OBJETIVO: Descrever prospectivamente o curso da doença de Vogt-Koyanagi-Harada (DVKH) com integração de parâmetros de atividades clínicos, estruturais e funcionais. MÉTODOS: Foram incluídos pacientes com diagnóstico da DVKH na fase aguda (parte I) e não aguda (tempo de doença maior que 12 meses; parte II). Os pacientes na fase aguda receberam tratamento inicial padronizado com pulsoterapia de metilprednisolona seguido de corticoterapia oral em doses lentamente regressivas, pelo período de 15 meses. As avaliações consistiram em exame clínico, retinografia, angiografias com fluoresceína (AGF) e indocianina verde (AIV) e tomografia de coerência óptica (TCO). Foram realizadas nos seguintes momentos: parte I, no diagnóstico e meses 1, 2, 4, 6, 9 e 12; parte II, na inclusão e a cada três meses. Eletrorretinograma campo total (ERGct) e eletrorretinograma multifocal (ERGmf) foram realizados na parte I, no 1.o mês e a cada seis meses e, na parte II, na inclusão e com 12 meses. A leitura dos exames, na parte I, foi efetuada por duas leitoras, não mascaradas; na parte II, foi realizada por três leitores mascarados e treinados, sendo considerada a leitura concordante entre, pelo menos, dois examinadores. As angiografias e TCO foram realizadas no aparelho Spectralis® (HRA+OCT, Heidelberg Engineering). Tratamento adicional com corticoterapia em doses imunossupressoras ou intensificação da imunossupressão sistêmica foi indicado nos casos com recidivas clínicas, na presença de sinais de atividade à AGF ou duas pioras consecutivas >= 30% no ERGct. Os sinais de atividade detectados na AGF, AIV e TCO foram denominados sinais subclínicos. RESULTADOS: Na parte I, foram incluídos nove pacientes (7F/2M) com idade mediana de 33 anos e intervalo mediano entre início dos sintomas e tratamento de 13 dias. Na apresentação inicial, sinais clínicos característicos da doença (coroidite difusa com hiperemia do disco óptico, descolamento seroso de retina e uveíte anterior acompanhados de sinais extraoculares) melhoraram dentro dos primeiros 30 dias em todos os casos. Os principais sinais subclínicos variaram no tempo de melhora ou desaparecimento: espessura de coroide (EC) subfoveal diminuiu para o valor mediano de 347u m aos 30 dias; dark dots diminuíram ao longo do seguimento, porém ainda estavam presentes aos 12 meses. Piora da inflamação foi observada em 17 de 18 olhos no tempo mediano de sete meses quando a redução do corticoide oral atingiu a dose média de 0,3mg/kg/d. Os sinais subclínicos mais frequentemente observados foram dark dots, fuzzy vessels e aumento da EC. Em 10 destes 17 olhos a piora foi acompanhada de queda da função pelo ERG. Três padrões de evolução puderam ser caracterizados: sem recidivas clínicas ou subclínicas (padrão A, 1 olho), com recidivas subclínicas somente (padrão B, 11 olhos) e com recidivas clínicas (padrão C, 6 olhos). Identificou-se que a EC aos 30 dias após início do tratamento >= 506u m teve sensibilidade e especificidade > 80% na detecção dos casos com recidivas clínicas (padrão C). A função pelo ERGct e ERGmf permaneceu alterada em relação ao grupo controle com 24 meses, apesar da melhora progressiva observada desde o início do tratamento. Na análise longitudinal dos pacientes, a função entre 12 e 24 meses permaneceu estável no grupo de doentes que recebeu tratamento adicional (8 olhos), enquanto no grupo que não o recebeu (4 olhos) houve deterioração da função ( < 0,001). Na análise dos grupos segundo padrão de recidiva, observou-se que os olhos com padrão B sem tratamento adicional tinham piora funcional maior em relação àqueles com padrão C ou B tratados (p < 0,001). Na parte II, foram incluídos 20 pacientes (17F/3M), com idade mediana ao diagnóstico de 31 anos, intervalo mediano entre início de sintomas e tratamento de 19 dias e tempo mediano de doença à inclusão de 55 meses. Na avaliação da concordância interobservador na leitura dos sinais subclínicos, EC teve concordância substancial (kappa=0,8), enquanto sinais angiográficos tiveram concordância sutil (kappa < 0,2). O curso da doença em 85% dos pacientes foi com recidiva clínica (padrão C, 11 casos) ou recidiva subclínica (padrão B, 6 casos). Nas 11 avaliações com detecção de células na câmara anterior (CA), sinais subclínicos de inflamação de segmento posterior foram concomitantemente observados em 64% dos olhos. Esta mesma concomitância de sinais subclínicos de inflamação de segmento posterior na presença de células na CA foi observada na parte I do estudo. Nos pacientes com padrão B, a variação da EC foi o principal sinal subclínico observado. A função pelo ERG foi realizada sequencialmente em 13 casos. Olhos com padrão C (7 pacientes), com grande comprometimento funcional desde a inclusão, evoluíram com piora mais acentuada do que aqueles com padrão B (5 pacientes). Ao se individualizar os olhos com padrão B, observou-se que esse diferencial (padrão B melhor que C) devia-se ao grupo padrão B com tratamento (p < 0,001). CONCLUSÕES: Neste estudo prospectivo de pacientes com DVKH em seguimento mínimo de 12 meses, desde as fases aguda e não aguda, três padrões de evolução foram observados, sendo que 94% (parte I) e 85% (parte II) dos pacientes apresentaram recidiva/piora clínica (padrão C) e/ou subclínica (padrão B). Na parte I do estudo, a piora da inflamação foi detectada aos sete meses de evolução durante dose regressiva do corticoide equivalente a 0,3mg/kg/d, apesar do tratamento inicial com corticoides em altas doses lentamente regressivo. A EC aferida 30 dias após o início do tratamento acima de 506 ?m mostrou-se um fator com sensibilidade e especificidade acima de 80% na identificação dos casos que evoluíram com recidivas clínicas. Dentre os sinais para detecção de inflamação subclínica, as alterações na EC são confiáveis, enquanto que sinais angiográficos devem ser interpretados com cautela. Exames sequenciais tornam a leitura mais confiável. A presença de células na CA comportou-se como a \"ponta do iceberg\" de uma inflamação mais difusa. O estudo eletrorretinográfico demonstrou resultado subnormal mesmo após 24 meses de seguimento na parte I; tratamento adicional pode evitar piora funcional nos pacientes com sinais subclínicos de inflamação. A pior função da retina em pacientes com inflamação clínica (padrão C) da parte II e dos pacientes com inflamação subclínica (padrão B) de ambas as partes do estudo sugerem que o tratamento ideal das recidivas inflamatórias ainda deve ser alvo de futuros estudos / OBJECTIVES: To describe the course of Vogt-Koyanagi-Harada disease (VKHD) prospectively, integrating clinical, structural and functional parameters. METHODS: Patients with VKHD in the acute (part I) and non-acute (more than 12 months from diagnosis) phases (part II) were included. Patients in the acute phase received a standard treatment with methylprednisolone pulsetherapy followed by high-dose oral corticosteroids with slow tapering during 15 months. Evaluations included clinical exams, fluorescein (FA) and indocyanine green (ICGA) angiographies and optical coherence tomography (OCT). In part I, they were performed at inclusion, then after 1,2,4,6,9,and 12 months; in part II, they were performed at inclusion then every 3 months for up to 12 months. Functional evaluation using electroretinography (ERG) was performed at inclusion and every 6 months in part I and at inclusion and at 12 months in part II. Two non-blinded readers analyzed the imaging exams in part I. In part II, three trained and blinded-readers performed the imaging exams analysis. For study`s purpose, at least two concordant readings were considered. Imaging exams utilized the Spectralis® (HRA+OCT, Heidelberg engineering). Inflammatory signs detected on FA, ICGA and OCT were denominated as subclinical signs. Additional treatment with high doses of corticosteroids or more intensive systemic immunosuppression was indicated in cases with clinical signs of inflammation, with subclinical signs on FA or with two consecutive worsening > 30% on ERG. RESULTS: Nine patients (7F/2M) were included in part I; median age was 33 years old and median time elapsed from onset of symptoms to treatment was 13 days. At disease presentation, classic signs (choroiditis, anterior uveitis, serous retinal detachment, optic disc hyperemia and extraocular manifestations) were observed; they improved in 30 days after treatment. Subclinical signs improved in variable periods of time: subfoveal choroidal thickness (CT) decreased to a median value of 347 ?m, 30 days after the beginning of treatment, dark dots diminished during the follow-up but they were still observed at 12 months. Relapse (worsening of inflammation) was noticed in 17 of 18 eyes at a median follow up time of seven months, when tapering schedule corticosteroid dosage reached the mean dose of 0.3mg/kg/d of prednisone. Dark dots, fuzzy vessels and choroid thickening were the most frequent subclinical signs. Relapses in 10 of 17 eyes were concomitant with worsening on ERG. Three patterns of evolution could be delineated: no signs of inflammation (pattern A, 1 eye), only subclinical signs of inflammation (pattern B, 11 eyes) and clinical signs of inflammation (pattern C, 6 eyes). CT>=506 ?m 30 days after the beginning of treatment was more than 80% sensitive and specific to detect more severe cases (pattern C). ERG parameters at 24 months were subnormal as compared to the control group, despite improvement during follow-up. Further long-term results after 24 month demonstrated stabilization of ERG parameters in patients that had received additional treatment, whereas there was worsening in those patients who had not received additional treatment (p<0.001). Moreover, pattern B patients without additional treatment had a further decrease on ERG values compared to results observed in pattern C or B patients with additional treatment (p<0.001). In Part II, 20 patients (17F/3M) were included; median age at diagnosis was 31 years old, median lag time from onset of symptoms and treatment was 19 days and median time after diagnosis was 55 months. The interobserver agreement for CT reading was substantial (kappa 0.8), whereas for angiographic signs was slight (kappa < 0.2). Recurrences, clinically (pattern C, 11 cases) or subclinically (pattern B, 6 cases) detected, were observed in 85% of cases. Concomitant inflammation of posterior segment detected by subclinical signs was present in 64% of cases with cells in anterior chamber. Simultaneous signs of subclinical inflammation of posterior segment and anterior uveitis were also observed in part I. CT change was the main subclinical sign observed in pattern B patients. ERG evaluation was performed in 13 cases. Pattern C cases (7 patients) presented worse results than pattern B cases (5 patients). Further analysis depicted that pattern B patients who had an additional treatment had better results than pattern B non-treated and pattern C (p<0.001). CONCLUSION: Three patterns of evolution were observed in VKHD patients during this prospective study, 94% (part I) and 85% (part II) presented recurrence/worsening with clinical (pattern C) or subclinical (Pattern B) signs of inflammation. In part I, worsening was observed at seven months after treatment start when reaching mean dose of 0.3mg/Kg/d of prednisone even after initial high-dose of corticosteroids followed by slow tapering. At day 30 after treatment, CT >= 506 ?m had a greater than 80% sensitivity and specificity to detect cases with pattern C evolution. Considering subclinical signs, CT increase reliably detected recurrence, whereas angiographic signs required cautious interpretation. Sequential analysis was more conclusive than an isolated exam. Anterior chamber cells seemed to be the \"tip of the iceberg\" of a more diffuse inflammation. ERG analysis was subnormal even after 24 months of follow up since disease onset; additional treatment could prevent functional worsening in patients with subclinical signs of inflammation. Worse retinal function in patients with clinical recurrences (pattern C) in part II and subclinical recurrences (pattern B) in parts I and II suggest that ideal treatment of recurrences should be further pursued

A toxoplasmose ocular é atribuída ao parasita, mas a auto-imunidade pode participar do processo. Soros humanos com IgG positiva para T. gondii mostraram níveis altos de IgG anti-retina para diferentes antígenos, se comparados com soros negativos para T. gondii, uveítes de outras origens também tiveram títulos elevados. Hamsters imunizados e/ou infectados não mostraram estes anticorpos sem mimetismo antigênico. A retinocoroidite por Toxoplasma induz resposta humoral auto-imune contra antígenos da retina, provavelmente piorando o efeito direto do agente. Estes anticorpos podem ser usados como marcadores de doença ocular em pacientes soropositivos para toxoplasmose pela triagem de lesão ocular. / Ocular toxoplasmosis is attributed to the parasite, but autoimmunity could have a role in this process. Human sera, positive of anti-T. gondii IgG, show high levels of anti-retina IgG, measured by several antigens, as compared to T. gondii seronegative samples. Sera from patients with uveitis from other origins also had higher anti-retina abs levels. Challenged and/or immunized hamsters showed low anti-retina abs levels, without antigen mimicry. Toxoplasmic retinochoroiditis presents a humoral anti-retina abs, probably worsening the parasite direct effect. Those antibodies could be used as markers of eye involvement in toxoplasmosis seropositive patients, as a screening for eye examination.

A toxoplasmose ocular é atribuída ao parasita, mas a auto-imunidade pode participar do processo. Soros humanos com IgG positiva para T. gondii mostraram níveis altos de IgG anti-retina para diferentes antígenos, se comparados com soros negativos para T. gondii, uveítes de outras origens também tiveram títulos elevados. Hamsters imunizados e/ou infectados não mostraram estes anticorpos sem mimetismo antigênico. A retinocoroidite por Toxoplasma induz resposta humoral auto-imune contra antígenos da retina, provavelmente piorando o efeito direto do agente. Estes anticorpos podem ser usados como marcadores de doença ocular em pacientes soropositivos para toxoplasmose pela triagem de lesão ocular. / Ocular toxoplasmosis is attributed to the parasite, but autoimmunity could have a role in this process. Human sera, positive of anti-T. gondii IgG, show high levels of anti-retina IgG, measured by several antigens, as compared to T. gondii seronegative samples. Sera from patients with uveitis from other origins also had higher anti-retina abs levels. Challenged and/or immunized hamsters showed low anti-retina abs levels, without antigen mimicry. Toxoplasmic retinochoroiditis presents a humoral anti-retina abs, probably worsening the parasite direct effect. Those antibodies could be used as markers of eye involvement in toxoplasmosis seropositive patients, as a screening for eye examination.

The eye contains closely related but widely different tissues, offering a unique opportunity to investigate the phenotype and function of monocyte-derived cell populations within functionally unique microenvironments in a single complex organ. The uveal tract and retina contain rich networks of immune cells that reside and traffic through the eye, these cells having been implicated in various ocular inflammatory processes and immune-mediated diseases. One such inflammatory condition is human posterior uveitis, an autoimmune disease mainly affecting the retina. As current treatments for posterior uveitis only serve to slow down disease progression, studies using animal models, namely, experimental autoimmune uveoretinitis (EAU), have focused on determining the key cellular and molecular mediators involved in disease initiation in order to expand the potential for novel therapeutic applications. The overall purpose of experiments in this thesis was to explore monocyte-derived cell populations of the uveal tract and retina, this being achieved by utilising a novel transgenic mouse model. Cx3cr1gfp/gfp transgenic mice on both BALB/c and C57Bl/6 backgrounds contain an enhanced green fluorescent protein (eGFP) encoding cassette knocked into the Cx3cr1 gene, disrupting its expression but facilitating GFP expression under the control of the Cx3cr1 promoter. Heterozygous (Cx3cr1+/gfp) mice were generated by crossing Cx3cr1gfp/gfp mice to wild-type (WT) mice. This transgenic model allowed for the exquisite visualisation of Cx3cr1-bearing monocyte-derived dendritic cells (DC) and macrophages in ocular tissues, whilst also enabling the investigation of a potential role for Cx3cr1 in recruiting monocyte-derived cells to the eye in steady-state and inflammatory conditions.

INTRODUÇÃO: O micofenolato de mofetila é uma droga imunomoduladora utilizada no tratamento de uveítes crônicas não infecciosas. No entanto, até 20% dos pacientes interrompem o tratamento devido aos efeitos colaterais sistêmicos. O ácido micofenólico é a droga ativa do micofenolato de mofetila e sua aplicação na cavidade vítrea pode ser uma alternativa complementar ao tratamento sistêmico. Entretanto, deve-se considerar o risco de efeitos tóxicos da droga na retina e em outras estruturas oculares. OBJETIVOS: Determinar a meia-vida do ácido micofenólico no vítreo de coelhos e avaliar os efeitos retinianos causados pela injeção intravítrea de diferentes doses de MPA através de avaliações clínica, funcional e morfológica. MÉTODOS: Para o estudo farmacológico, a suspensão de ácido micofenólico (1 mg em 0,1 mL de veículo) foi injetada no vítreo de 16 coelhos albinos New Zealand. Como controle, o olho contralateral de cada coelho foi injetado com 0,1 mL do veículo usado na preparação da suspensão. Os animais foram sacrificados após 1, 7, 15 e 30 dias e as concentrações de ácido micofenólico no vítreo e no sangue foram determinadas por cromatografia líquida de alta eficiência. Para a determinação dos efeitos retinianos do ácido micofenólico foram utilizados 20 coelhos albinos New Zealand, que foram divididos em 5 grupos com quatro animais em cada grupo. Cada animal recebeu injeção de 0,005, 0,05, 0,2, 1 e 10 mg de ácido micofenólico em 0,1 mL de veículo no olho direito e 0,1 mL do veículo no olho esquerdo. Exames de biomicroscopia com lâmpada de fenda e oftalmoscopia binocular indireta foram realizados antes da injeção e 30 dias após. A avaliação funcional da retina foi feita por eletrorretinografia, que foi realizada antes e 7, 15 e 30 dias após a injeção. Os animais foram sacrificados 30 dias após as injeções intravítreas e a avaliação histológica foi feita por microscopia de luz (hematoxilina-eosina). RESULTADOS: A meia-vida do ácido micofenólico no vítreo do coelho foi de 5,0±0,3 dias e o ácido micofenólico foi detectável no vítreo por 29 dias. O ácido micofenólico não foi detectado no vítreo do olho contralateral e no sangue em nenhum tempo estudado. Sinais inflamatórios de pouca intensidade foram observados em pelo menos um olho de cada grupo e não tiveram relação com a dose de ácido micofenólico injetada. A análise da eletrorretinografia não mostrou diferenças significativas da amplitude e tempo implícito da onda-a e da onda-b nas condições escotópica e fotópica após a injeção intravítrea nos cinco grupos avaliados. A análise da relação entre a amplitude da onda-b versus intensidade do estímulo luminoso mostrou diminuição da sensibilidade retiniana após a injeção intravítrea do ácido micofenólico nas doses de 0,05, 0,2, 1 e 10 mg. O estudo histológico não mostrou alterações estruturais da retina após a injeção intravítrea de ácido micofenólico nas cinco doses avaliadas. CONCLUSÕES: O ácido micofenólico tem meia-vida de 5 dias e foi detectável no vítreo de coelhos até 29 dias após a injeção intravítrea. A avaliação funcional mostrou que a injeção intravítrea de 0,05 a 10 mg de ácido micofenólico causou diminuição da sensibilidade retiniana. As doses entre 0,005 e 10 mg de ácido micofenólico não provocaram alterações histológicas na área analisada da retina de coelhos. / INTRODUCTION: Mycophenolate mofetil is a potent immunomodulatory agent used in the treatment of patients with chronic non-infectious uveitis. However, systemic side effects are the main reason for discontinuation, occurring in up to 20% of patients. Mycophenolic acid is the active form of mycophenolate mofetil and its intraocular delivery may avoid the side effects observed with systemic therapy. However, local side effects in the retina and other ocular structures must be considered. PURPOSE: The aims of this study were to determine the half-life of mycophenolic acid in the rabbit vitreous after intravitreal injection, and to determine the clinical, functional, and morphological retinal effects of the intravitreal injection of five different doses of mycophenolic acid. METHODS: For the pharmacological study, mycophenolic acid 1 mg was injected in the vitreous of 16 New Zealand albino rabbits. Animals were sacrificed at different time points after injections (1, 7, 15, and 30 days) and vitreous and blood samples underwent high performance liquid chromatography. For functional and histological studies, 20 New Zealand albino rabbits were divided in 5 groups of 4 animals each, according to the dose of MPA injected in the vitreous (0.005, 0.05, 0.2, 1, and 10 mg in 0.1 mL of vehicle). As control, contralateral eyes were injected with 0.1 mL of the aqueous vehicle. Electroretinograms were recorded before injection and on days 7, 15, and 30. Slit-lamp examination and indirect fundus ophthalmoscopy were performed before injection and after 30 days. Animals were sacrificed and retinas were analyzed by light microscopy (hematoxylin and eosin). RESULTS: Mycophenolic acid half-life in the rabbit vitreous was 5.0±0.3 days and the drug was detectable in the vitreous for 29 days. Mycophenolic acid was not detected either in the serum or in contralateral eyes. Signs of intraocular inflammation were detected in at least one eye of each group and had no correlation with the dose injected. Electroretinogram analysis did not show signifficant differences on a and b-wave amplitude and implicit time on scotopic and photopic conditions after intravitreal injection. The analysis of the b-wave amplitude versus light intensity curves in the dark-adapted state showed decrease in retinal sensitivity in eyes injected with mycophenolic acid 0.05, 0.2, 1 and 10 mg of the drug. No morphological change was found in any dose tested. CONCLUSION: Mycophenolic acid half-life in the rabbit vitreous is 5 days. Electroretinography shows that intravitreal injection of doses from 0.05 to 10 mg of mycophenolic acid decrease retina sensitivity. Intravitreal injection of doses from 0.005 to 10 mg of mycophenolic acid does not cause histological changes in the analysed area in the rabbit retina.

Lors d’uvéite non infectieuse (UNI), des événements environnementaux vont provoquer une rupture de la tolérance immune périphérique et une activation des cellules résidentes oculaires. Plusieurs données attestent de l’importance du rôle joué par les signaux de danger lors de ces deux phases clefs d’activation pathologique. Si une place capitale a été donnée aux signaux de danger exogènes, notamment microbiens, l’importance des signaux de danger endogènes commence à émerger. A ce titre, les nucléotides constituent une famille importante de signaux de danger endogènes puisque en situation pathologique, ils vont être libérés de façon massive dans l’espace extracellulaire où ils peuvent avoir de nombreux effets en activant des récepteurs P2X et P2Y. Le but de ce travail est d’investiguer si les récepteurs P2Y2 jouent un rôle de récepteurs de danger lors d’UAI. Pour ce faire, nous avons d’abord étudié in vitro l’effet des nucléotides extracellulaires sur la production d’IL-8 (cytokine connue pour son rôle chimiotactique lors d’UNI) par des cellules de l’EPR. Nous avons pu montrer que les nucléotides ATPγS, UTP et UDP, stimulent la sécrétion d’IL-8 tant basale qu’induite par le TNFα en activant la voie intracellulaire d’ERK1/2 via l’activation des récepteurs P2Y2 et P2Y6. Ensuite, in vivo, nous avons comparé le développement d’uvéites autoimmunes expérimentales entre des souris génétiquement déficientes pour le récepteur P2Y2 (P2Y2-/-) et des souris sauvages (P2Y2+/+) et avons pu montrer que le groupe P2Y2-/- était moins affecté par la maladie que le groupe sauvage contrôle. De même, après transfert adoptif de lymphocytes T autoréactifs semi-purifiés, les souris P2Y2-/- étaient moins malades que les souris P2Y2+/+ et le transfert adoptif de lymphocytes T autoréactifs semi purifiés de souris P2Y2-/- induisait moins de maladie que le transfert adoptif de cellules contrôles. En accord avec ces dernières données, nous avons mis en évidence que les LT autoréactifs semi-purifiés issus des souris P2Y2-/- immunisées proliféraient moins et sécrétaient moins de cytokines proinflammatoires que ceux issus des souris P2Y2+/+. Une série de co-cultures nous a permis de démontrer que ce déficit de prolifération provenait d’un défaut au niveau des CPA des souris P2Y2-/-. En conclusion, notre travail de thèse a mis en évidence que la stimulation des récepteurs P2Y augmente l’activation de l’EPR et des lymphocytes T autoréactifs, favorisant ainsi le développement d’UNI. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Les uvéites non infectieuses sont considérées actuellement comme une des plus importantes cause de déficience visuelle dans la population des jeunes adultes. Les uvéites non infectieuses sont des atteintes inflammatoires de la rétine et de l’uvée et sont généralement considérées comme autoimmunes et initiées par la perte de la tolérance immune aux protéines rétiniennes. Elles sont orchestrées d’une part, systémiquement par deux sous populations lymphocytaires dont la signature cytokinique est l’IFNγ (Th1) et l’IL-17 (Th17) et d’autre part, localement, par l’activation du tissu rétinien. Néanmoins, la vision systémique actuelle est plus complexe et fait intervenir une activation pathologique de l’immunité innée, donnant une composante d’autoinflammation aux uvéites non infectieuses. En plus de ce volet systémique, de nombreux travaux attestent de l’importance de l’activation des cellules rétiniennes dans le développement d’uvéites non infectieuses. Loin de jouer un rôle passif durant la maladie, elles vont être stimulées par une série de molécules pro-inflammatoires et ainsi permettre le recrutement et l’activation de cellules immunocompétentes. <p>Notre travail de thèse s’inscrit précisément dans ce contexte du rôle de l’activation des cellules rétiniennes et plus spécifiquement de celles de la barrière hémato rétinienne (BHR) dans le développement d’uvéite non infectieuses.<p>Lors de ce travail, nous avons tout d’abord caractérisé in vivo, dans deux modèles expérimentaux, l’expression de la molécule d’adhésion VCAM-1 (Vascular Adhesion Molecule) sur les cellules de la BHR. VCAM-1 est une molécule d’adhésion qui facilite l’extravasation des leucocytes du sang vers les tissus. Nous avons montré que VCAM-1 n’est pas exprimé dans l’œil sain mais est induit progressivement lors de la maladie et que l’intensité et l’extension de son expression étaient dépendantes de la sévérité de la maladie. Par ailleurs, nous avons montré que VCAM-1 pouvait être induit sur l’ensemble des cellules de la BHR. <p>Nous avons ensuite analysé in vitro, sur les cellules de l’EPR (Epithélium Pigmentaire Rétinien) qui forment la partie externe de la BHR, les effets antagonistes du TNFα sur l’induction des molécules de CMH de classe II par l’IFNγ. Durant le processus inflammatoire, l’EPR est la cible d’un ensemble de cytokines secrétées par les cellules inflammatoires. Il a été donc intéressant d’étudier les effets d’autres cytokines présentes lors de l’inflammation sur l’induction du CMHII par l’IFNγ au niveau de l’EPR. Nous avons démontré que le TNFα inhibe l’expression du CMH II induit par l’IFNγ sur les ARPE par régulation négative du CIITA (Class II Transactivator). Comme l’activation des lymphocytes T par les cellules de l’EPR dépend de leur niveau d’expression du CMH II, notre étude soutient l’idée que le TNFα possède des propriétés immunomodulatrices sur l’activation de ces cellules, et participe ainsi à la phase de résolution de l’inflammation.<p>Enfin, nous avons étudié les effets du blocage de l’activation des cellules rétiniennes par l’IFNγ en surexprimant le gène SOCS1 (Suppressor Of Cytokine Signaling) in vivo et in vitro.<p>Nous avons surexprimé le gène SOCS1 au niveau rétinien et étudier l’effet de cette surexpression sur le développement de l’UAE. L’analyse des grading clinique n’a pas montré de différence significative entre les yeux injectés par l’AAV2-SOCS1 versus l’AAV2-EGFP contrôle. Afin de normaliser par rapport à la diversité inter-individuelle de la maladie, nous avons calculé pour chaque souris un ratio des grades cliniques de l’œil injecté sur l’œil non-injecté. L’analyse de la moyenne de ces ratios montre un effet à la limite de la significativité entre le groupe SOCS1 et le groupe EGFP en terme de grades cliniques. La différence devient par contre significative lorsque l’analyse de ces ratios est faite sur les grades histologiques. Nos expériences mènent donc plutôt à la conclusion que l’expression de SOCS1, médié par injection intravitréenne de l’AAV2 ne protège globalement pas les yeux du développement d’une UAE.<p>Cette absence d’effet peut avoir comme explication que l’injection intravitréenne conduit à une infection relativement limitée des cellules rétiniennes impliquées dans le développement de l’UAE. Il se pourrait également que le niveau d’expression de la protéine SOCS1 soit trop faible pour obtenir un effet protecteur ou que la surexpression de SOCS1 affecte uniquement l’activation des cellules de la rétine par l’IFNγ mais pas par d’autres cytokines telles le TNFα, l’IL-17, ou l’IL-22 qui jouent aussi un rôle important dans le développement d’UAE. C’est cette dernière hypothèse que nous avons choisi d’investiguer in vitro. Nos résultats montrent que la surexpression de ce même gène SOCS1 dans les cellules d’EPR a un effet inhibiteur sur leur activation par l’IFNγ mais pas par le TNFα.<p>Ce travail met tout d’abord en évidence l’importante expression, in vivo, de VCAM1 par les cellules de la BHR lors d’UAE et in vitro les effets antagonistes du TNFα et de l’IFNγ sur la régulation de l’expression de molécules du CMHII à la surface de l’EPR. Nos expériences démontrent que la surexpression du gène SOCS1 après injection intravitréenne du vecteur AAV-CAG-SOCS1 n’a que peu d’effet sur le développement de la maladie. Par ailleurs, la surexpression de ce même gène SOCS1 dans les cellules d’EPR a un effet inhibiteur sur leur activation par l’IFNγ mais pas par le TNFα et l’IL-17.<p> / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2014-07-29T15:25:16Z (GMT). No. of bitstreams: 1 Tese Alan Ricardo Rassi.pdf: 1456250 bytes, checksum: c7e131ba3c1d15cc824df69f5c3dc3f6 (MD5) Previous issue date: 2011-10-08 / The objective of this study was to determine the levels of toxicity of two and three intravitreous injections of infliximab to the retina and choroid of albino rabbits by means of histological, electroretinographic and clinical ophthalmological tests. Twelve New Zealand albino rabbits (24 eyes) were used in the study. Each eye was given two (n=10) or three (n=10) serial intravitreous 2 mg injections of infliximab dissolved in 0.06 ml of saline, at monthly intervals. A separate group of rabbits (n=4 eyes) served as a control group. Ninety days after the first injection, the rabbits underwent electroretinographic and clinical ophthalmological tests. After being enucleated, the eyes underwent histological examination. No clinical ophthalmologic abnormalities were detected in the 24 eyes studied. The histological change noted was the presence of rare lymphocytes and eosinophiles in the posterior vitreous of four eyes subjected to two injections and six eyes subjected to three injections of infliximab, but it was not considered clinically significant. One clinically significant abnormality was found, a severe inflammatory reaction with vitreous exudates and ganglion cell edema in both eyes of a single rabbit, subjected to two to three injections of infliximab. The electroretinographic tests showed amplitudes that were on the average 12% smaller than those obtained before the treatment. However, there were no statistically significant differences when comparing amplitude or the implicit time between the pre and post-treatment electroretinographic findings, in all groups examined. Then, two and three intravitreous 2 mg injections of infliximab in eyes of rabbits at monthly intervals did not cause any changes after a 90-day follow-up, according to histological, electroretinographic tests and clinical ophthalmological evaluation. It was concluded that serial intravitreous infliximab doses to rabbits is a safe procedure. / O objetivo deste trabalho foi determinar os níveis de toxicidade de duas e três aplicações intravítreas de infliximabe na retina e coroide de coelhos albinos, por meio de exames clínicos oftalmológicos, eletrorretinográficos e histológicos. Foram utilizados doze coelhos albinos (24 olhos) da raça New Zealand. Cada olho recebeu duas (n=10 olhos) ou três (n=10 olhos) injeções intravítreas seriadas de 2 mg de infliximabe dissolvidos em 0,06 ml de solução salina, em intervalos mensais. Um grupo separado de olhos (n=4 olhos) serviu como controle. Noventa dias após a primeira injeção, os coelhos foram novamente submetidos a exames clínicos oftalmológicos e eletrorretinográfico e, após enucleados, os olhos foram submetidos a exame histológico. Nos 24 olhos estudados, não foram detectadas alterações clínicas oftalmológicas. A alteração histológica notada foi a presença de raros linfócitos e eosinófilos na região posterior do vítreo de quatro olhos submetidos a duas aplicações e de seis olhos que receberam três aplicações de infliximabe, mas sem significado clínico. Foi encontrada uma única alteração clinicamente significante, caracterizada como reação inflamatória grave, com presença de exsudatos vítreos nos dois olhos de um coelho, que foi submetido a duas e três aplicações de infliximabe. Os exames eletrorretinográficos mostraram amplitudes em média 12% menores do que aquelas obtidas antes do tratamento, porém sem diferenças estatisticamente significantes, comparando-se a amplitude ou o tempo implícito entre os achados eletrorretinográficos pré e pós-tratamento em todos os grupos examinados. Assim, duas e três aplicações intravítreas de infliximabe em olhos de coelhos em intervalos mensais, na dosagem de 2 mg, não provocaram alterações após seguimento de noventa dias, quer no exame histológico, na eletrorretinografia ou na avaliação clínica oftalmológica. Conclui-se que doses seriadas de infliximabe por via intravítrea em coelhos é um procedimento seguro.

Introduction. La toxoplasmose oculaire (TO) est la première cause d’uvéite postérieure. L’évolution de cette pathologie dépend d’une balance entre la régulation de la réponse immunitaire et la limitation de la prolifération parasitaire.Méthodes. Le but de nos travaux est de déterminer le spectre des cytokines dans l’humeur aqueuse de patients une To ou une autre inflammation intraoculaire (uvéite d’étiologies virales ou secondaires à des maladies inflammatoires systémiques et endophtalmie bactérienne). Pour mieux appréhender les mécanismes immunitaires mis en jeu lors d’une TO, des dosages de transcrits par RT-PCR et de protéines inflammatoires par immunoessaimultiplexe sont réalisés à partir de modèles murins de TO. Enfin, l’effet de l’injection intraoculaire d’anticorps (AC) anti-IL-17A sur l’inflammation intraoculaire et la prolifération parasitaire est étudié.Résultats. Les spectres de cytokines observés dans l’humeur aqueuse diffèrent nettement en fonction de la cause de l’inflammation. Plus particulièrement, IL-17A semble jouer un rôle primordial dans la pathogénicité de la TO humaine et murine. Chez la souris infectée, l’inflammation intraoculaire et le nombre de parasites intraoculaires sont diminués parl’administration d’AC anti-IL-17A. Les niveaux d’ARNm de T-bet et Foxp3, ainsi que la concentration en IFN-γ (marqueurs de l’immunité cellulaire de type Th1 et Treg), sont augmentés après l’injection d’AC anti-IL-17A.Discussion. Les AC anti-IL-17A modèrent la réponse inflammatoire intraoculaire et limitent la prolifération parasitaire en antagonisant les cellules Th17, probablement via l’induction des cellules Th1 et Treg, secrétant IL-10 et IL-27. Ces résultats préliminaires suggèrent une nouvelle approche thérapeutique in vivo lors d’une toxoplasmose oculaire. / Purpose. Toxoplasmosis is the most common cause of posterior uveitis in immunocompetent subjects. Taking into account the opposing needs of limiting parasite multiplication and minimizing tissue destruction, the immune imbalance implies especially Th17 and T regulatory (Treg) cells.Methods. In a prospective clinical study of acute intraocular inflammation including ocular toxoplasmosis, viral uveitis, systemic inflammatory disease related uveitis and bacterial endophthalmitis we evaluated the cytokine pattern in aqueous humors of affected patients. To further study the immunological mechanisms involved during ocular toxoplasmosis, weevaluated the intraocular inflammation, the parasite load and the immunological response characterized on mRNA and protein level in a mouse model. To evaluate the role of IL-17A, anti IL-17A monoclonal antibodies (mAbs) were administered concomitantly with the parasite.Results. Cytokines networks are different, depending on the cause of intraocular inflammation. In OT, we observed severe ocular inflammation and cytokine patterns comparable to human cases, including IL-17A production. Neutralizing IL-17A decreased intraocular inflammation and parasite load in mice. Detailed studies revealed upregulation of Treg and Th1 pathways. When IFN-γ was neutralized concomitantly, the initial parasite multiplication rate was partially restored.Conclusions. Local IL-17A production plays a central role in pathology of OT. The balance of Th17 and Th1 responses (especially IFN-γ) is crucial for the outcome of infection. These data open new in vivo therapeutic approaches by repressing inflammatory pathways using intravitreal injection of IL-17A mAbs.

Introduction:Uveitis in an ocular inflammation affecting mostly people of working age. Uveitis is responsible for severe visual impairment despite of expanding new therapeutics. The animal models of uveitis were established, because the wide clinical variability of uveitis limits the studies in human medicine. The goal our project was to establish a reproducible model of experimental autoimmune uveitis in Czech Republic, and further on this model to observe the frequency of CD3+ and F4/80+ cells in retina, to assess the influence of microbial environment on intensity of intraocular inflammation and to test the therapeutical possibilities. Material and methods: The C57BL/6J mice were immunized by retinal antigen (IRBP 1-20, interphotoreceptor retinoid binding protein), enhanced by complete Freund's adjuvant and pertussis toxin and mild posterior autoimmune uveitis was induced. The mice were bred in conventional and germ-free (gnotobiotic) conditions. The uveitis intensity was evaluated in vivo biomicroscopically and post mortem histologically on hematoxylin eosin stained sections according to the standard protocol. The histological eye specimen were analyzed also by imunohistochemisty and by flow cytometry. Each experiment was performed for 35 days. The conventional mice with uveitis were treated...