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B cells play crucial roles in the development of the two human autoimmune diseases, type 1 diabetes (T1D) and systemic lupus erythematosus (SLE). In the past decade, numerous studies showed positive responses of B cell depletion therapies in these two diseases. However, the beneficial effects are temporary and accompanied with adverse events. In this dissertation, we aimed to identify novel targets for a better modulation of disease development using mouse models. These diseases have circulating autoantibodies that are mostly mutated with an IgG isotype, indicating B cells that are producing them have been through the process of affinity maturation. Activation-induced cytidine deaminase (AID) is a core enzyme that regulates somatic hypermutation (SHM) and class switch recombination (CSR), the two key mechanisms in affinity maturation. We showed that genetic ablation of AID significantly inhibited the development of TID in NOD mice. Homologous recombination (HR) pathway is important for the repair of AID-induced DNA double strand breaks during CSR. 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid, also known as DIDS, is a small molecule that inhibits HR pathway and subsequently leads to apoptosis of class switching cells. DIDS treatment remarkably retarded the progression of TID, even when started at a relatively late stage, indicating the potential of this treatment for disease reversal. In both approaches, we observed a notable expansion of CD73+ B cells, which exerted an immunosuppressive role and could be responsible for T1D resistance. Next we examined the effect of targeting affinity maturation through these two approaches in lupus-prone mice. The genetic abrogation of AID in BXSB mice significantly ameliorated lupus nephritis and prolonged their lifespan. AID-deficient mice also exhibited improvement on disease hallmarks with increased marginal zone B cells and more normal splenic architecture. DIDS treatment notably reduced class switching when B cells were stimulated in vitro. However, the administration of DIDS did not strikingly alter the course of SLE in either BXSB mice or MRL/lpr mice. These findings demonstrated that affinity maturation could be a potential target for T1D and SLE, while further explorations into targeting other components in the repair pathway are warranted for SLE. Lastly, we assessed the effect of maternal AID modulation on the SLE development in the offspring using BXSB mouse model. Interestingly, the absence of maternal AID resulted in offspring that developed significantly more severe lupus nephritis compared to control. The offspring born to AID-deficient dams also exhibited elevated levels of pathogenic autoantibodies and exacerbated disease features. Therefore, the modulation of maternal AID could influence the SLE development in the offspring, and future investigations are needed to determine the underlying mechanisms responsible for the disease acceleration. / Doctor of Philosophy / The failure of the immune system to differentiate self from non-self leads to the development of autoimmune diseases. Type 1 diabetes (T1D) and systemic lupus erythematosus (SLE) are complex autoimmune diseases affecting millions of people in the world. Despite intensive research regarding these two diseases, no known cure is available indicating an imperative need for the development of novel therapies. With the importance of B cells in the pathogenesis of these two diseases, intensive research focused on whole B cell depletion therapies. However, these therapies exhibited high risks of infections as a result of depleting all the B cells. In this dissertation, we sought to selectively target specific B lymphocyte subsets that are crucial contributing factors in the development of T1D and SLE. While the effect of therapeutic treatment varied among different mouse models, the genetic manipulation of specific B cells successfully retarded the progression of both T1D and SLE and extended the lifespan of the mice. Further studies shed light on the possible mechanisms that are responsible for the disease inhibition. These data proved that targeting specific B cell compartment could be a potential disease management in T1D and SLE patients. In addition, using the established mouse model, we demonstrated the modulation of maternal factors significantly impact the SLE development in the offspring. Future experiments to identify the underlying mechanisms could provide more targets for the therapeutic development.

Le récepteur DcR3 (Decoy receptor 3) est un membre de la famille des récepteurs aux facteurs de nécrose tumorale (TNF). Il est fortement exprimé dans les tissus humains normaux ainsi que les tumeurs malignes. DcR3 est un récepteur pour trois ligands de la famille du TNF tels que FasL, LIGHT et TL1A. Étant une protéine soluble donc dépourvue de la portion transmembranaire et intracytoplasmique, le récepteur DcR3 est incapable d’effectuer une transduction de signal intracellulaire à la suite de son interaction avec ses ligands. De ce fait, DcR3 joue un rôle de compétiteur pour ces derniers, afin d’inhiber la signalisation via leurs récepteurs fonctionnels tels que Fas, HVEM/LTbetaR et DR3. Lors de nos précédentes études, nous avons pu démontrer, que DcR3 pouvaist moduler la fonction des cellules immunitaires, et aussi protéger la viabilité des îlots de Langerhans. À la suite de ces résultats, nous avons généré des souris DcR3 transgéniques (Tg) en utilisant le promoteur du gène β-actine humaine afin d’étudier plus amplement la fonction de ce récepteur. Les souris Tg DcR3 ont finalement développé le syndrome lupus-like (SLE) seulement après l’âge de 6 mois. Ces souris présentent une variété d'auto-anticorps comprenant des anticorps anti-noyaux et anti-ADN. Elles ont également manifesté des lésions rénales, cutanées, hépatiques et hématopoïétiques. Contrairement aux modèles de lupus murin lpr et gld, les souris DcR3 sont plus proche du SLE humain en terme de réponse immunitaire de type Th2 et de production d'anticorps d'anti-Sm. En péus, nous avons constaté que les cellules hématopoïétiques produisant DcR3 sont suffisantes pour causer ces pathologies. DcR3 peut agir en perturbant l’homéostasie des cellules T pour interférer avec la tolérance périphérique, et ainsi induire l'autoimmunité. Chez l'humain, nous avons détecté dans le sérum de patients SLE des niveaux élevés de la protéine DcR3. Chez certains patients, comme chez la souris, ces niveaux sont liés directement aux titres élevés d’IgE. Par conséquent, DcR3 peut représenter un facteur pathogénique important du SLE humain. L’étude des souris Tg DcR3, nous a permis aussi d’élucider le mécanisme de protection des îlots de Langerhans. Le blocage de la signalisation des ligands LIGHT et TL1A par DcR3 est impliqué dans une telle protection. D'ailleurs, nous avons identifié par ARN microarray quelques molécules en aval de cette interaction, qui peuvent jouer un rôle dans le mécanisme d’action. Nous avons par la suite confirmé que Adcyap1 et Bank1 joue un rôle critique dans la protection des îlots de Langerhans médiée par DcR3. Notre étude a ainsi élucidé le lien qui existe entre la signalisation apoptotique médiée par Fas/FasL et la pathogénèse du SLE humain. Donc, malgré l’absence de mutations génétiques sur Fas et FasL dans le cas de cette pathologie, DcR3 est capable de beoquer cette signalisation et provoquer le SLE chez l’humain. Ainsi, DcR3 peut simultanément interférer avec la signalisation des ligands LIGHT et TL1A et causer un phénotype plus complexe que les phénotypes résultant de la mutation de Fas ou de FasL chez certains patients. DcR3 peut également être utilisé comme paramètre diagnostique potentiel pour le SLE. Les découvertes du mécanisme de protection des îlots de Langerhans par DcR3 ouvrent la porte vers de nouveaux horizons afin d'explorer de nouvelles cibles thérapeutiques pour protéger la greffe d'îlots. / Decoy receptor 3 (DcR3) is a member of the tumor necrosis factor (TNF) receptor family, and is widely expressed in human normal tissues and malignant tumors. It is a decoy receptor of three TNF family members, i.e., FasL, LIGHT and TL1A. The interaction of DcR3 and its ligands will not transmit signal into cells via DcR3 because DcR3 is a soluble protein without a transmembrane and intracellular segment. Thereby, DcR3 competitively inhibits signaling through three functional receptors, i.e., Fas, HVEM/LTbetaR and DR3. In previous studies, we found that DcR3 could modulate immune cell function, and protect islet viability. Herein, we generated DcR3 transgenic (Tg) mice driven by the human β-actin promoter to further investigate the function of DcR3. Interestingly, the DcR3 Tg mice developed a lupus-like syndrome at 6 months of age. They presented a variety of autoantibodies including anti-nucleus and anti-dsDNA antibodies. They also manifested renal, dermal, hepatic and hematopoietic lesions. Compared to lpr and gld mouse lupus models, DcR3 Tg mice more closely resembled human SLE in terms of Th2-biased immune response and anti-Sm antibody production. Furthermore, we found that DcR3-producing hematopoietic cell were sufficient to cause these pathological changes. Mechanistically, DcR3 may break T-cell homeostasis to interfere with peripheral tolerance, and then induce autoimmunity. In humans, we detected high DcR3 levels in SLE patient sera. The high DcR3 levels were related to elevated IgE titer in some SLE patients, as was the case in the mouse model. Therefore, DcR3 may represent an important pathogenetic factor of human SLE. Utilizing the DcR3 Tg mouse, we further elucidated the mechanism by which DcR3 protected islets from primary nonfunction (PNF). Blocking of LIGHT and TL1A signaling by DcR3 are involved in such protection. Moreover, by mRNA microarray we identified possible downstream molecules, which may mediate such protection. We confirmed that Adcyap1 and Bank1 played critical roles in mediating DcR3’s effect in islet protection. Our studies resolved a puzzle about the relationship between the Fas/FasL apoptosis signaling pathway and the pathogenesis of human SLE. DcR3 can block Fas/FasL pathway even if there is no genetic mutation in Fas and FasL. DcR3 can simultaneously interfere with LIGHT and TL1A signaling to cause a more complex phenotype than the simple Fas or FasL mutation in patients. DcR3 can also be employed as a potential diagnostic parameter for SLE. The discovery of the mechanism of DcR3 in protecting islets allows us to explore novel therapeutic targets to protect islet graft.

L’acide mycophénolique (MPA) est un immunosuppresseur très prometteur dans le traitement des maladies auto-immunes (MAI) telles que le lupus érythémateux disséminé (LED) et les vascularites à ANCA, et disponible sous deux formes pharmaceutiques : le mycophénolate mofétil (MMF) et le mycophénolate sodique (EC-MPS). Les études menées chez les patients transplantés recommandent le dosage plasmatique et le suivi pharmacocinétique (PK) du MPA, dans un objectif d’optimisation thérapeutique. A ce jour, ce suivi est encore inexistant dans les MAI, et les données de corrélation concentrations-efficacité thérapeutique, sur lesquelles se base l’optimisation, demeurent toujours rares dans ce domaine. Les travaux présentés dans cette thèse s’inscrivent dans l’étude des corrélations PK/pharmacodynamie (PD) du MPA dans les MAI. Ces travaux ont permis de proposer des schémas et des outils d’optimisation des traitements à base de MPA pour ces patients. Pour cela, les concentrations plasmatiques du MPA et de son métabolite 7-O-glucuronide (MPAG) ont été déterminées pour 53 patients présentant de manifestations extra-rénales de MAI à l’aide d’une méthode de chromatographie couplée à la spectrométrie de masse. Les paramètres PK ont été estimés pour MMF et EC-MPS dans les deux groupes de MAI. D’après ces travaux, l’optimisation du MMF chez les patients atteints de MAI peut reposer sur le suivi de la concentration à 12 h (C12) en MPA. Un seuil de 3 mg/L est proposé afin de maintenir la rémission dans le LED, mais reste à définir dans les vascularites. Pour EC-MPS, une stratégie de prélèvements limités basée sur la mesure de la concentration maximale et la C12 est nécessaire pour estimer l’aire sous la courbe des concentrations entre 0 et 12 h du MPA. / Mycophenolic acid (MPA), the active form of both mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), is an immunosuppressant increasingly used in the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE) and ANCA-associated vasculitis. In transplant recipients, therapeutic drug monitoring (TDM) of MPA is widely used to prevent acute organ rejection. However, MPA TDM is currently not available in autoimmune diseases, as data on the pharmacokinetic (PK)/pharmacodynamic (PD) relationships are very sparse in this indication. Our aim was to study the possible PK/PD relationships of MPA in patients with non-renal manifestations of SLE or ANCA-associated vasculitis. An assay based on liquid chromatography coupled with mass spectrometry was applied to the PK study of MPA and its major glucuronide metabolite (MPAG) in 53 SLE and vasculitis patients receiving either MMF or EC-MPS. According to our results, in SLE patients with non-renal manifestations, TDM based on the measurement of MPA 12-h trough concentration (C12) would allow optimizing therapies with MMF. A 3-mg/L efficacy threshold could be proposed to prevent clinical flares under MMF maintenance therapy. For EC-MPS, a limited sampling strategy including MPA maximum concentration and C12 is necessary to estimate the area under the curve between 0 and 12-h of MPA.

Introdução: A ativação do eixo hormônio liberador de corticotropina (CRH) e da pró-opiomelanocortina (POMC) leva a produção de vários derivados bioativos que incluem o hormônio adrenocorticotrófico (ACTH) e o hormônio estimulador de melanócito alfa (alfa-MSH). Estudos avaliando a participação desse eixo no lúpus eritematoso sistêmico (LES) são escassos, particularmente no envolvimento cutâneo da doença. Objetivo: Avaliar a participação do CRH e das melanocortinas (MCs) na fisiopatologia do lúpus eritematoso sistêmico com envolvimento cutâneo. Métodos: Dezessete pacientes com LES com envolvimento cutâneo foram avaliados clinicamente e biópsias da pele afetada e não afetada e do sangue periférico foram obtidas. Dezessete indivíduos saudáveis foram pareados por idade e gênero. Os fragmentos de pele foram submetidos à análise imuno-histoquímica para avaliação da expressão de CRH, ACTH, alfaMSH, e receptor de melanocortina tipo 1 (MC-1R). Os níveis séricos de alfa-MSH, IL-1, IL-1ra, IL-6, IL-10, IL-12p70, IL-17, TNF-alfa, e IFN-y foram determinados pelo método Multiplex. Resultados: A pele afetada de pacientes com LES apresentaram maior expressão CRH na derme profunda quando comparada à pele não afetada dos mesmos doentes e a pele saudável dos controles (p = 0,024). Níveis séricos de alfa-MSH foram similares entre LES e controles. Dentre as citocinas avaliadas, IFN-y, TNF-alfa e IL-6 foram mais elevadas nos pacientes com LES em relação aos controles (p = 0,041, p = 0,001 e p = 0,049, respectivamente). Embora não significativamente, os níveis de IL-17 também foram mais altos nos pacientes (p = 0,099). A expressão tecidual de ACTH, cortisol, alfa-MSH e seu receptor MC-1R foram semelhantes entre os pacientes e controles. Conclusões: Nossos resultados mostram, pela primeira vez a participação do eixo CRH-POMC na patogênese das lesões cutâneas do LES / Introduction: Corticotropin-releasing hormone (CRH) and pro-opiomelanocortin (POMC) axis activation leads to the production of several bioactive hormones including adrenocorticotrophic hormone (ACTH) and the neuropeptide alfa-melanocyte stimulating hormone (alfa-MSH). There are scarce data regarding their role in systemic lupus erythematosus (SLE) particularly in cutaneous involvement of this disease. Objective: To evaluate the role of CRH and melanocortins (MCs) in the pathophysiology of systemic lupus erythematosus with skin involvement. Methods: Seventeen patients with SLE with skin involvement were evaluated clinically and biopsies of affected and unaffected skin and peripheral blood were obtained. Seventeen healthy subjects were matched for age and gender. The skin fragments were subjected to immunohistochemical analysis for the expression of CRH, ACTH, alfa-MSH and melanocortin receptor type 1 (MC-1R). Serum levels of alfa-MSH, IL-1, IL-1ra, IL-6, IL-10, IL-12p70, IL-17, TNF-alfa and IFN-y were determined by multiplex. Results: The affected skin of SLE patients exhibited greater CRH expression in the deep dermis compared to unaffected skin of the same patients and the control\'s healthy skin (p = 0.024). alfa-MSH were similar between SLE and controls. Among the evaluated cytokines, IFN-y, TNF-alfa and IL-6 were significantly higher in SLE patients compared to controls (p = 0.041, p = 0.001 and p = 0.049, respectively). Although not significant, levels of IL-17 were also higher in patients (p = 0.099). Tissue expression of ACTH, cortisol, alfa-MSH and its receptor MC-1R were similar between patients and controls. Conclusions: Our results show for the first time the involvement of CRH-POMC axis in the pathogenesis of SLE cutaneous lesions through interactions between the brain-skin axis

Introdução: O lúpus eritematoso sistêmico (LES) é uma doença crônica autoimune com predomínio no sexo feminino e com evidente impacto em sua fertilidade. Por sua vez, em homens com LES foi observado alterações nos parâmetros seminais e nos níveis de hormônios sexuais. A análise seminal somente apresenta baixa correlação com potencial de fertilidade dos pacientes. Recentemente, a análise da integridade do DNA do espermatozoide tem mostrado melhor capacidade prognóstica para predizer a fertilidade do que os parâmetros seminais convencionais. Objetivo: Avaliar a fragmentação do DNA espermático de homens com LES sem azoospermia. Métodos: Vinte e oito pacientes homens, consecutivos, com LES (pelos critérios da ACR) e 34 controles foram avaliados conforme dados demográficos e de exposição ambiental, avaliação urológica, perfil hormonal e avaliação seminal (incluindo a fragmentação do DNA espermático). Aspectos clínicos, escores de atividade e dano cumulativo da doença e aspectos do tratamento também foram analisados. Resultados: Mediana da idade [33(20-52) vs. 36.5(25-54) anos, p=0.329] e frequência de varicocele (25% vs. 32%, p=0.183) foram similares entre o grupo de pacientes e o grupo controle. Na análise da fragmentação do DNA do espermatozoide observou-se quantidades significativamente mais altas de células classe III [44(9-88) vs. 16.5(0-80)%,p=0.001] e células classe IV [10.5(3-86) vs. 7(0-36)%,p=0.039] no grupo com LES. O índice de fragmentação do DNA espermático também foi significativamente mais alto em pacientes com LES [62(31-97) vs. 25.5(0-100)%, p < 0.001]. Parâmetros seminais convencionais (incluindo contagem espermática, motilidade e morfologia) foram similares em ambos os grupos. Dentro de grupo de pacientes com LES não foi observada correlação entre o índice de fragmentação do DNA espermático com idade, duração da doença, SLEDAI-2K e SLICC/ACR-DI ou dose cumulativa de predinisona, hidroxicloroquina, metotrexato, azatioprina, micofenolato mofetil ou ciclofosfamida intravenosa (CIC) (p > 0.05). Análises adicionais evidenciaram que motilidade espermática total foi significativamente menor no grupo que fez uso de CIC [64%(15-83) vs. 72%(57-86)%, p=0.024]. O índice de fragmentação do DNA espermático foi semelhante nos dois grupos [52.5(31-95) vs. 67.5(34-97)%, p=0.185]. Conclusões: Homens com SLE sem azoospermia apresentam maior índice de fragmentação do DNA espermático sem alteração dos parâmetros seminais ou hormonal. CIC não parece ter papel significativo nesta alteração. Estudos prospectivos futuros são necessários para determinar o impacto desta alteração na fertilidade destes pacientes / Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a female predominance and have clear impact on fertility. In male SLE has been shown to alter seminal parameters and sexual hormonal levels. Recently, sperm DNA integrity analysis has shown better prognostic performance to predicting male fertility than seminal parameters. Objective: To evaluate sperm DNA fragmentation in non-azoospermic male SLE patients. Methods: Twenty-eight consecutive male SLE patients (ACR criteria) and 34 healthy controls were evaluated for demographic/exposures data, urologic evaluation, hormone profile and seminal analysis (including sperm DNA fragmentation). Clinical features, disease activity/damage scores and treatment were also assessed. Results: Median age [33(20-52) vs. 36.5(25-54) years, p=0.329] and frequency of varicocele (25% vs. 32%, p=0.183) were similar in SLE patients and healthy controls. Sperm DNA fragmentation showed significantly higher levels of cells class III [44(9-88) vs. 16.5(0-80)%, p=0.001] and cell class IV [10.5(3-86) vs. 7(0-36)%,p=0.039] in SLE. Sperm DNA fragmentation Index was also significantly higher in SLE patients [62(31-97) vs. 25.5(0-100)%, p < 0.001]. Conventional sperm parameters (including sperm count, motility and morphology) were similar in both groups. In SLE patients no correlations were observed between sperm DNA fragmentation index with age, disease duration, SLEDAI-2K and SLICC/ACR-DI scores, and cumulative dose of prednisone, hydroxychloroquine, intravenous cyclophosphamide (IVCYC), methotrexate, azathioprine and mycophenolate mofetil (p > 0.05). Further analysis of SLE patients treated with and without IVCYC showed that total sperm motility was significantly lower in the former group [64%(15-83) vs. 72%(57-86)%, p=0.024]. Sperm DNA fragmentation index was alike in both groups [52.5(31-95) vs. 67.5(34-97)%, p=0.185]. Conclusions: To our knowledge, this is the first demonstration that male non-azoospermic SLE patients have increased sperm DNA fragmentation without evident gonadal dysfunction. IVCYC does not seem to be a major determinant for this abnormality. Future prospective study is necessary to determine the impact of this alteration in these patients\' fertility

Objetivos: Determinar a incidência de perda de massa óssea em um ano em pacientes com lúpus na pré-menopausa e o valor preditor dos marcadores do metabolismo ósseo para essa complicação. Métodos: Sessenta e três pacientes foram avaliadas à entrada no estudo e após um ano de seguimento. Variações na densidade mineral óssea (DXA) acima da mínima variação significativa (MVS) foram consideradas significativas, como recomendado pela Sociedade Internacional de Densitometria Clínica (International Society for Clinical Densitometry). Os níveis séricos dos marcadores do metabolismo ósseo foram determinados no início do estudo: propeptídeo N-terminal do pro-colágeno tipo 1 (P1NP) e telopeptídeo C-terminal do colágeno tipo 1 (CTX) por eletroquimioluminescência; osteoprotegerina (OPG) e ligante do receptor ativador do fator nuclear kB (RANKL) por ELISA. Resultados: 36,5% dos pacientes apresentaram perda de massa óssea e 17,5% ganho de massa óssea na coluna lombar e/ou fêmur. Os pacientes foram divididos em três grupos: perda de massa óssea (P), massa óssea estável (E) e ganho de massa óssea (G). Pacientes com P e E tomaram doses cumulativa, média e máxima de glicocorticoide semelhantes durante o estudo, mas pacientes com G receberam doses menores (G vs. P e G vs. E, p < 0,05). Os níveis séricos basais de P1NP foram diferentes nos três grupos (P: 36,95 ± 23,37 vs. E: 54,63 ± 30,82 vs. G: 84,09 ± 43,85 ng/ml, p=0,001). Análises de múltiplas comparações demonstraram diferenças significativas nos níveis de P1NP entre P vs. E, p=0,031; P vs. G, p < 0,001 e E vs. G, p=0,039. Não houve diferença entre os grupos com relação aos níveis de CTX, OPG/RANKL, fatores de risco para osteoporose ou parâmetros relacionados à doença. Após análise multivariada, apenas níveis baixos de P1NP permaneceram como fator de risco independente para perda de massa óssea (p < 0,013). Conclusão: Este estudo fornece evidência original que níveis mais baixos de P1NP, o marcador de formação óssea mais específico, são preditores de perda de massa óssea em um ano em mulheres com lúpus na pré-menopausa / Objective: To determine the one-year incidence of bone loss in premenopausal lupus patients and the value of bone markers as predictors of this complication. Methods: Sixty-three premenopausal SLE patients were evaluated at baseline and after one-year of follow-up. Bone mineral density changes (DXA) above the least significant change (LSC) were considered significant, as recommended by International Society for Clinical Densitometry. Serum levels of bone markers were determined at baseline: N-terminal propeptide of type 1 collagen (P1NP) and C-terminal telopeptide of type 1 collagen (CTX) by electrochemiluminescence; osteoprotegerin (OPG) and receptor activator of nuclear factor kB ligand (RANKL) by ELISA. Results: 36.5% of patients presented bone loss and 17.5% bone gain at lumbar spine and/or femur. Patients were divided in three groups: bone mass loss (BL), no bone mass change (NC) and bone mass gain (BG). Patients with BL e NC took similar cumulative, mean and maximum GC doses during the study, but patients with BG took lower doses (BG vs. BL and BG vs. NC, p < 0.05). Baseline P1NP levels were different in the three groups (BL: 36.95 ± 23.37 vs. NC: 54.63 ± 30.82 vs. BG: 84.09 ± 43.85 ng/ml, p=0.001). Further multiple comparison analysis demonstrated significant differences in P1NP between BL vs. NC, p=0.031; BL vs. BG, p < 0.001 and NC vs. BG, p=0.039. No difference was observed concerning the levels of CTX, OPG/RANKL, risk factors for osteoporosis or disease related parameters. After multivariate analysis only lower P1NP levels remained as an independent risk factor for bone loss (p < 0.013). Conclusion: This study provides original evidence that lower levels of P1NP, the most specific bone formation marker, are predictive of bone loss in the next year in premenopausal SLE patients

INTRODUÇÃO: Estudos indicam uma prevalência de 20 a 30% de hiperprolactinemia discreta em pacientes com lúpus eritematoso sistêmico (LES), sugerindo um possível papel da prolactina (PRL) na sua etiopatogenia. Como a expressão do gene da PRL é encontrada na maioria das células do sistema imunológico, onde atua como citocina, de forma parácrina e autócrina, a origem linfocitária desta PRL tem sido aventada. OBJETIVOS: estudar a expressão do gene da PRL linfocitária de pacientes com LES em atividade e inatividade de doença e de controles normais, e sua atividade biológica em bioensaios com células Nb2 e Ba/F-LLP; Determinar o nível sérico de PRL e a prevalência de macroprolactinemia numa população nossa com LES. MÉTODOS: grupo 1, composto de 73 pacientes (66 mulheres e 7 homens), sendo 28 pacientes com LES em atividade e 45 em inatividade de doença, onde foi avaliado o nível de PRL sérica e a prevalência da macroprolactinemia; grupo 2, derivado do grupo 1, com 30 pacientes: 18 com LES em atividade e 12 em inatividade e um grupo controle com 10 indivíduos normais, dos quais foram extraídos linfócitos do sangue periférico e colocados em cultura por 72 horas. Em seguida, o sobrenadante da cultura foi utilizado como amostra de PRL linfocitária em ensaios com células Nb2 (heterólogo) e Ba/F-LLP (homólogo) para avaliação da bioatividade. Os RNAs totais destes linfócitos foram extraídos e usados na RT-PCR em tempo real (método quantitativo), para comparar a expressão do gene da PRL em linfócitos de pacientes com LES em atividade e inatividade, utilizando pool de indivíduos normais como calibrador. RESULTADOS: hiperprolactinemia discreta foi encontrada em 21,9% (16 de 73 pacientes do grupo 1): 7 de 28 pacientes com LES em atividade (25%), e 9 de 45 em inatividade (20%). A presença de macroprolactinemia foi encontrada em 3 pacientes, todos com LES em inatividade. O nível de PRL sérica: grupo 1 (LES em atividade) teve mediana de 10,8 (4,9 38,9) ng/mL e o grupo 1 (LES em inatividade) mediana de 7,6 (1,9 49,6) ng/mL, não havendo diferença significante entre os dois subgrupos (p=0,123). No entanto, quando consideramos apenas o nível sérico da PRL monomérica, a mediana da PRL do grupo 1 (LES em inatividade) caiu para 7,3 ng/mL (1,9 20,6) ng/mL e assim, quando comparado novamente ao grupo 1 (LES em atividade), observamos que além de uma porcentagem maior dos casos em atividade apresentarem hiperprolactinemia, a mediana da PRL monomérica nesses pacientes é significantemente maior que nos pacientes em inatividade de doença (p= 0,016). Os bioensaios foram realizados com as amostras do grupo 2 (subgrupo do grupo 1): no ensaio com células Nb2, a bioatividade da PRL linfocitária foi semelhante, não havendo diferença significante entre os pacientes com LES em atividade e inatividade e desses com o grupo controle. Já o bioensaio com a Ba/F-LLP não mostrou sensibilidade adequada, portanto não sendo confiável para avaliação da PRL linfocitária. O RT-PCR em tempo real apresentou expressão gênica também semelhante entre os pacientes avaliados (grupo 2). CONCLUSÕES: A expressão do gene da PRL linfocitária e a sua bioatividade foram semelhantes nos pacientes com LES em atividade e inatividade. A prevalência de hiperprolactinemia nos nossos pacientes com LES foi de 21,9%, sendo maior nos pacientes com atividade de doença. A macroprolactinemia só foi encontrada em pacientes com LES inativo, sugerindo um possível efeito protetor deste achado. / INTRODUCTION: Studies point to a prevalence of 20-30% of discrete hyperprolactinemia in patients with systemic lupus erythematosus (SLE), suggesting a possible implication of prolactin (PRL) in the pathogenesis of this disorder. As the lymphocytic PRL gene expression is found in the majority of the immune cells, where it acts as citokine, by paracrine and autocrine regulation, the lymphocytic source of this PRL has been suggested. OBJECTIVES: 1) to study the lymphocytic PRL gene expression of patients with active and inactive SLE and normal controls, as well as its biological activity in bioassays using Nb2 (heterologous) and Ba/F-LLP cells (homologous); 2) To assess serum PRL level and the prevalence of macroprolactinemia in our population with SLE. METHODS: group 1, composed of 73 patients (66 women and 7 men), 28 patients with active and 45 with inactive SLE, where the serum PRL level and prevalence of macroprolactinemia were evaluated; group 2, a subset of group 1, with 30 patients: 18 with active and 12 with inactive SLE and 10 normal individuals as control group, from whom lymphocytes were extracted from peripheral blood and were set on culture for 72 hours. After that, the supernatant was taken as lymphocytic PRL samples in bioassays with Nb2 cells (heterologous) and Ba/FLLP (homologous) in order to assess its bioactivity. Total RNA from these lymphocytes was extracted and a comparison was made between lymphocytic PRL gene expression of patients with active and inactive SLE by real time RT-PCR, using normal pool as calibrator. RESULTS: mild hyperprolactinemia was found in 21.9% (16 of 73 patients of group 1), 7 of 28 patients in activity (25%), and 9 of 45 in inactivity (20%). Macroprolactinemia was found in 3 patients, all with inactive SLE. Regarding serum PRL levels group 1 (active SLE) had median of 10.8 (4.9 38.9) ng/mL and the group 1 (inactive SLE) median of 7.6 (1.9 49.6) ng/mL, without significant difference between the two sub-groups (p=0.123). However, when only monomeric PRL level was considered, the median of group 1 (inactive SLE) dropped to 7.3 ng/mL (1.9 20.6) ng/mL and, when compared again with group 1 (active SLE), we observed that beyond a bigger percentage of the cases in activity to present hyperprolactinemia, the medium of serum PRL level in patients with active SLE is significantly greater of that in the ones in inactivity (p= 0.016). The bioassays with the samples of group 2 (sub-group of group 1): The assays with Nb2 cells showed similar lymphocytic PRL bioactivity. They did not have significant difference between patients with SLE active and inactive and these with normal control group. The assays with Ba/F-LLP cells did not show adequate sensitivity, so not trustworthy for lymphocytic PRL evaluation. The real time RT-PCR also presented similar gene expression between patients (group 2). CONCLUSIONS: The gene lymphocytic PRL expression and its bioactivity were similar in patients with SLE in activity and inactivity of illness and the normal controls. The prevalence of hyperprolactinemia in our population of patients with SLE was of 21.9%, being greater in patients with active SLE. The macroprolactinemia was found only in patients with inactive SLE, suggesting a possible protective effect of this finding.

INTRODUÇÃO: A periodontite é uma doença infecciosa associada à inflamação crônica dos tecidos do dente. Os mediadores inflamatórios e citocinas podem influenciar o curso de doenças reumáticas. O objetivo deste trabalho foi verificar se o tratamento da doença periodontal (DP) possue correlação com a atividade do lúpus eritematoso sistêmico (LES). MÉTODOS: Foram avaliados 42 pacientes com LES (ACR, 1997) e consecutivamente randomizados em dois grupos. Os critérios de exclusão foram SLEDAI < 2 e/ou Índice de Sangramento Sulcular (ISS) igual a zero, obtendo-se o grupo TRATADO com 19 pacientes e o CONTROLE com 17 pacientes. Todos os pacientes estavam sob tratamento com ciclofosfamida endovenosa. Estes grupos foram pareados para idade, sexo, e raça. Foram determinados a velocidade de hemossedimentação (VHS), a proteína C reativa (PCR) e o SLEDAI. A graduação da doença periodontal foi aferida através da profundidade da bolsa periodontal (PB), do ISS e do nível de inserção (NI). O tratamento odontológico objetivou a desinfecção oral completa. O grupo TRATADO iniciou o tratamento odontológico imediatamente após a visita inicial e foram reavaliados após três meses. O grupo CONTROLE iniciou o tratamento odontológico após três meses de observação. Assim, todos os pacientes com SLEDAI ? 2 e/ou ISS ? zero, passaram a pertencer ao grupo TRATADO (n=32). Esses pacientes foram reavaliados após três meses do término do tratamento odontológico. RESULTADOS: Os grupos foram similares na visita inicial para o ISS (40,75 ± 30,98 vs. 40,72 ± 36,19%, p=0,89), PB (1,73 ± 1,80 vs. 1,48 ± 0,59mm, p=0,80) e NI (2,47 ± 1,9 vs. 1,91 ± 1,34mm, p=0,18) e para a VHS (20,69 ± 23,88 vs. 23,41 ± 21,92 mm/h, p=0,80), a PCR (4,7 ± 4,61 vs. 4,21 ± 5,86mg/dl, p=0,34) e o SLEDAI (5,94 ± 4,24 vs. 6,29 ± 4,35, p=0,73). A eficácia do tratamento odontológico foi atestada pela redução dos índices periodontais no grupo TRATADO: ISS (40,75 ± 30,98 vs. 15,19 ± 17,22%, p<0,01), PB (1,73 ± 1,80 vs. 1,10 ± 0,29 mm, p<0,01) e NI (2,47 ± 1,96 vs. 1,68 ± 0,90 mm, p<0,01). Os níveis de VHS e PCR não se alteraram ao longo do tratamento para os grupos. O grupo TRATADO apresentou redução do SLEDAI (5,94 ± 4,24 vs. 3,38 ± 3,30, p=0,04). O grupo CONTROLE não alterou os parâmetros odontológicos e nem clínicos nos 3 meses em observação. CONCLUSÃO: O tratamento odontológico propiciou a melhora da doença periodontal nos pacientes com lúpus eritematoso sistêmico, associada a uma redução significativa na atividade da doença aferida pelo SLEDAI. / INTRODUCTION: Periodontal disease is an infectious disease associated to a chronic inflammation of dental tissues. Inflammatory mediators and cytokines determine the course of rheumatic diseases. The aim of this study is to evaluate if periodontal disease (PD) treatment influences systemic lupus erythematosus (SLE) activity. METHODS: Forty-two SLE patients (ACR, 1997) were evaluated and consecutively randomized in two groups. Exclusion criteria were SLEDAI < 2 and/or bleeding gingival index (BGI) of zero, resulting in TREATED group with 19 patients and CONTROL with 17 patients. All SLE patients were under IV cyclophosphamide therapy. Both groups were matched for age, gender, and race. Erythrocyte sedimentation rate (ESR), C- reactive protein (CRP) and SLEDAI were determined. Periodontal disease graduation was defined according to probing depth (PD), bleeding gingival index (BGI) and probing attachment level (PAL). Odontological treatment focused a complete mouth desinfection. TREATED group have odontologial treatment immediately at entry and evaluated after three months. CONTROL started odontological treatment after 3 months of observation period. At the end, all SLE patients with SLEDAI ³ 2 and/or BGI > zero, entered in the TREATED group (n=32). These patients were evaluated 3 months after the end of odontological treatment. RESULTS: At entry, both groups had similar BGI (40.75 ± 30.98 vs. 40.72 ± 36.19%, p=0.89), PD (1.73 ± 1.80 vs. 1.48 ± 0.59mm, p=0.80), and PAL (2.47 ± 1.9 vs. 1.91 ± 1.34mm, p=0.18) and also for ESR (20.69 ± 23.88 vs. 23.41 ± 21.92 mm/h, p=0.80), CRP (4.7 ± 4.61 vs. 4.21 ± 5.86mg/dl, p=0.34) and SLEDAI (5.94 ± 4.24 vs. 6.29 ± 4.35, p=0.73). Efficacy of odontological treatment was identified in the reduction of all indexes of TREATED group: BGI (40.75 ± 30.98 vs. 15.19 ± 17.22%, p<0.01), PD (1.73 ± 1.80 vs. 1.10 ± 0.29 mm, p<0.01) and PAL (2.47 ± 1.96 vs. 1.68 ± 0.90 mm, p<0.01). ESR and CRP did not alter during treatment in both groups. TREATED group had a significant reduction in SLEDAI (5.94 ± 4.24 vs. 3.38 ± 3.30, p=0.04) after odontological treatment whereas CONTROL had similar odontological and clinical parameters during the 3 months of observation. CONCLUSION: SLE patients after odontological treatment had a significant reduction of SLEDAI with a better control of the inflammation of the disease.

INTRODUÇÃO. A síndrome antifiosfolípide (SAF) é uma condição trombofílica autoimune associada a títulos elevados e persistentes de anticorpos antifosfolípides. Caracteriza-se por tromboses em diversos órgãos, incluindo os testículos. OBJETIVO. Realizar uma avaliação global da função gonadal em pacientes masculinos com SAF primária (SAFP) e SAF associada ao lúpus eritematoso sistêmico (SAF-LES). MÉTODOS. Estudo transversal realizado em 22 pacientes (12 com SAFP e 10 com SAF-LES) e 20 controles saudáveis pareados por sexo e idade. Os pacientes foram avaliados em relação a dados demográficos, exame urológico, ultrassonografia testicular, perfil hormonal, análise do sêmen, anticorpos antiespermatozóides e características clínicas e laboratoriais. RESULTADOS. A mediana da idade atual foi semelhante nos pacientes com SAFP e controles (p=0,27), assim como naqueles com SAF-LES e controles (p=0,31). Disfunção erétil foi significantemente maior nos pacientes com SAFP comparado aos controles (25% vs. 0%, p=0,044), e nos SAF-LES comparado aos controles (30% vs. 0%, p=0,029). Com relação à antropometria do pênis, a análise dos subgrupos de pacientes com (n=7) e sem (n=5) tromboses arteriais prévias demonstrou que a mediana da circunferência do pênis foi significantemente menor em SAFP com trombose arterial versus sem trombose arterial [8,1 (6-10) vs. 10,2 (10-11) cm, p=0,007], bem como também observado em pacientes com SAF-LES com (n=2) e sem (n=8) eventos arteriais prévios [7,5 (7-8) vs. 9,18 (8-10,5) cm, p=0,039]. A mediana da circunferência do pênis foi significantemente menor nos pacientes com SAFP com disfunção erétil versus sem essa alteração [7,5 (6-9,5) vs. 9,5 (7,5-11) cm, p=0,039], assim como no grupo de SAF-LES [8,17 (8-8,5) vs. 9,14 (7-10,5) cm, p=0,0397]. Com relação à avaliação da função testicular, todos os parâmetros foram semelhantes nos pacientes com SAFP e controles (p>0,05). Por sua vez, as medianas de concentração e de mobilidade dos espermatozóides foram significantemente menores nos pacientes com SAF-LES comparado aos controles [41,1 (0-145) vs. 120,06 (34,5-329) x 106/mL, p=0,003; 47,25 (0-87,5) vs. 65,42 (43-82)%, p=0,047; respectivamente], assim como a frequência de oligo/azoopermia (40% vs. 0%, p=0,007). A análise dos pacientes com SAF-LES mostrou que as medianas da concentração e da contagem total de espermatozóides foram significantemente menores nos que usaram ciclofosfamida endovenosa versus os que não usaram tal medicação [6,87 (0-23,5) vs. 63,9 (7,5-145) x 106/mL, p=0,04; 16,12 (0-55,5) vs. 226,25 (8,5-471) x 106, p=0,035; respectivamente]. CONCLUSÕES. Diminuição do tamanho peniano nos pacientes com SAFP e SAF-LES com disfunção erétil associada foi evidenciada, além de disfunção testicular secundária ao uso de agentes alquilantes nos pacientes com SAF-LES / INTRODUCTION. Antiphospholipid syndrome (APS) is an autoimmune thrombophilic condition associated with persistent high titers of antiphospholipid antibodies. It is characterized by thrombosis in various organs including the testes. OBJECTIVE. To perform a global testicular assessment in male primary antiphospholipid syndrome (PAPS) and secondary systemic lupus erythematosus-APS (SLE-APS) patients, and healthy controls. METHODS. A cross-sectional study was conducted in 22 APS (12 PAPS and 10 SLE-APS) male patients, and 20 healthy controls. They were assessed by demographic data, systematic urological examination, testicular ultrasound, hormone profile, sperm analysis, antisperm antibodies, clinical features and treatment. RESULTS. The median of current age was similar in PAPS patients and controls (p=0.27), likewise in SLE-APS and controls (p=0.31). Erectile dysfunction was significantly higher in PAPS patients compared than controls (25% vs. 0%, p=0.044), and in SLE-APS and controls (30% vs. 0%, p=0.029). Regarding the penile anthropometry, the analysis of subgroups of PAPS patients with (n=7) and without (n=5) previous arterial thrombosis demonstrated that the median circumference penis was significantly lower in PAPS with arterial thrombosis versus without [8.1 (6-10) vs. 10.2 (10-11) cm, p=0.007], as also observed in SLE-APS patients with (n=2) and without (n=8) previous arterial events [7.5 (7-8) vs. 9.18 (8-10.5) cm, p=0.039]. In addition, the median penis circumference was significantly lower in PAPS patients with erectile dysfunction versus without this alteration [7.5 (6-9.5) vs. 9.5 (7.5-11) cm, p=0.039], likewise in SLE-APS patients [8.17 (8-8.5) vs. 9.14 (7-10.5) cm, p=0.0397]. Regarding gonadal evaluation, these parameters were uniformly normal in PAPS versus controls (p>0.05). In contrast, the median of sperm concentration and sperm motility were significantly lower in SLE-APS patients compared to controls [41.1 (0-145) vs. 120.06 (34.5-329) x 106/mL, p=0.003; 47.25 (0-87.5) vs. 65.42 (43-82)%, p=0.047; respectively], likewise the frequency of oligo/azoopermia (40% vs. 0%, p=0.007).The analysis of SLE-APS patients showed that the median of sperm concentration and total sperm count were significantly lower in SLE-APS patients treated with intravenous cyclophosphamide versus untreated [6.87 (0-23.5) vs. 63.9 (7.5-145) x 106/mL, p=0.04; 16.12 (0-55.5) vs. 226.25 (8.5-471) x 106, p=0.035; respectively]. CONCLUSIONS. We have identified reduced penile size in PAPS and SLE-APS patients with deleterious erectile function, and testicular dysfunction due to alkylating agents in SLE-APS patients