This thesis examines the influence of stress on the activity of hippocampal CA1 area. The main task was to determine whether the stress load affects the changes of the local metabolism of glucocorticoids, and whether the levels of corticosteroid receptors in the CA1 hippocampus are modulated in response to stress. In order to answer these questions, the experiments were carried out using three different rat strains - Fisher, Lewis and Wistar which differ in their activities of hypothalamic-pituitary-adrenal axis. Our results demonstrate that stress has no effect on expression of MR mRNA. Conversely, stress reduces the levels of GR mRNA in CA1 area of the dorsal hippocampus. Moreover, we confirmed that the Lewis and Wistar rats didn't change metabolism of glucocorticoids after stress response. By the Fisher rats increased levels of 11β-HSD1 mRNA expression and therefore increased the metabolism of corticosterone.

L‟obésité est principalement du à la surconsommation d‟aliments riches en énergie, en particulier les aliments hyperlipidiques (HL). En plus des comorbidités comme le diabète et les maladies cardiovasculaires, l‟obésité est associée à des troubles émotionnels et cognitifs. La prévalence de l‟obésité chez les enfants et les adolescents augmente sans cesse et ceci est inquiétant car ce sont des périodes cruciales pour la maturation de structures cérébrales comme l‟hippocampe et l‟amygdale, indispensables à la mise en place des processus cognitifs et émotionnels pour le restant de la vie. Il est donc déterminant d‟évaluer l‟impact de la consommation d‟un régime HL obésogène durant ces périodes développementales sur les processus cognitifs et émotionnels. Dans une première étude nous mettons en évidence chez le rat que l‟exposition à un régime HL durant la période périnatale (gestation et lactation) entraine l‟atrophie dendritique des neurones pyramidaux du CA1 de l‟hippocampe et de l‟amygdale basolatérale (BLA) des descendants adultes. Ces résultats sur l‟hippocampe enrichissent les données comportementales déjà existantes indiquant des altérations de la mémoire dépendante de l‟hippocampe suite au régime HL périnatal. Nous montrons également que les changements morphologiques du BLA s‟accompagnent d‟un déficit spécifique de la mémoire aversive olfactive, qui dépend fortement de l‟intégrité du BLA. Ceci démontre pour la première fois les effets délétères, cellulaires et comportementaux, d’un régime HL périnatal sur les fonctions amygdaliennes. La seconde étude se focalise sur les perturbations de la mémoire induites par la consommation d‟un régime HL pendant l‟adolescence et vise à approfondir la compréhension des mécanismes impliqués. Dans un premier temps, nous montrons que la consommation d‟un régime HL du sevrage à l‟âge adulte (couvrant l‟adolescence) chez la souris perturbe la consolidation de la mémoire de reconnaissance d‟objet (MRO) lorsque le contexte est nouveau lors de l‟apprentissage. Ceci s‟accompagne d‟une libération plus importante de glucocorticoïdes au niveau circulant et d‟endocannabinoides (eCB, anandamide en particulier) au niveau hippocampique chez les souris HL. Le blocage des récepteurs aux glucocorticoïdes (GR) ou des récepteurs aux cannabinoïdes de type 1 (CB1R) juste après l‟apprentissage améliore la MRO à long-terme des souris HL. Ces traitements normalisent également la sur-activation c-Fos de l‟hippocampe suite à l‟apprentissage chez les souris HL soulignant l‟importance de cette structure. En effet, la délétion spécifique des CB1R hippocampique améliore fortement la MRO à long-terme des souris HL et nous mettons en évidence que la plasticité synaptique in vivo de la voie CA3-CA1 hippocampique représente un mécanisme perturbé par l‟activation des CB1R suite à l‟apprentissage chez ces souris. Enfin l‟inactivation des neurones glutamatergique hippocampique par une approche pharmacogénétique (DREADD-Gi) améliore la MRO à long-terme chez les souris HL, de façon similaire au blocage ou à la délétion des CB1R suggérant une levée d‟inhibition de ces neurones par l‟activation des CB1R chez les souris HL qui conduirait à leur déficit de MRO à long-terme. Nos résultats indiquent que la consommation d’un régime HL pendant l’adolescence modifie le système eCB de l’hippocampe conduisant à des perturbations de la plasticité synaptique et de la consolidation de la mémoire. Dans leur ensemble ces données permettent d‟améliorer notre compréhension des effets délétères de l‟exposition précoces aux régimes HL obésogènes sur les fonctions mnésiques. / Clinical and experimental studies have established that obesity, resulting mainly from consumption of energy-dense food such as high-fat diet (HFD), is associated with adverse cognitive and emotional outcomes. The prevalence of obesity during childhood and adolescence has reached epidemic levels. This is particularly worrisome since these periods are crucial for hippocampal and amygdala maturation, two brain structures necessary for shaping memory and emotional functions. It is thus critical to determine the impact of HFD exposure during these early developmental periods on memory and emotional processes. First, we show that perinatal HFD exposure (throughout gestation and lactation), leads to dendritic shrinkage of pyramidal neurons in the CA1 of the hippocampus but also in the basolateral amygdala (BLA) in adult rats. These results add to the growing literature indicating changes in hippocampal-dependent memory after perinatal HFD exposure. Regarding amygdala, perinatal HFD exposure specifically impairs odor aversion memory, a task highly dependent on BLA function, without affecting olfactory or malaise processing. These results are the first to show that perinatal HFD exposure impairs amygdala functions, at cellular and behavioral levels. Next, we investigated the cellular mechanisms underlying memory impairment induced by adolescent HFD consumption. We first show that HFD consumption from weaning to adulthood (covering adolescence) impairs long-term, but not short-term, object recognition memory (ORM) in novel context condition which was associated with higher circulating corticosterone and enhanced hippocampal endocannabinoid levels (anandamide in particular) in HFD-fed mice. Systemic post-training blockade of glucocorticoid receptors (GR) or cannabinoid receptors type 1 (CB1R) prevented HFD-induced memory deficits. These treatments also normalized training-induced c-Fos over-activation specifically in hippocampus in HFD group stressing the importance of this structure. Indeed, hippocampal CB1R deletion improved memory in HFD-fed mice. Moreover, we identified changes of in vivo hippocampal synaptic plasticity after training as a potential mechanism impaired by HFD in a CB1R-dependent manner. Finally, chemogenetic inhibition of hippocampal glutamatergic cells improved memory in HFD group similarly to CB1R deletion or blockade suggesting CB1R-dependent disinhibition of these neurons in HFD-fed mice. These results indicate that high-fat diet consumption during adolescence alters the hippocampal eCB system leading to impairment of hippocampal synaptic plasticity and deficit in recognition memory consolidation. Taken together, our results provide new evidences of how HFD consumption during early developmental periods exerts its deleterious effects on cognitive functions and identify the endocannabinoid system as a potential target for treating cognitive impairment associated with obesity.

Introdução: Estudos avaliando a prevalência de síndrome de Evans (SE) no lúpus eritematoso sistêmico juvenil (LESJ) bem como possíveis fatores associados são restritos a poucos relatos de caso. Objetivos: Avaliar a prevalência de SE em uma grande população de LESJ, assim como sua possível associação com dados demográficos, manifestações clínicas, características laboratoriais, atividade/dano cumulativo da doença e tratamento. Métodos: Um estudo de coorte multicêntrico retrospectivo foi realizado em 10 serviços de Reumatologia Pediátrica provenientes do Grupo Brasileiro de Lúpus e incluiu 850 pacientes com LESJ. SE foi avaliada ao diagnóstico do LES e definida pela combinação de púrpura trombocitopênica autoimune (PTI) e anemia hemolítica autoimune (AHAI). Os pacientes foram divididos em dois grupos para a avaliação das associações propostas: pacientes que apresentaram SE e pacientes sem SE. Todos foram avaliados ao diagnóstico do LES. Resultados: SE foi observada em 11/850 pacientes de LESJ ao diagnostico (1,3%). A maioria deles tinha doença ativa (82%) e apresentaram manifestações hemorrágicas (58%). Todos os pacientes com SE foram hospitalizados e não houve nenhum óbito. As comparações entre pacientes LESJ com e sem SE ao diagnóstico demonstrou frequências similares do sexo feminino, envolvimento de múltiplos órgãos, perfil de auto-anticorpos semelhantes e complemento baixo (p > 0,05). Pacientes com SE tinham frequências menores de eritema malar (9% vs. 53%, p=0,003) e envolvimento músculo-esquelético (18% vs. 69%, p=0,001) do que aqueles sem esta complicação. A frequência de pulsoterapia com metilprednisolona (82% vs. 43%, p=0,013) e uso de gamaglobulina endovenosa (64% vs. 3%), p < 0,0001) foram significativamente maiores no grupo com SE, com dose atual de prednisona semelhante entre os dois grupos [1,1 (0,76-1,5) vs. 1,0 (0-30) mg/kg/dia, p=0,195]. Conclusões: Este foi o primeiro estudo que evidenciou a possível relação de SE como uma manifestação inicial rara e grave do LESJ, porém com bom prognóstico. O diagnóstico se torna o principal desafio devido à falta de sinais e sintomas característicos de lúpus e a dificuldade de se excluir diagnósticos diferenciais como infecção e imunodeficiência primária / Introduction: Studies evaluating the prevalence of Evans Syndrome (ES) in childhood-onset systemic lupus erythematosus (cSLE) as well as possible associated factors has been rarely reported and restricted to case reports. Objectives: To evaluate the prevalence of ES in a large population of cSLE, and the association with demographic data, clinical manifestations, laboratory characteristics, disease activity, cumulative damage, and treatment. Methods: A retrospective multicenter cohort study was performed in 10 Pediatric Rheumatology services and included 850 patients with cSLE. ES was evaluated at the diagnosis of cSLE and defined as the combination of autoimmune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA). The patients were divided into two groups for the evaluation of the proposed associations: patients who presented ES and patients without ES. All were assessed at the cSLE diagnosis. Results: ES was observed in 11/850 (1.3%) cSLE patients. The majority of them had hemorrhagic manifestations (58%) and active disease (82%). All patients with ES were hospitalized and none died. Comparisons of cSLE patients with and without ES at diagnosis revealed similar frequencies of female gender, multi-organ involvement, autoantibodies profile and low complement (p > 0.05). Patients with ES had a lower frequency of malar rash (9% vs. 53%, p=0.003) and musculoskeletal involvement (18% vs. 69%, p=0.001) than those without this complication. The frequencies of intravenous methylprednisolone (82% vs. 43%, p=0.013) and intravenous immunoglobulin use (64% vs. 3%, p < 0.0001) were significantly higher in the former group, with similar current prednisone dose between groups [1.1 (0.76-1.5) vs. 1.0 mg/kg/day (0-30), 0.195]. Conclusions: This was the first study that evidenced the possible relationship of ES as a rare and severe initial manifestation of cSLE, but with a good prognosis. Diagnosis becomes the main challenge due to the lack of signs and symptoms characteristic of lupus and the difficulty of excluding differential diagnoses such as infection and primary immunodeficiency

Introdução: As infecções são importantes causas de morbidade e mortalidade em pacientes com lúpus eritematoso sistêmico juvenil (LESJ). No entanto, estudos avaliando somente infecções fúngicas invasivas (IFI) em pacientes com LESJ são restritos a relatos de casos ou série de casos, sem qualquer avaliação sistemática dos possíveis fatores de risco ou desfechos associados. A escassez de dados referentes às IFI em pacientes com LESJ e seu impacto sobre as características da doença em uma grande população levou ao desenvolvimento deste estudo multicêntrico. Objetivos: Estudar a prevalência, fatores de risco e mortalidade de IFI em pacientes com LESJ. Método: Um estudo de coorte multicêntrico retrospectivo foi realizado com 852 pacientes com LESJ de 10 Serviços de Reumatologia Pediátrica do Estado de São Paulo. Uma reunião foi realizada e todos os pesquisadores foram treinados para o preenchimento do banco de dados. As IFI foram diagnosticadas de acordo com as definições revisadas pelo grupo de consenso EORTC/MSG (comprovadas, prováveis ou possíveis). Foram coletados dados acerca de dados demográficos, características clínico-laboratoriais, atividade da doença (SLEDAI-2K), dano cumulativo (SLICC/ACR-DI) e tratamento, além de características e complicações das IFI. Resultados: IFI foram diagnosticadas em 33/852 (3,9%) pacientes com LESJ. IFI comprovadas foram diagnosticadas em 22 pacientes, IFI prováveis em 5 e IFI possíveis em 6. Os tipos de IFI encontradas foram: candidíase em 20 pacientes, aspergilose em 9, criptococose em 2, histoplasmose disseminada em um e paracoccidioidomicose em um. A mediana de duração da doença foi menor (1,0 vs. 4,7 anos, p < 0,0001), com maiores escores de SLEDAI-2K atual [19,5 (0-44) vs. 2 (0-45), p < 0,0001] e dose atual de prednisona [50 (10-60) vs. 10 (2-90) mg/dia, p < 0,0001] em pacientes com IFI em comparação com os pacientes sem IFI. A frequência de óbito foi maior no grupo com IFI (51% vs. 6%, p < 0,0001). A análise de regressão logística revelou que SLEDAI-2K atual (OR=1,108, IC 95%=1,057- 1,163, p < 0,0001), dose atual de prednisona (OR=1,046, IC 95%=1,021-1,071; p < 0,0001) e duração da doença (OR=0,984, IC 95%=0,969-0,998, p=0,030) foram fatores de risco independentes para IFI (R2 Nagelkerke 0,425). Conclusão: Este foi o primeiro estudo que caracterizou IFI em pacientes com LESJ. Identificou-se que a atividade da doença e uso de glicocorticoides foram os principais fatores de risco para estas infecções potencialmente graves, principalmente nos primeiros anos de curso da doença e com uma elevada taxa mortalidade / Introduction: Infections are an important cause of morbidity and mortality in childhoodonset systemic lupus erythematosus (cSLE) patients. However, studies evaluating solely invasive fungal infections (IFI) in cSLE patients are restricted to case reports or case series without any systematic evaluation of the possible associated risk factors and outcome in pediatric lupus population. The scarcity of data regarding IFI in cSLE patients and its impact on disease characteristics in a large population led to the development of this multicenter study. Objective: To study the prevalence, risk factors and mortality of IFI in cSLE patients. Methods: A retrospective multicenter cohort study was performed in 852 cSLE patients from 10 Pediatric Rheumatology services. An investigator meeting was held and all participants received database training. IFI were diagnosed according to EORTC/MSG Consensus Group criteria (proven, probable and possible). Demographic data, clinical, laboratorial, disease activity (SLEDAI-2K), cumulative damage (SLICC/ACR-DI) and treatment were collected. IFI were characterized and its outcome were also evaluated. Results: IFI were observed in 33/852 (3.9%) cSLE patients. Proven IFI was diagnosed in 22 cSLE patients, probable IFI in 5 and possible IFI in 6. Types of IFI were: 20 candidiasis, 9 aspergillosis, 2 cryptococcosis, one disseminated histoplasmosis and one paracoccidioidomycosis. The median of disease duration was lower (1.0 vs. 4.7 years, p < 0.0001), with a higher current SLEDAI-2K [19.5 (0-44) vs. 2 (0-45), p < 0.0001] and current prednisone dose [50 (10-60) vs. 10 (2-90) mg/day, p < 0.0001] in patients with IFI compared to those without IFI. The frequency of death was higher in the former group (51% vs. 6%, p < 0.0001). Logistic regression analysis revealed that current SLEDAI-2K (OR=1.108; 95%CI=1.057-1.163; p < 0.0001), prednisone current dose (OR=1.046; 95%CI=1.021-1.071; p < 0.0001) and disease duration (OR=0.984; 95%CI=0.969-0.998; p=0.03) were independent risk factors for IFI (R2 Nagelkerke 0.425). Conclusion: This was the first study that characterized IFI in cSLE patients. We identified that disease activity and glucocorticoid use were the main risk factors for these life-threatening infections, mainly in the first years of disease course and with a high rate of fatal outcome

Les triterpènes sont un groupe important et structurellement diversifié de produits naturels issus du squalène. Les progestatifs et les glucocorticoïdes sont des triterpènes oxygénés ayant un squelette tétracyclique et reconnus pour leurs diverses propriétés thérapeutiques. Par ailleurs, l'érythrodiol et l'uvaol sont des triterpènes pentacycliques reconnus pour leurs effets bénéfiques dans l'alimentation humaine. Dans ce travail, l'interaction avec les membranes des vésicules lipidiques et la liaison à la sérum albumine humaine de ces molécules sont étudiées dans le but de mieux comprendre leurs propriétés pharmacologiques. Les vésicules lipidiques ont été caractérisées par DSC, spectroscopie Raman, FTIR et polarisation de fluorescence du DPH pour comprendre l'effet des molécules sélectionnées sur la fluidité membranaire.Également, nous avons étudié la liaison du cholestérol, d'une série de progestatifs et de glucocorticoïdes à l'albumine humaine par la spectroscopie de fluorescence.Les résultats ont démontré que les progestatifs, les glucocorticoïdes, l'érythrodiol et l'uvaol altèrent les propriétés physiques de la bicouche lipidique.Les progestatifs et les glucocorticoïdes démontrent un attachement modéré à l'albumine. Par ailleurs, la dydrogestérone présente la constante de liaison la plus importante. Enfin, notre étude a démontré que la constante de liaison du cholestérol à l'albumine est faible en comparaison avec les autres molécules étudiées. Notre étude a conduit à une connaissance approfondie des mécanismes moléculaires et des caractéristiques structurales impliqués dans l'interaction des triterpènes avec les protéines et les membranes synthétiques / The triterpenoids are a large and structurally diverse group of natural products derived from squalene. Progesterone derivatives and glucocorticoids are a group of oxygenated triterpenes having a tetracyclic skeleton and identified for their therapeutic properties. Whereas, erythrodiol and uvaol are pentacyclic triterpenes, known for their beneficial effects on human diet. In this thesis, we studied their interaction with the membranes of lipid vesicles and with human serum albumin to better understand their pharmacological properties. DSC, Raman spectroscopy, FTIR and fluorescence polarization of DPH were used to investigate the effect of triterpenes on the membrane fluidity. Besides, we used fluorescence spectroscopy to study the binding of cholesterol, a series of progesterone derivatives and another series of glucocorticoids to albumin. The results revealed that progesterone derivatives, glucocorticoids, erythrodiol and uvaol changed the physical properties of the bilayers. Progesterone derivatives and glucocorticoids have been proven to bind moderately to albumin. Dydrogesterone showed the highest binding constant. Finally, our study demonstrated that cholesterol exhibited a much weaker interaction with albumin compared to progesterone derivatives and glucocorticoids.Our work has led to a better understanding of triterpenes molecular mechanisms of their interaction with proteins and biological membranes and structural features controlling these interactions

Introdução: As infecções são importantes causas de morbidade e mortalidade em pacientes com lúpus eritematoso sistêmico juvenil (LESJ). No entanto, estudos avaliando somente infecções fúngicas invasivas (IFI) em pacientes com LESJ são restritos a relatos de casos ou série de casos, sem qualquer avaliação sistemática dos possíveis fatores de risco ou desfechos associados. A escassez de dados referentes às IFI em pacientes com LESJ e seu impacto sobre as características da doença em uma grande população levou ao desenvolvimento deste estudo multicêntrico. Objetivos: Estudar a prevalência, fatores de risco e mortalidade de IFI em pacientes com LESJ. Método: Um estudo de coorte multicêntrico retrospectivo foi realizado com 852 pacientes com LESJ de 10 Serviços de Reumatologia Pediátrica do Estado de São Paulo. Uma reunião foi realizada e todos os pesquisadores foram treinados para o preenchimento do banco de dados. As IFI foram diagnosticadas de acordo com as definições revisadas pelo grupo de consenso EORTC/MSG (comprovadas, prováveis ou possíveis). Foram coletados dados acerca de dados demográficos, características clínico-laboratoriais, atividade da doença (SLEDAI-2K), dano cumulativo (SLICC/ACR-DI) e tratamento, além de características e complicações das IFI. Resultados: IFI foram diagnosticadas em 33/852 (3,9%) pacientes com LESJ. IFI comprovadas foram diagnosticadas em 22 pacientes, IFI prováveis em 5 e IFI possíveis em 6. Os tipos de IFI encontradas foram: candidíase em 20 pacientes, aspergilose em 9, criptococose em 2, histoplasmose disseminada em um e paracoccidioidomicose em um. A mediana de duração da doença foi menor (1,0 vs. 4,7 anos, p < 0,0001), com maiores escores de SLEDAI-2K atual [19,5 (0-44) vs. 2 (0-45), p < 0,0001] e dose atual de prednisona [50 (10-60) vs. 10 (2-90) mg/dia, p < 0,0001] em pacientes com IFI em comparação com os pacientes sem IFI. A frequência de óbito foi maior no grupo com IFI (51% vs. 6%, p < 0,0001). A análise de regressão logística revelou que SLEDAI-2K atual (OR=1,108, IC 95%=1,057- 1,163, p < 0,0001), dose atual de prednisona (OR=1,046, IC 95%=1,021-1,071; p < 0,0001) e duração da doença (OR=0,984, IC 95%=0,969-0,998, p=0,030) foram fatores de risco independentes para IFI (R2 Nagelkerke 0,425). Conclusão: Este foi o primeiro estudo que caracterizou IFI em pacientes com LESJ. Identificou-se que a atividade da doença e uso de glicocorticoides foram os principais fatores de risco para estas infecções potencialmente graves, principalmente nos primeiros anos de curso da doença e com uma elevada taxa mortalidade / Introduction: Infections are an important cause of morbidity and mortality in childhoodonset systemic lupus erythematosus (cSLE) patients. However, studies evaluating solely invasive fungal infections (IFI) in cSLE patients are restricted to case reports or case series without any systematic evaluation of the possible associated risk factors and outcome in pediatric lupus population. The scarcity of data regarding IFI in cSLE patients and its impact on disease characteristics in a large population led to the development of this multicenter study. Objective: To study the prevalence, risk factors and mortality of IFI in cSLE patients. Methods: A retrospective multicenter cohort study was performed in 852 cSLE patients from 10 Pediatric Rheumatology services. An investigator meeting was held and all participants received database training. IFI were diagnosed according to EORTC/MSG Consensus Group criteria (proven, probable and possible). Demographic data, clinical, laboratorial, disease activity (SLEDAI-2K), cumulative damage (SLICC/ACR-DI) and treatment were collected. IFI were characterized and its outcome were also evaluated. Results: IFI were observed in 33/852 (3.9%) cSLE patients. Proven IFI was diagnosed in 22 cSLE patients, probable IFI in 5 and possible IFI in 6. Types of IFI were: 20 candidiasis, 9 aspergillosis, 2 cryptococcosis, one disseminated histoplasmosis and one paracoccidioidomycosis. The median of disease duration was lower (1.0 vs. 4.7 years, p < 0.0001), with a higher current SLEDAI-2K [19.5 (0-44) vs. 2 (0-45), p < 0.0001] and current prednisone dose [50 (10-60) vs. 10 (2-90) mg/day, p < 0.0001] in patients with IFI compared to those without IFI. The frequency of death was higher in the former group (51% vs. 6%, p < 0.0001). Logistic regression analysis revealed that current SLEDAI-2K (OR=1.108; 95%CI=1.057-1.163; p < 0.0001), prednisone current dose (OR=1.046; 95%CI=1.021-1.071; p < 0.0001) and disease duration (OR=0.984; 95%CI=0.969-0.998; p=0.03) were independent risk factors for IFI (R2 Nagelkerke 0.425). Conclusion: This was the first study that characterized IFI in cSLE patients. We identified that disease activity and glucocorticoid use were the main risk factors for these life-threatening infections, mainly in the first years of disease course and with a high rate of fatal outcome

Introdução: Estudos avaliando a prevalência de síndrome de Evans (SE) no lúpus eritematoso sistêmico juvenil (LESJ) bem como possíveis fatores associados são restritos a poucos relatos de caso. Objetivos: Avaliar a prevalência de SE em uma grande população de LESJ, assim como sua possível associação com dados demográficos, manifestações clínicas, características laboratoriais, atividade/dano cumulativo da doença e tratamento. Métodos: Um estudo de coorte multicêntrico retrospectivo foi realizado em 10 serviços de Reumatologia Pediátrica provenientes do Grupo Brasileiro de Lúpus e incluiu 850 pacientes com LESJ. SE foi avaliada ao diagnóstico do LES e definida pela combinação de púrpura trombocitopênica autoimune (PTI) e anemia hemolítica autoimune (AHAI). Os pacientes foram divididos em dois grupos para a avaliação das associações propostas: pacientes que apresentaram SE e pacientes sem SE. Todos foram avaliados ao diagnóstico do LES. Resultados: SE foi observada em 11/850 pacientes de LESJ ao diagnostico (1,3%). A maioria deles tinha doença ativa (82%) e apresentaram manifestações hemorrágicas (58%). Todos os pacientes com SE foram hospitalizados e não houve nenhum óbito. As comparações entre pacientes LESJ com e sem SE ao diagnóstico demonstrou frequências similares do sexo feminino, envolvimento de múltiplos órgãos, perfil de auto-anticorpos semelhantes e complemento baixo (p > 0,05). Pacientes com SE tinham frequências menores de eritema malar (9% vs. 53%, p=0,003) e envolvimento músculo-esquelético (18% vs. 69%, p=0,001) do que aqueles sem esta complicação. A frequência de pulsoterapia com metilprednisolona (82% vs. 43%, p=0,013) e uso de gamaglobulina endovenosa (64% vs. 3%), p < 0,0001) foram significativamente maiores no grupo com SE, com dose atual de prednisona semelhante entre os dois grupos [1,1 (0,76-1,5) vs. 1,0 (0-30) mg/kg/dia, p=0,195]. Conclusões: Este foi o primeiro estudo que evidenciou a possível relação de SE como uma manifestação inicial rara e grave do LESJ, porém com bom prognóstico. O diagnóstico se torna o principal desafio devido à falta de sinais e sintomas característicos de lúpus e a dificuldade de se excluir diagnósticos diferenciais como infecção e imunodeficiência primária / Introduction: Studies evaluating the prevalence of Evans Syndrome (ES) in childhood-onset systemic lupus erythematosus (cSLE) as well as possible associated factors has been rarely reported and restricted to case reports. Objectives: To evaluate the prevalence of ES in a large population of cSLE, and the association with demographic data, clinical manifestations, laboratory characteristics, disease activity, cumulative damage, and treatment. Methods: A retrospective multicenter cohort study was performed in 10 Pediatric Rheumatology services and included 850 patients with cSLE. ES was evaluated at the diagnosis of cSLE and defined as the combination of autoimmune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (AIHA). The patients were divided into two groups for the evaluation of the proposed associations: patients who presented ES and patients without ES. All were assessed at the cSLE diagnosis. Results: ES was observed in 11/850 (1.3%) cSLE patients. The majority of them had hemorrhagic manifestations (58%) and active disease (82%). All patients with ES were hospitalized and none died. Comparisons of cSLE patients with and without ES at diagnosis revealed similar frequencies of female gender, multi-organ involvement, autoantibodies profile and low complement (p > 0.05). Patients with ES had a lower frequency of malar rash (9% vs. 53%, p=0.003) and musculoskeletal involvement (18% vs. 69%, p=0.001) than those without this complication. The frequencies of intravenous methylprednisolone (82% vs. 43%, p=0.013) and intravenous immunoglobulin use (64% vs. 3%, p < 0.0001) were significantly higher in the former group, with similar current prednisone dose between groups [1.1 (0.76-1.5) vs. 1.0 mg/kg/day (0-30), 0.195]. Conclusions: This was the first study that evidenced the possible relationship of ES as a rare and severe initial manifestation of cSLE, but with a good prognosis. Diagnosis becomes the main challenge due to the lack of signs and symptoms characteristic of lupus and the difficulty of excluding differential diagnoses such as infection and primary immunodeficiency

O tráfego de leucócitos é um processo complexo, dependente da ação de inúmeras substâncias químicas, além da perfeita interação celular. Desta forma, este estudo teve como objetivo avaliar a ação dos GCe e da ANXA1 sobre o eixo SDF-1&#945;/CXCR4 e IL-17/IL-23/G-CSF e sobre a expressão de moléculas de adesão CD18, CD49d e CD62L. Foram utilizados camundongos machos Balb/C selvagens (WT) ou ANXA1-/-. As avaliações foram realizadas em condições basais, na presença de altas concentrações de GCe e na vigência de processo inflamatório, induzidos pela administração de ACTH (5 &#181;g/animal, i.p.) ou pela injeção de LPS (100 &#181;g/kg, i.p.), respectivamente, ou na ausência da ação dos GCe, pela ação do RU 38486 (RU, 10 mg/kg, i.p.). A participação da ANXA1 e do receptor FPR2 foi avaliada pelo pré-tratamento com Ac2-26 (1 mg/Kg, i.p.) ou com BOC2 (10 &#181;g/animal, i.p.) durante 4 dias, 1 vez ao dia. A quantificação total e diferencial das células foi realizada em câmara de Neubauer e em esfregaços corados por May-Grunwald ou citometria de fluxo. As quantificações de CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L e maturação granulocítica (CD11b/Ly6G) em células da medula e da circulação foram realizadas por citometria de fluxo. A expressão de ANXA1 nos tecidos do estomago e do baço foi realizada por western blotting e nas células da medula óssea e sangue circulante foi realizada por imunofluorescência. As quantificações de IL-17, IL-23, G-CSF, SDF-1&#945; e corticosterona foram realizadas por ELISA. A quimiotaxia de neutrófilos da medula óssea e sangue periférico foi ensaiada na placa de quimiotaxia com filtro de poro de 8 &#181;m. A fagocitose de neutrófilos apoptóticos por macrófagos da medula óssea foi avaliada por ensaio in vitro. Para verificar os efeitos do ACTH na migração de neutrófilos no processo inflamatório, foi empregado o modelo de bolsa de ar (100 &#181;g/mL; LPS); e o comportamento dos leucócitos circulantes de animais tratados com ACTH foi avaliado pela técnica de microscopia intravital. Os resultados obtidos, que estão apresentados em quatro temáticas, mostraram que: 1) neutrófilos da medula óssea e sangue periférico expressam ANXA1 no citoplasma e membrana, bem como o receptor FPR2, constitutivamente, e a expressão de ambos é regulada pelos GCe. A ANXA1, via receptor FPR2 expresso em células da medula óssea, controlam a maturação neutrofílica e o tráfego destas células da medula óssea para o sangue. A ANXA1, via interação ao FPR2, controla o clearance de neutrófilos do sangue para a medula óssea, modulando o eixo SDF-1&#945;/CXCR4; 2) A administração do ACTH causa neutrofilia e os neutrófilos circulantes são ANXA1+, CD18+, CD49d+, CD62L+, mostrando que injeção do ACTH in vivo altera o fenótipo destas células na circulação. Estas modificações alteram o comportamento dos neutrófilos na circulação, bem como a migração para a bolsa de ar na vigência de inflamação e para os tecidos de clearance. Estes efeitos podem ser dependentes, pelo menos em parte, da inibição de migração orientada, já que quimiotaxia frente ao fMLP ou ao SDF-1&#945; estavam reduzidas. Ainda, o clearance de neutrófilos é reduzido em animais tratados com o ACTH pela menor atividade fagocítica e secretora dos macrófagos medulares; 3) Animais tratados com RU 38486 e ANXA1-/- mobilizam granulócitos da medula óssea para o sangue circulante e, deste compartimento para o foco de inflamação com maior intensidade que o observado em animais controles. O eixo IL-17/IL-23/G-CSF parece estar envolvido na granulopoiese e na mobilização de neutrófilos para o sangue durante a inflamação, mas não é alvo de ação da ANXA1 e o GCe nesta etapa do processo inflamatório. Adicionalmente, foi observado que na vigência de peritonite, as moléculas de adesão, CD49d e CD62L estão envolvidas no processo de migração de neutrófilos da medula óssea para o sangue. Os resultados aqui obtidos permitem concluir que os GCe e a ANXA1 são relevantes para granulopoiese e tráfego dos neutrófilos da medula óssea em condições fisiológicas e na vigência de processo inflamatório. Ainda, em conjunto com os dados da literatura, os nossos resultados podem sugerir a participação da ANXA1 dos GCe na plasticidade fenotípica dos neutrófilos de acordo com os estímulos a que são submetidos, e podem auxiliar na compreensão dos novos conceitos sobre a produção, tempo de vida, localização e funções de neutrófilos. / The traffic leukocytes is a complex process dependent on the action of severals chemical mediators, in addition to perfect cell interaction. Therefore, this study aimed to evaluate the effect of GCe and ANXA1 on SDF-1&#945;/CXCR4 and IL-17/IL-23/G-CSF and on the expression of adhesion molecules CD18, CD49d and CD62L. Balb/C wild type and ANXA1-/- male mice were employed. The analysis were performed at physiological conditions, in the presence of high concentrations of GCe and during of inflammatory process induced by ACTH administration (5 &#181;g/animal, i.p.) or LPS injection (100 &#181;g/kg, i.p.), respectively or in the absence of GCe action, by the action of RU 38486 (RU, 10 mg/kg , i.p.). The involvement of the receptor FPR2 and ANXA1 was assessed by pre-treatment with Ac2-26 (1 mg/kg, i.p.) or BOC2 (10 &#181;g/animal, i.p.) for 4 days, once a day. The quantification of total and differential cell was performed in a Neubauer chamber and stained smears by May-Grunwald and flow cytometry. Quantification of expression of CXCR2, CXCR4, FPR2, CD18, CD49d, CD62L and granulocytic maturation (CD11b/Ly6G) in the bone marrow and circulation were performed by flow cytometry. The expression of ANXA1 on tissues was performed by western blotting and on cells from bone marrow and blood by immunocytochemistry. Quantification of IL-17, IL-23, G-CSF, SDF-1&#945; and corticosterone were performed by ELISA. The chemotaxis of neutrophils from the bone marrow and blood was tested in the chemotaxis chamber with filter pore of 8 microns. The phagocytosis of apoptotic neutrophils by bone marrow macrophages was assessed by in vitro assay. To investigate the effects of ACTH in the migration of neutrophils in the inflammatory process, the model employed was air pouch (100 &#181;g/ ml, LPS), and the behavior of circulating leukocytes from animals treated with ACTH were evaluated by intravital microscopy. The results obtained, which are presented in three sections, showed that: 1) neutrophils from the bone marrow and blood expressed ANXA1 in the cytoplasm and membrane, as well as FPR2, constitutively and the expression of both is regulated by GCe. The ANXA1 via FPR2 receptor expressed in bone marrow cells, controls the neutrophilic maturation and traffic of these cells from the bone marrow into the blood. The ANXA1 via interaction to FPR2 controls the clearance of neutrophils from the blood to the bone marrow by modulating the SDF-1&#945;/CXCR4 axis; 2) the administration of ACTH induces neutrophilia and the circulating neutrophils are ANXA1+, CD18+, CD49d+ and CD62L+, showing that the injection of ACTH in vivo alters the phenotype of these cells in the blood. These modifications alter the behavior of neutrophils in the blood, as well as the migration to the air pouch in the presence of inflammation and to the tissue clearance, and these effects may be dependent, at least in part, on inhibition of migration oriented events, as chemotaxis in response to fMLP or SDF-1&#945; were reduced. Further, the clearance of neutrophils is reduced in animals treated with ACTH due to the lower phagocytic and secretory activity of medullary macrophages; 3) Animals treated with RU 38486 and ANXA1-/- mobilize granulocytes from bone marrow into the blood, and from this compartment to the focus of inflammation with higher intensity than that observed in the control group. The axis IL-17/IL-23/G-CSF seems to be involved in granulopoiesis and mobilization of neutrophils into the blood during inflammation, but it is not the target of action of ANXA1 and GCe at this step of inflammatory process. Additionally, it was observed that in the presence of peritonitis, the adhesion molecules, CD49d and CD62L are involved in the migration of neutrophils from the bone marrow into the blood. The results obtained allow concluding that the GCe and ANXA1 are relevant to the granulopoiesis and the traffic of neutrophils from bone marrow under physiological conditions and in the presence of inflammation. Furthermore, together with literature data, the data presented here may suggest the involvement of ANXA1 the GCe in phenotypic plasticity of neutrophils according to the stimuli that are submitted, and may support to understand the new concepts of production, half-life, location and function of neutrophils.

Les glucocorticoïdes (GC) sont des hormones stéroïdes synthétisées par les glandes surrénales. La production de ces GC est une des réponses de l’organisme pour rétablir l’homéostasie grâce à différentes actions comprenant des effets centraux sur le comportement et la douleur. C’est ce dernier qui a fait l’objet de mes travaux dans le cadre de cette thèse.Les afférences sensorielles primaires véhiculent les informations de la périphérie dans les cornes dorsales de la moelle épinière. Ces informations qui peuvent être nociceptives sont modulées par un réseau de neurones spinal avant d’être transmises et intégrées. Nous avons montré que les GC sont impliqués dans la maturation et le fonctionnement de la transmission inhibitrice faisant intervenir le neurotransmetteur GABA. Dans les cornes dorsales, cette inhibition est cruciale pour limiter les mécanismes de transmission de l’information nociceptive. / Glucocorticoids (GC) are steroid hormones synthesized in adrenals following HPA axis activation. GC production is a response of the organism to alleviate homeostasis perturbations through different actions. One of them involves central neuronal modulation of behavior and pain perception.Primary afferents convey peripheral sensory information in the dorsal horns of the spinal cord. This information can be nociceptive and are modulated by a spinal neuronal network before being transmitted and integrated. We showed that GC are implied in the maturation and functioning of the inhibitory transmission involving GABA neurotransmitter. In the dorsal horns this inhibitory transmission is of major importance, limiting the processing of nociceptive information.

No description available.