR-590-5p) ou neutralidade no 3º grupo (miR-15b, miR-100, miR-181a e miR-483-5p) Resposta antagônica foi observada para o miR-25 e miR-485- 3p, diminuídos no grupo AcP e aumentados no DM1A 2- 5. Tais miRNAs estão relacionados com resposta imunológica, secreção de insulina, lesão de células ? e glicotoxicidade, à semelhança do observado para o miR-101-3p, validado por ensaios individuais numa casuística maior. CONCLUSÃO: nossos dados sugerem que miRNAs circulantes podem estar envolvidos na patogênese do DM1A / INTRODUCTION: Autoimmune type 1 diabetes (T1D) is associated with changes in innate and adaptive immunity. The organ-specific autoimmune aggression determines the destruction of beta-cells in the pancreas and the deficient insulin production. The inflammatory infiltration of the lymphomononuclear type, configuring the insulite, and the scarcity or the absence of the beta cells, define the histological picture of T1D. Autoantibodies against beta-cell antigens, which usually develop in the preclinical phase, confer predisposition to T1D. However, it is difficult to define when and which individuals will progress to overt diabetes, justifying the search for other biomarkers that could be indicative of preventive treatments. In this context, it is known that the microRNAs (miRNAs) - small RNAs that act post transcription - play a crucial role in regulating genes and in integrating genetic and environmental factors, influencing the function of organs and tissues in a punctual or systemic way. OBJECTIVES: to evaluate the biological involvement and relevance of miRNA expression in the immune response and ?-cell function in the pathogenesis of T1D. METHODS: we analyzed the profile of serum miRNAs of 4 groups, namely: patients with T1D up to 6 months after diagnosis (recent T1D), (n = 30); patients with T1D lasting 2-5 years (T1D 2- 5) (n = 26) and individuals expressing pancreatic autoantibodies without diabetes (AbP) (n = 25), which were compared to healthy controls (n = 29). Expression of the microRNAs was obtained with individual assays TaqMan® MicroRNA Assays 5x primers and TaqMan MicroRNA Human Array Card A (Applied Biosystems-Forster City CA, USA), consisting of 377 targets and 4 endogenous. The expression data was analyzed in the Cloud Software (Thermo Fisher Scientific) and Limma (Linear Models for Microarray and RNASeq Data) program. RESULTS: There was no difference in demographic characteristics, such as age, self-reported color, and sex among groups (p > 0.05). Patients with T1D (both recent and 2-5 years), similar to each other, differed from the control group by high glucose, glycated hemoglobin levels, pancreatic autoantibody titers, and lower C peptide values (p < 0.05) . Pancreatic autoantibodies (AbP) carriers had intermediate characteristics among the groups: lower HbA1c and anti-tyrosine phosphatase antibody (anti- IA2) values and higher C-peptide levels than the two groups with diabetes. They differed from controls only by the higher titers of anti-insulin (IAA) and anti-decarboxylase of glutamic acid 65 (anti-GAD65) autoantibodies. The frequency of high risk HLA alleles for diabetes (-DR3 or -DR4 and -DQ2 or DQ- 8) decreased from the recent T1D and T1D 2-5 groups to the AbP and controls. We evaluated 135 miRNAs that were expressed in 20% or more of the samples from the four groups analyzed. Higher expression was observed in 13, 4 and 33 miRNAs of the Abp, recent T1D and T1D 2-5 groups respectively and lower in 11, 7 and 31 miRNAs of these groups. Of these, 4 miRNAs were differentially expressed in the AbP, recent T1D and T1D 2-5 groups in relation to the control group.The miRNAs: miR -16, miR-195 and miR-454, related to endocrine regeneration of the pancreas, anti-inflammatory effect and response to beta-cell injury were decreased in these 3 groups. miR-200a, implicated in beta-cell apoptosis, was increased in the recent and decreased AbP and T1D groups in patients with longer duration of diabetes (T1D 2-5y), possibly due to the shortage of these cells. Another eight miRNAs showed different expression of the control group in two of the evaluated groups, and a similar trend in the third group, four of them high (miR-193a-5p, miR-323-3p, miR-423-5p, and miR- 92a ) and four, decreased (miR-191, miR-19a, miR-376a, miR-590-5p) or neutrality in the 3rd group (miR-15b, miR-100, miR-181a and miR-483-5p) was observed for miR-25 and miR-485-3p, decreased in the AbP group and increased in T1D 2-5y. Such miRNAs are related to immune response, insulin secretion, ?-cell damage and glycotoxicity, similar to that observed for the miR- 101-3p, validated by individual trials in a larger cohort. CONCLUSION Our data suggests that circulating miRNAs may be involved in the pathogenesis of T1D

Diabetes Mellitus tipo 1A (DM1A), doença autoimune clássica, decorrente da quebra de tolerância imune por fatores ambientais em indivíduos geneticamente predispostos, é caracterizada pela infiltração pancreática de linfócitos T e B, macrófagos e células dendríticas. As células T auxiliadoras 17 (Th17) são células potentes, altamente inflamatórias, que produzem a interleucina 17A (IL-17A), citocina mediadora de várias desordens imunológicas como, artrite reumatóide, esclerose múltipla, encefalite experimental autoimune, psoríase e asma, e em animais, o diabetes autoimune. No entanto, seu papel na patogênese do DM1A em humanos não está definido O objetivo de nosso estudo foi avaliar a influência da IL-17A na predisposição ao DM1A através da identificação de variantes alélicas no gene da IL-17A (por sequenciamento automático) e da determinação dos níveis séricos de IL-17A (por ELISA) e da expressão do seu receptor em linfócitos T periféricos (por citometria de fluxo). Foram analisados 103 pacientes com DM1A (idade 15,15 ± 10,38) e 102 controles normais (idade 18,29 ± 10,83). O estudo da expressão do receptor da IL-17A em linfócitos T periféricos bem como o da proteína sérica foram conduzidos em 24 pacientes com DM1A recente (duração inferior a 6 meses) e 23 controles normais. Resultados: Nos 3 exons da IL-17 A analisados, a freqüência das 14 variantes alélicas já descritas em bancos de dados e de três novas variantes alélicas na região não codificadora do exon 3 (3UTR) não diferiu entre diabéticos e controles. Detectamos, pela primeira vez, diminuição estatisticamente significativa da expressão proporcional do receptor de IL-17A em células TCD3+ (p = 0,041) e TCD4+ (p = 0,0019) periféricas de pacientes com DM1A de início recente quando comparados com controles normais. As concentrações séricas de IL-17A foram menores nos diabéticos. Não observamos correlação entre a expressão dos receptores com a resposta humoral (níveis de autoanticorpos pancreáticos anti-GAD65 e anti-IA2) ou com variáveis metabólicas (glicemia e HbA1c). Nossos resultados sugerem que mutações ou polimorfismos no gene da IL-17A não estão implicadas na predisposição ao DM1A em humanos. A reduzida expressão dos receptores de IL-17A em linfócitos T CD3+ e CD4+ periféricos e das concentrações séricas de IL-17A nos pacientes diabéticos não indicam a participação ativa da via Th17 na periferia na patogênese do DM1A em humanos. No entanto, não descartamos a possibilidade de que, ao estudarmos variáveis na periferia e não do local de agressão imune (as ilhotas pancreáticas), tenhamos obtido valores que não expressem o processo adequadamente. Um eventual mecanismo de regulação negativa da via Th17, na tentativa de proteção do organismo contra o processo inflamatório autoimune, poderia explicar a diminuição de expressão de IL-17RA nos linfócitos periféricos / Type 1A diabetes mellitus (T1AD), a classical autoimmune disease related to the loss of immune tolerance is determined by environmental factors in genetically predisposed individuals. Pancreatic infiltration of T and B lymphocytes, macrophages and dentric cells characterize the process. T helper 17 (Th17) cells are potent, highly inflammatory cells, which initiate tissue inflammation and induce infiltration of other inflammatory cells in target organs. They produce the Interleukin 17A (IL-17A), considered a mediator of various immune disorders such as rheumatoid arthritis, multiple sclerosis, experimental autoimmune encephalitis, psoriasis and asthma, and in animals, autoimmune diabetes. However, its role in T1AD pathogenesis in humans is not defined. The aim of our study was to evaluate the influence of IL-17A in T1AD predisposition in humans. The allelic variants of IL-17A gene (by automatic sequencing), the expression of IL-17A receptors in peripheral lymphocytes (by flow cytometry assay) and the serum levels of IL-17A (by ELISA) were analyzed. Our casuistic was composed of 103 patients with T1D (15,15 ± 10,38 years) and 102 normal controls (18,29 ± 10,83 years). The expression of IL-17A receptor in peripheral lymphocytes and the serum concentration of IL-17A were determined in a subgroup of 24 recent-onset T1D (less than 6 months) and 23 normal controls. Results: The frequency of the 14 allelic variants on the 3 exons of IL- 17A gene already described on data bases did not differ between patients with diabetes and controls. We detected three new allelic variants at the final non-coding region of exon 3. Their frequency was also similar between patients and controls. We detected for the first time a statistically significant decrease in the proportional expression of the receptor of IL-17 on CD3+ (p=0,041) and CD4+ (p=0,0019) T lymphocytes in patients with recent-onset type 1A diabetes. IL- 17A serum concentrations were also lower in patients. There was no correlation between the expression of IL-17A receptor and titles of pancreatic autoantibodies (anti-GAD65 or anti-IA2) or metabolic variables (glucose and HbA1c levels). Our results suggest that mutations or polymorphisms of IL-17A gene are not implicated in the pathogenesis of T1AD in humans. The reduced expression of IL-17A receptors in peripheral T lymphocytes and of IL-17A serum concentrations in patients with diabetes did not indicate a role of Th17 via at the periphery in the autoimmune process. There is however the possibility that by studying the peripheral and not the local immune aggression (pancreatic islets) we have obtained values that do not adequately express the process. A possible mechanism of negative regulation of receptors in an attempt to protect the organism against autoimmune inflammatory process could explain the decrease of IL-17A levels and of IL-17RA expression in peripheral lymphocytes

L’encéphalite limbique à auto-anticorps anti-CASPR2 est une atteinte du système nerveux central, caractérisée par la présence des auto-anticorps (autoAcs) dirigé contre CASPR2 dans le sérum et fluide céphalorachidien. La pathologie affecte majoritairement des hommes âgés présentant l’épilepsie comme symptôme prédominant. CASPR2 est une molécule d’adhésion neuronale, connue pour son rôle d’assemblage des canaux Kv1, régulateurs de l’excitabilité neuronale, à la région juxtaparanodale du nœud de Ranvier, une organisation essentielle pour la conduction saltatoire des flux nerveux. Un nombre croissant de données dans la littérature suggère un rôle pour CASPR2 dans des fonctions synaptiques et l’activité neuronale. Ceci pourrait expliquer l’épilepsie, un symptôme neurologique qui trouve son origine dans la perturbation de l’activité neuronale, observée chez les patients avec de l’encéphalite limbique anti-CASPR2. Dans ce travail de thèse, j’ai utilisé des autoAcs de patients comme outil pour investiguer le rôle de CASPR2 dans des neurones normalement développés en culture, permettant aussi d’évaluer l’effet des autoAcs des patients sur les fonctions synaptiques et de révéler des mécanismes physiopathologiques possibles sous-jacents à la maladie. Je me suis d’abord intéressée aux effets des autoAcs des patients sur l’expression et la distribution en surface de CASPR2 et sur l’expression des canaux Kv1.2 dans des neurones hippocampiques matures in vitro. J’ai montré que les neurones inhibiteurs sont positifs pour les canaux Kv1.2 et CASPR2 en surface, et que les autoAcs de patients augmentent l’expression de Kv1.2 et n’induisent pas l’internalisation de CASPR2. Dans un second temps, j’ai analysé les effets des autoAcs de patients sur les synapses excitatrices et inhibitrices dans des neurones hippocampiques immatures et matures in vitro. Dans les neurones immatures, la densité des épines dendritiques et le contenu des récepteurs AMPA sont augmentés, tandis que dans les neurones matures l’altération de la géphyrin suggère une perturbation de la transmission neuronale après traitement avec des autoAcs de patients. Mes résultats permettent de mieux comprendre les fonctions de CASPR2 dans les processus synaptiques et révèlent des mécanismes pathologiques possibles des autoAcs anti-CASPR2 menant à la présentation clinique des patients atteints d’encéphalite limbique anti-CASPR2 / Anti-CASPR2 autoimmune limbic encephalitis is a central nervous system disorder, characterized by the presence of autoantibodies (autoAbs) directed against CASPR2 in the serum and cerebrospinal fluid. Elderly men are mostly affected, with epilepsy being the predominant symptom. CASPR2 is a neuronal cell adhesion molecule, known for its role in gathering Kv1 channels, regulators of neuronal excitability, at the juxtaparanodal region of the node of Ranvier, an essential organization for saltatory conduction of nervous influxes. Increasing sets of data in literature point out a role for CASPR2 in synaptic functions and neuronal activity. This could explain the observed epilepsy, a neurological symptom that finds its origin in disturbed neuronal activity, in patients with anti-CASPR2 autoimmune limbic encephalitis. In this work, I used patients’ autoAbs as a tool to investigate the role of CASPR2 in normally developed cultured neurons which also allowed me to assess the effects of patients’ autoAbs on synaptic functions and reveal possible physiopathological mechanisms underlying the disease. I first assessed the effects of patients’ autoAbs on CASPR2 surface expression and distribution and on Kv1.2 channel expression in mature in vitro hippocampal neurons. I provided evidence that inhibitory neurons are positive for both Kv1.2 channels and surface CASPR2, and that patients’ autoAbs increase Kv1.2 expression and do not induce CASPR2 internalization. Secondly, I analyzed effects of patients’ autoAbs on excitatory and inhibitory synapses in vitro, in immature and mature hippocampal neurons. In immature neurons, dendritic spine densities and AMPA receptor content are increased, while in mature neurons alteration of gephyrin suggests disturbed neuronal transmission after treatment with patients’ autoAbs. My results allow for a better understanding of CASPR2 functions in synaptic processes and unravel possible pathological mechanisms regarding how anti-CASPR2 autoAbs lead to the clinical presentation of patients with anti-CASPR2 autoimmune limbic encephalitis

Bei Verdacht auf Vorliegen einer systemischen Autoimmunerkrankung wird für die serologische Routinediagnostik ein Zwei-Stufen-Verfahren empfohlen. Zuerst werden Autoantikörpern (AAK) mittels sensitiver zellbasierter indirekter Immunfluoreszenz (IIF)-Teste bestimmt. Ein positives Ergebnis muss aufgrund der Möglichkeit von falsch-positiven Ergebnissen mit einem weiteren, spezifischen Test bestätigt werden. Dieses sukzessive Vorgehen ist notwendig, da zurzeit keine Assaytechnik die notwendigen Anforderungen an ein einstufiges Verfahren hinsichtlich Sensitivität und Spezifität erfüllt. Im Sinne einer effektiven AAK-Diagnostik kann heute schon eine simultane Bestimmung von mehreren AAK mittels multiparametrischer Bestätigungstests die Diagnosefindung im Vergleich zu konventionellen, monoparametrischen Tests wesentlich verkürzen. Jedoch erlauben die verfügbaren multiparametrischen AAK-Nachweismethoden nicht die Kombination von Screening- und Bestätigungstesten. Deshalb wurde basierend auf der digitalen Fluoreszenz mit der hier vorgestellten CytoBead Technologie ein neuer Ansatz entwickelt. Ziel war die Kombination der empfohlenen Stufendiagnostik bestehend aus sensitivem Screening und spezifischer Bestätigungsdiagnostik in einer Reaktionsumgebung und darüber hinaus die Möglichkeit der Adaption auf die serologische Diagnostik mehrerer Autoimmunerkrankungen. Durch a) die Nutzung von Standardglasobjektträgern, b) die Kombination von nativen zellulären oder Gewebesubstraten mit antigenbeladenen fluoreszierenden Mikropartikeln (Beads) in einer Reaktionsumgebung, c) die Möglichkeit der manuellen und automatischen Auswertung mittels IIF und d) die Erhebung von quantitativen Fluoreszenzmessergebnissen konnten die Nachteile der bisher bestehenden Testsysteme überwunden werden. Das neue Prinzip ist auf verschiedene multiparametrische AAK-Nachweise wie zum Beispiel die Bestimmung von antinukleären Antikörpern und AAK gegen entsprechende nukleäre und zytoplasmatische autoantigene Zielstrukturen anwendbar. Damit wurde weiterhin die Basis für die simultane AAK-Multiparameterbestimmung für die Serologie der Zöliakie und von ANCA-assoziierten systemischen Vaskulitiden geschaffen.

Tau protein, a microtubule-associated protein localized in axonal projections of neurons, is a key molecule in the pathology of Alzheimer's disease (AD), the most common cause of dementia in the elderly population. Tau belongs to the group of natively unfolded proteins without globular structure and is prone to numerous posttranslational modifications (PTMs). Under pathological conditions, abnormal PTMs and misfolding of tau protein occurs and leads to oligomerization and aggregation into paired helical filaments forming neurofibrillary tangles, the histopathological hallmark of AD. Currently available drugs applied in AD treatment can only slow the disease progression and those, which halt the AD-specific neurodegenerative processes, are still missing. Very promising and evolving therapeutic approach is immunotherapy, and even immunomodulation by administration of intravenous immunoglobulin (IVIG) products, a reservoir of natural antibodies from the plasma of healthy donors, has been already tested. The discovery of naturally occurring antibodies directed to tau (nTau-Abs) in body fluids of both AD and healthy subjects and their presence in IVIG begin the investigation of their therapeutic potential. Considering a wide range of possible modifications of tau and of various tau species (oligomers,...

While clearly different in their aims and means, classification and diagnosis both try to accurately label the disease patients are suffering from. For systemic lupus erythematosus (SLE), this is complicated by the multi-organ nature of the disease and by our incomplete understanding of its pathophysiology. Hallmarks of SLE are the presence of antinuclear antibodies (ANA), and multiple immune-mediated organ symptoms that are largely independent. In an attempt to overcome limitations of the current sets of SLE classification criteria, a new fourphase approach is being developed, which is jointly supported by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR). This review attempts to delineate the performance of the current sets of criteria, the reasons for the decision for classification, and not diagnostic, criteria, and to provide a background of the current approach taken.

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Les anticorps anti-phospholipides (aPL), tels que les anticoagulants lupiques (LAC), sont associés au développement récurrent de thromboses chez les patients atteints du lupus érythémateux disséminé (LED). Il a été observé que des titres élevés d’auto-anticorps antilamine B1 (anti-LB1), chez des patients porteurs de LAC, diminuent le risque de ces manifestations thrombotiques. Toutefois, la relation existant entre la lamine B1 (LB1), les anti-LB1 et la thromboprotection n’est toujours pas expliquée. Dans cette étude, nous avons donc cherché à comprendre comment la LB1 et les anti-LB1 induisent cette thromboprotection. Nous avons testé les effets d'anti-LB1 purifiés et de LB1 recombinante sur l'activation des cellules endothéliales et des plaquettes. Nous avons été en mesure de déterminer que la LB1, contrairement aux anti-LB1, possède une activité anti-plaquettaire. En effet, la LB1 réduit l’activation et l’agrégation plaquettaires in vitro et in vivo. Cette activité est due à une liaison directe de la LB1 aux plaquettes, suivie par une internalisation rapide dans des vésicules de clathrine. Par co-immunoprécipitation, nous avons découvert que la LB1 interagit avec le récepteur de l’insuline situé sur la membrane plaquettaire. La liaison de la LB1 à ce récepteur entraîne vraisemblablement son internalisation et l'inhibition d'une des cascades de signalisation normalement induite par le récepteur de l’insuline, menant éventuellement à l’inhibition des fonctions plaquettaires. L’ajout d’anti-LB1 purifiés dans nos expériences a permis d'augmenter de façon significative la persistance de la LB1 dans les plaquettes, une observation confirmée par la détection de LB1 uniquement dans les lysats de plaquettes prélevées chez des patients anti-LB1 positifs. iv Nos résultats suggèrent que la LB1 prend part aux mécanismes régulateurs des processus d’hémostase chez des sujets sains et que la présence d’anti-LB1, chez les patients lupiques, prolonge la persistance de cet auto-antigène dans les plaquettes, les empêchant ainsi de s’activer. Ce mécanisme expliquerait la diminution du risque de thrombose chez les patients LAC positifs porteurs d’anti-LB1 circulants. / Anti-phospholipid antibodies such as lupus anticoagulant antibodies (LAC) are associated with recurrent thrombotic events in systemic lupus erythematosus (SLE) patients. However, the risk of thrombosis in LAC positive patients is markedly reduced in the presence of high titers of autoantibodies to lamin B1 (anti-LB1). To date, the implication of lamin B1 (LB1) and anti-LB1 in thromboprotection remains unclear. Our objective was to examine the mechanism whereby LB1 and anti-LB1 induced thromboprotection. Functional platelet and endothelial cell activation assays were used to determine the effects of recombinant LB1 and affinity purified anti-LB1 on these two cell types. LB1, contrarily to anti-LB1, was found to possess an intrinsic anti-platelet activity. This protein reduced the activation and aggregation of platelets in vitro and in vivo. This activity was likely due to the direct binding of LB1 to platelets, followed by its rapid internalization within clathrin coated-pits. Coimmunoprecipitation revealed that LB1 interacted with the insulin receptor located within the platelet membrane. The binding of LB1 to this receptor induced its internalization and inhibited at least one of the phosphorylation cascade stimulated by the receptor, which in turn inhibited platelet functions. The addition of affinity-purified anti-LB1 in our model markedly increased the persistence of LB1 within platelets, a finding supported by the detection of LB1 only in platelets from anti-LB1 positive SLE patients. Our results suggest that LB1 regulates haemostasis in normal subjects. The presence of anti-LB1 in SLE patients prolongs the persistence of LB1 within platelets, thus possibly vi preventing further platelet activation. This mechanism likely explains the reduced risk of thrombotic events in LAC positive SLE patients with circulating anti-LB1 autoantibodies.

L'épilepsie est une maladie neurologique chronique caractérisée par des crises spontanées et récurrentes. Les crises sont générées par un déséquilibre dans le fonctionnement des neurotransmetteurs et des canaux ioniques qui contrôlent l'excitabilité. L'épileptogenèse est majoritairement associée à des pertes neuronales, une gliose, une inflammation plus ou moins importants. Un tiers des patients deviennent réfractaires. Récemment, plusieurs équipes ont montré une association entre les épilepsies focales pharmacorésistantes et la rupture de la barrière hémato-encéphalique (BHE). De plus, une implication du système immunitaire ainsi qu'une cause auto-immune de l'épilepsie ont été suggérées. Dans cette thèse, nous avons observé dans le tissu de patients atteints d'épilepsie pharmacorésistante du lobe temporal (ELT), des fuites d'Immunoglobulines G (IgG) dans le parenchyme et leur accumulation dans les neurones présentant des signes de neurodégénérescence. Le récepteur d'IgG de grande affinité FcyRI est surexprimé sur les cellules ayant une morphologie de type microglie/ macrophages, tandis que le récepteur de faible affinité FcyRIII et le récepteur inhibiteur FcγRII sont moins présents. Dans ce même tissu nous avons noté que les protéines du complément C3c et C5b9 sont exprimées. Ensuite, nous avons étudié si le modèle murin d'épilepsie focale induite par injection intra-amygdalienne de kaïnate reproduit la physiopathologie de l'ELT associée à une rupture de la BHE. ZO-1, la principale protéine des jonctions serrées, présente un marquage discontinu indiquant que la BHE a été affectée. Nous avons remarqué des fuites d'IgGs et d'albumine ainsi que leur accumulation dans le parenchyme coïncidant avec la survenue des crises. La présence d‘IgG dans l'épilepsie pourrait également avoir une cause auto-immune. Nous avons utilisé des puces à protéines pour identifier des antigènes qui induisent une réponse immunitaire, dans le plasma des patients atteints d'ELT, Nous avons sélectionné 19 auto-anticorps spécifiques qui peuvent servir de potentiels biomarqueurs diagnostiques L'ensemble de ces résultats suggère que les fuites d'IgG sont associées à une déficience neuronale, conduisant à des changements immunologiques dans le foyer épileptique qui participent à la pathogénèse de l'ELT. Nous pensons qu'une meilleure interprétation des profils de ces auto-anticorps pourrait offrir de nouvelles perspectives thérapeutiques. / Epilepsy is a chronic neurologic disorder characterized by recurrent unprovoked seizures. Seizures are generated by an imbalance in the functioning of neurotransmitters and ion channels that control excitability. Epileptogenesis is mostly associated with neuronal loss, gliosis, and inflammation more or less important. A third of patients become drug refractory. Recently, several teams have shown an association between drug-resistant focal epilepsy and disruption of the blood-brain barrier (BBB). In addition, a possible role of the immune system and an autoimmune nature in epilepsy has been suggested. In this thesis, in the tissue of patients with drug-resistant temporal lobe epilepsy (TLE), leakage of immunoglobulin G (IgG) into the parenchyma and IgG accumulation in neurons with attendant signs of neurodegeneration was observed. In addition, the high affinity IgG receptor, FcγRI was expressed on microglia/macrophage shaped cells. The expression of the low affinity IgG receptor, FcγRIII and the inhibitory IgG receptor, FcγRII was decreased. In the same tissue the complement proteins C3c and C5b9 were present on astrocyte/ microglia and macrophage/ microglia shaped cells respectively. Then, we evaluated whether the mouse model of focal epilepsy induced by intra-amygdala microinjection of kainic acid reproduced a pathophysiology of TLE associated with BBB impairment. ZO-1, the main tight junction protein presented discontinuous staining indicating that BBB was affected. Both IgG and albumin extravasations from blood vessels were noted and its parenchymal accumulation was concomitant with seizure occurrence. Another hypothesis of IgG presence in epilepsy incriminates an auto-immune cause. Protein microarray technology was used for identification in pooled plasma samples, of antigens that bind plasma antibody from TLE patients. 19 potential autoantibodies were identified as potential diagnostic biomarkers. Together, these observations suggest that IgG leakage is associated with neuronal impairment, leading to immunological changes in epileptic focus involved in the pathogenesis of TLE. A better interpretation of the profiles of these autoantibodies could offer new therapeutic and diagnostic perspectives.