Introdução: O envolvimento hepático na doença celíaca (DC) é amplamente reconhecido e atualmente é uma das manifestações extra-intestinais mais freqüentes. Com o advento de marcadores sorológicos de elevada especificidade e sensibilidade, sobretudo o anticorpo antiendomísio (EMA), a DC tem sido descrita em associação a várias hepatopatias. Objetivos: caracterizar as formas de triagem de DC em portadores de hepatopatia crônica; caracterizar e estudar os pacientes cujo diagnóstico de DC foi realizado durante a investigação de uma doença hepática; pesquisar a reatividade do antiendomísio em pacientes com hepatite auto-imune, cirrose biliar primária, colangite esclerosante primária e hipertensão portal não cirrótica; avaliar o comportamento da doença hepática na vigência de dieta sem glúten. Métodos: Os pacientes foram triados pela detecção dos anticorpos anti-reticulina e anticorpo antimatriz extracelular durante a rotina de imunofluorescência de pesquisa dos auto-anticorpos hepáticos; pela presença de manifestações de DC em hepatopatas crônicos, pelo aspecto endoscópico sugestivo de DC e pela pesquisa sistemática do EMA nas patologias referidas anteriormente. Todos os pacientes foram submetidos à pesquisa do EMA, anti-reticulina IgG ou antimatriz de fibroblastos IgG na presença de deficiência de IgA. Em caso de positividade desses marcadores, foram submetidos à endoscopia digestiva alta para biópsia intestinal e caracterizados do ponto de vista clínico, laboratorial e histopatológico. A evolução desses dados permitiu a caracterização da evolução da doença hepática e da doença celíaca a partir da introdução da dieta sem glúten. Resultados: Foram identificados 43 pacientes com auto-anticorpos relacionados à DC (em 42 o EMA IgA e em um o antimatriz extracelular IgG em associação com deficiência de IgA). A rotina de pesquisa de auto-anticorpos hepáticos identificou 31 pacientes; seis apresentavam hepatopatia crônica e manifestação de DC; em três o exame endoscópico foi sugestivo de DC e a pesquisa sistemática do EMA foi positiva em três casos. O diagnóstico de DC foi confirmado em 37 de 40 pacientes (92,5%) em que a biópsia intestinal foi realizada. A idade dos pacientes variou de 2 a 68 anos, com mediana de 35 anos. Houve maior prevalência de acometimento no sexo feminino (65%). A DC foi mais prevalente na raça branca (87%), mais foi identificada em quatro mulatos e um negro. As doenças hepáticas mais freqüentes foram hipertransaminasemia criptogênica, hepatite auto-imune, hiperplasia nodular regenerativa e hepatite pelo vírus C. Conclusões: 1) A reatividade do anti-reticulina, a presença de diarréia inexplicada e a análise endoscópica da mucosa duodenal foram as formas de seleção mais efetivas de se identificar a DC em hepatopatas crônicos. 2) A ausência de manifestações clínicas de DC nesse grupo de pacientes foi bastante expressiva. 3) A pesquisa sistemática do EMA em cirrose biliar primaria, hepatite auto-imune, colangite esclerosante primária não contribuiu para o diagnóstico de DC em um número significativo de pacientes, ao contrário do observado no grupo de hipertensão portal não cirrótica, especialmente hiperplasia nodular regenerativa 4) As doenças hepáticas em que mais freqüentemente foi diagnosticada a DC foram a hiperplasia nodular regenerativa, hepatite auto-imune, hipertransaminasemia criptogênica, hepatite pelo vírus C e cirrose biliar primária antimitocôndria negativo. 5) A retirada do glúten da dieta contribuiu de maneira efetiva para normalização das enzimas hepáticas nos casos de hipertransaminasemia criptogênica. Nos grupos de hiperplasia nodular regenerativa, hepatite B e C, cirrose biliar primária, álcool e hepatite auto-imune, o papel da dieta foi de difícil avaliação em razão da interferência da instituição do tratamento específico e da evolução natural da doença hepática de base. / Introduction: The hepatic involvement in Celiac Disease (CD) is well known and widely regarded as a frequent extra-intestinal manifestation. With the advent of highly specific and sensitive serological markers, especially the antiendomysial antibody (EMA), CD has been described in association with several liver conditions. Objectives: To characterize ways for screening patients with chronic liver conditions in order to diagnose CD, to characterize and study patients whose CD diagnoses were performed when investigating hepatic diseases, to test the reactivity of EMA in patients with autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis and non-cirrhotic portal hypertension, to evaluate the course of the hepatic disease in gluten-free diet. Methods: Patients were selected by the detection of antireticulin and anti-extracellular matrix antibodies during routine immunoflourescence determination for hepatic autoantibodies, by the presence of CD manifestations in chronic patients with liver diseases, by the endoscopic aspects suggestive of CD and by systematic search for EMA in the above mentioned pathologies. All patients were submitted to tests for EMA, antireticulin IgG or antimatrix of IgG fibroblasts in IgA deficiency. When testing positive for these markers, patients were submitted to upper digestive endoscopy for intestinal biopsy, and were also characterized from the clinical, laboratorial and histological point of view. The assessment of these data enabled the characterization of the hepatic condition as well as the CD from the onset of a gluten-free diet. Results: 43 patients with autoantibodies related to CD were identified (42 tested positive for IgA EMA and 1 for IgG extracellular antimatrix in the presence of IgA deficiency). Routine determination of hepatic autoantibodies identified 31 patients. Of those, 6 presented chronic liver diseases and CD manifestations. In 3 patients, the endoscopic exam was suggestive of CD; systematic EMA determination was positive in all three cases. The CD diagnosis was confirmed in 37 out of 40 patients (92.5%) that performed intestinal biopsy. Patients aged between 2 and 68 years (median: 35 years). Female patients were most affected (65%). CD was more prevalent in white patients (87%), but was also found in four mulattoes and 1 black person. The most common liver disorders were cryptogenic hypertransaminasemia, autoimmune hepatitis, nodular regenerative hyperplasia and chronic hepatitis C. Conclusions: 1) The reactivity of antireticulin, the presence of unexplained diarrhea and the endoscopic analysis of the duodenal mucous were the most effective ways to identify CD in chronic liver diseases. 2) The absence of CD clinical manifestations in this group of patients was impressive. 3) Contrary to what was observed in the group with non-cirrhotic portal hypertension, especially regenerative nodular hyperplasia, the systematic determination of EMA in primary biliary cirrhosis, autoimmune hepatitis and primary sclerosing cholangitis did not contribute to CD diagnosis in a significant number of patients. 4) CD was most frequently diagnosed in the following liver diseases: cryptogenic hypertransaminasemia, autoimmune hepatitis, nodular regenerative hyperplasia and chronic hepatitis C and negative antimitochondrial primary biliary cirrhosis. 5) The removal of gluten from the diet contributed effectively to bring the hepatic enzymes levels back to normal in cases of cryptogenic hypertransaminasemia. However, the role of diet was difficult to evaluate in nodular regenerative hyperplasia, autoimmune hepatitis, alcohol disease and primary biliary cirrhosis groups due to the nature of the specific treatments and the natural course of the hepatic conditions.

INTRODUÇÃO: A urticária crônica é caracterizada pelo aparecimento de placas eritêmato-edematosas, pruriginosas, que perduram por mais de seis semanas. A etiologia é desconhecida na maioria dos pacientes sendo definida como idiopática (UCI). A desregulação imunológica na UCI pode ser devido a distúrbios na produção de citocinas e quimiocinas. OBJETIVOS: Avaliar o perfil citocinas e quimiocinas em pacientes submetidos ao teste de soro autólogo (ASST) avaliando os soros, a expressão de RNAm e a expressão intracelular de células mononucleares do sangue periférico (CMN) induzidas por estímulos policlonais. METODOLOGIA: Pacientes com UCI (n=37) foram selecionados do Ambulatório de Dermatologia do HC-FMUSP e submetidos ao ASST. O grupo controle foi constituído por indivíduos saudáveis (n=33). Os níveis séricos de citocinas e quimiocinas foram determinados por citometria de fluxo ou por ELISA e a expressão de RNAm de citocinas foi determinada por Real-Time PCR. RESULTADOS: Uma elevação dos níveis séricos de TNF-, IL-6, IL-1, IL-12p70 e IL-10 foi detectada nos pacientes com UCI comparados ao grupo controle, independente da resposta ao ASST. A secreção in vitro de citocinas por CMN estimuladas por fitohemaglutinina (PHA) mostrou aumento da produção de IL-2 nos pacientes com UCI e de IL-17A e IL-10 no grupo ASST positivo em relação ao grupo controle. A expressão de RNAm para IL-10 em CMN, foi diminuída no grupo ASST negativo comparado ao grupo controle. Além disto, um aumento da capacidade linfoproliferativa ao mitógeno Pokeweed foi observado nos pacientes ASST positivo em relação aos indivíduos controles. Os níveis séricos de CXCL8, CCL2, CXCL10 e CXCL9 foram encontrados elevados nos pacientes com UCI em relação aos controles. A secreção de quimiocinas in vitro, foi observado aumento dos níveis basais de CCL2 pelas CMN dos pacientes em relação aos controles, que se elevaram em resposta a enterotoxina A de Staphylococcus aureus (SEA). Já o estímulo com PHA promoveu aumento na produção de CXCL8 e CCL5 pelas CMN dos pacientes. A expressão intracelular de CXCL8 foi detectada principalmente nas células CD14+. A intensidade média de fluorescência (MFI) e a porcentagem da expressão de CXCL8 em CD14+ nos níveis basais e estimulados com SEA encontram-se diminuídos nos pacientes com UCI comparado ao grupo controle. A expressão intracelular de CCL2 em células CD14+ mostrou uma queda na porcentagem dos níveis basais somente nos pacientes ASST negativo em relação ao grupo controle. Além disto, em condições basais de cultura houve um aumento na porcentagem da expressão de CCR5 em células T CD8+ de pacientes com UCI, em função do aumento no grupo ASST positivo. CONCLUSÕES: Os resultados enfatizam o conceito de desequilíbrio imunológico na UCI, independente da resposta ao ASST, evidenciado pelo aumento na secreção de quimiocinas e citocinas pró-inflamatórias. Estes dados sugerem que na UCI, os linfócitos e monócitos estão ativados, os quais podem contribuir para a imunopatogênese da doença / INTRODUCTION: Chronic urticaria is skin disorder characterized by recurrent and transitory itchy weals occurring regularly for more than 6 weeks. The aetiology is not identified in most patients being considered as idiopathic (CIU). The immunological dysregulation in CIU could be due to a disturbed cytokines and chemokines production. OBJECTIVES: To evaluate the pattern of cytokine and chemokine in CIU patients who undergone autologous serum skin test (ASST), assessing sera, mRNA expression and intracellular expression of peripheral blood mononuclear cells (PBMC) through the secretion upon induced by policlonal stimuli. METHODS: CIU patients (n=37) were selected from the Dermatological Outpatient Clinic of the Hospital das Clínicas de São Paulo (HC-FMUSP) and submitted to the ASST. The control group consisted of healthy subjects (n=33). Cytokine and chemokine levels were assessed by flow cytometer and ELISA and mRNA expression was analyzed by Real-Time PCR. RESULTS: Elevated levels of TNF-, IL-6, IL-1, IL-12p70 and IL-10 were observed in sera from CIU patients compared to healthy control group. CIU patients also showed increased IL-2 production by PBMC stimulated with phytohemagglutinin (PHA). Moreover, it was observed higher IL-17A and IL-10 levels in the ASST+ group compared to control group. The IL-10 mRNA expression was diminished in the ASST- group compared to control group. Furthermore, an increased lymphoproliferative response to Pokeweed mitogen was observed in the ASST+ patients compared to healthy subjects. Seric levels of CXCL8, CCL2, CXCL10 and CXCL9 were higher in CIU patients. Regarding the in vitro chemokines secretion, it was detected higher basal levels of CCL2 in CIU patients, which was increased by Staphylococcus aureus enterotoxin A (SEA). Stimulation with PHA increased the CXCL8 and CCL5 production by CIU mononuclear cells. The main source of CXCL8 was the CD14+ cells. CIU CD14+ cells showed decreased mean fluorescence intensity and percentage of CXCL8 expression with and without SEA stimuli. The percentage of CD14+ producing CCL2 was lower in ASST- patients compared to healthy control subjects. Furthermore, in the absence of stimuli the percentage of CCR5-expressing CD8+ T cells was higher in CIU patients, mainly due to an increased expression by the ASST+ group. CONCLUSIONS: These results indicate an immunological dysregulation in CIU, without association to ASST response, which was evidenced by the increased production of pro-inflammatory cytokines and chemokines. The data suggest a higher activation of monocytes and lymphocytes in CIU, which may contribute to its immunopathogenesis

Orientadores: Cássia Raquel Teatin Juliato, Cristina Laguna Benetti-Pinto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T01:05:51Z (GMT). No. of bitstreams: 1 Mayrink_Jussara_M.pdf: 620094 bytes, checksum: e3d5f6dee5c7be899f7f699f6c82fdfe (MD5) Previous issue date: 2013 / Resumo: Introdução: A síndrome dos ovários policísticos (SOP) é uma doença que acomete aproximadamente 10% das mulheres em idade reprodutiva. Está associada a diversos distúrbios metabólicos, como a resistência insulínica (RI) e a síndrome metabólica (SM). A resistência insulínica, por sua vez, está relacionada a disfunções tireoidianas (hipotireoidismo clínico e subclínico). Alguns estudos têm relacionado essas disfunções à ocorrência de SOP, bem como a maior prevalência de tireoidite crônica autoimune nessas mulheres. Entretanto, esses trabalhos ainda são escassos. Objetivos: comparar a prevalência de tireoidite crônica autoimune em mulheres com e sem SOP, comparar a presença de anticorpos antitireoidianos antiperoxidase (ATPO) e antitireoglobulina (ATG) e achados ecográficos tireoidianos sugestivos de tireoidite crônica autoimune em mulheres com e sem SOP. Sujeitos e métodos: foi um estudo de corte transversal, incluindo 130 mulheres entre 18 e 40 anos dos ambulatórios de ginecologia endocrinológica e de planejamento familiar do Departamento de Ginecologia e Obstetrícia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas - entre os meses de agosto de 2012 e agosto de 2013 -, sendo 65 com SOP, em conformidade com o Consenso de Rotterdam de 2004, e 65 mulheres com ciclos menstruais regulares e sem doenças crônicas previamente diagnosticadas. Para todas foram avaliados: índice de massa corpórea (IMC, kg/m2), graus de hirsutismo através do índice de Ferriman-Gallwey, TSH, T4L, T3L, glicemia e insulinemia de jejum, avaliação de resistência insulínica através do índice de HOMA, antiperoxidase e antitireoglobulina e ultrassom da glândula tireóide. Análise Estatística: Para análise dos dados foram usados os testes de Mann- Whitney, t de student, qui quadrado e exato de Fischer. Os dados foram apresentados como média ±DP. Resultados: As 130 mulheres participantes deste estudo eram jovens, com idade entre 18 e 40 anos e média de 27,8 ±6,9 anos no grupo com SOP e 33,5 ±5,7 anos no grupo sem SOP. As mulheres com e sem SOP apresentaram média de IMC de 34,8±8,9 e 28,4±4,8 kg/m2, respectivamente (p<0,0001). O índice de Ferriman-Gallwey para os grupos com e sem SOP foi 8±3,1 e 5±0,7, respectivamente (p<0,0001). Os valores de insulinemia, glicemia e índice de HOMA também foram significativamente maiores na presença de SOP. Em relação ao TSH, os valores para mulheres com e sem SOP foram de 2,4 ±1,8 mIU/L e 2,1±1,2 mIU/L, respectivamente (p=0,0133). Não houve diferença na ocorrência de ATPO e ATG comparando-se os dois grupos. Não houve diferença entre os grupos para o volume tireoidiano avaliado pela ecografia. Entretanto, as glândulas eram mais hipoecoicas no grupo com SOP (26,8% versus 15,4%; p=0,05). A prevalência de HSC foi de 16,9% (11/65mulheres) no grupo com SOP e 6,2% (4/65 mulheres) no grupo sem SOP. Em relação ao diagnóstico de tireoidite crônica autoimune, houve diferença significativa em sua prevalência, com 43,1% de tireoidite no grupo com SOP e 26,2% no grupo sem SOP (p=0,04). Conclusão: A maior prevalência de tireoidite crônica autoimune e de alteração na função tireoidiana (HSC) das mulheres com SOP indicam a necessidade de manter-se vigilância periódica sobre a função tireoidiana, embora não aponte a necessidade de pesquisa rotineira de anticorpos antitireoidianos / Abstract: Introduction: Polycystic ovary syndrome (PCOS) is a condition that affects approximately 10% of women of reproductive age. It's associated with various metabolic disorders such as insulin resistance (IR) and metabolic syndrome (MS). Insulin resistance, in turn, is related to thyroid dysfunction (clinical and subclinical hypothyroidism). Some studies have linked these disorders to the occurrence of PCOS as well as a higher prevalence of autoimmune thyroiditis. However, these studies are still scarce. Objectives: To compare the prevalence of autoimmune thyroiditis , the occurrence of anti -thyroid peroxidase antibodies (ATPO) and anti - thyroglobulin (ATG) and thyroid ultrasound findings suggestive of autoimmune thyroiditis in subjects with and without PCOS. Subjects and methods: It was a cross-sectional study including 130 women between 18 and 40 years of outpatient gynecological endocrinology and family planning at the Department of Obstetrics and Gynecology, School of Medical Sciences, State University of Campinas, between the months of August 2012 and August 2013, 65 with PCOS, in accordance with the Rotterdam criteria, and 65 women with regular menstrual cycles and without previously diagnosed chronic diseases. For all evaluated: body mass index (BMI, kg/m2), degree of hirsutism by Ferriman - Summary xii Gallwey, TSH , FT4 , FT3, blood glucose and fasting insulin, assessment of insulin resistance by HOMA index, ATPO and ATG and ultrasound of the thyroid gland. Statistical analysis: For data analysis we used the Mann- Whitney test, Student's t, chi-square and exact Fisher. Data were presented as mean ± SD. Results: 130 women participating in this study were young, between 18 and 40 years, 27.8 ± 6.9 years in the PCOS group and 33.5 ± 5.7 years in the group without PCOS. Women with and without PCOS had a mean BMI of 34.8 ± 8.9 and 28.4 ± 4.8 kg/m2 , respectively ( p < 0.0001 ) . The Ferriman - Gallwey index for the groups with and without PCOS was 8 ± 5 and 3.1 ± 0.7, respectively (p < 0.0001). The amounts of insulin, glucose and HOMA index were also significantly higher in the presence of PCOS. The TSH values for women with and without PCOS were 2.4 ± 1.8 mIU / L and 2.1 ± 1.2 mIU / L, respectively (p = 0.0133). There was no difference in the occurrence of ATPO and ATG comparing the two groups. There was no difference between groups for thyroid volume measured by ultrasound. However, the glands were more hypoechoic in the PCOS group (26.8% vs. 15.4%, p = 0.05). The prevalence of SCH was 16.9 % (11/65 women) in the PCOS group and 6.2% (4/65 women) in the group without PCOS. The prevalence of autoimmune thyroiditis in the PCOS group was 43.1% and 26.2% in the group without PCOS (p = 0.04). Conclusion: The higher prevalence autoimmune thyroiditis and SCH in women with PCOS indicates the necessity of keeping surveillance on their periodic thyroid function, but do not point the necessity of routine investigation of antithyroid antibodies / Mestrado / Fisiopatologia Ginecológica / Mestra em Ciências da Saúde

Antibodies are a class of proteins that are made by the immune system to recognize harmful organisms and molecules. Their exceptional capability of specifically recognizing molecules has been investigated for over a century and information thereof has been utilized for a variety of applications including vaccine and generation of therapeutic antibodies. Occasionally, instead of protecting the host against pathogens, antibodies can recognize constituents of the host and thereby cause an autoimmune reaction that eventually can lead to a disease. Therefore, it is of great interest to understand what the antibodies bind to and their specificities.   The last decades of technical development and availability of protein and peptide microarrays have enabled large-scale profiling of antibodies and precise determination of their specificities through epitope mapping. In this thesis the aim was to use affinity proteomics tools to profile antibodies, determine their specificities, and discover potential associations of autoantigens to disease by analyzing blood-derived samples with microarray-based methods.   In Paper I, 57 serum samples from patients with the suggested autoimmune disease narcolepsy, were analyzed on planar antigen microarrays with 10,846 human protein fragments. Verification on an independent sample collection consisting of serum samples from 176 individuals, revealed METTL22 and NT5C1A as two potential autoantigens. In Paper II, antibodies from 53 plasma samples from patients with first-episode psychosis, a condition suggested to have a partial autoimmune component, were analyzed on planar antigen microarrays with 2,304 human protein fragments. After a follow-up study of the patients, antibodies toward an antigen representing the three proteins, PAGE2, PAGE2B, PAGE5, was found associated to an increased risk of developing schizophrenia. In Paper III, serum and plasma samples from patients with the autoimmune diseases multiple sclerosis and narcolepsy, were epitope mapped on high-density peptide microarrays with approximately 2.2 million peptides. Technical and biological verification, by using other microarray technology and analyzing  samples from 448 patients, revealed one peptide for multiple sclerosis and narcolepsy, representing the proteins MAP3K7 and NRXN1, with higher antibody reactivity towards in each group, respectively. In Paper IV, purified polyclonal antibodies raised against a surface antigen found on malaria-infected erythrocytes, were profiled on the peptide microarrays representing all proteins found on malaria-infected erythrocytes derived from Plasmodium falciparum. Then, different Plasmodium falciparum strains were analyzed by immunofluorescence microscopy and western blots, using the epitope mapped antibodies. The performance of the immunoassays were compared to the identified epitopes, and validated by RNA sequencing.   In conclusion, these investigations describe multiplex methods to identify and characterize antibodies, their disease association and epitopes. Follow-up studies are needed to determine their potential use and clinical value. / <p>QC 20170905</p>

La Polyarthrite Rhumatoïde (PR) est une maladie auto-immune chronique qui détruit les articulations. Les auto-anticorps (AAc) les plus spécifiques sont dirigés contre des protéines citrullinées (ACPA). Environ 30% des patients ont des AAc dirigés contre la protéine B-Raf. Les ACPA sont des IgG produits sans réponse lymphocytaire T (LT) détectable contre les protéines citrullinées. Les enzymes PAD, responsables de la citrullination, sont la cible d’AAc dans la PR. Les LT pourrait être dirigés contre les PAD. Les LB produiraient des IgG contre les PAD et les protéines citrullinées, du fait qu’elles soient fixées aux PAD durant leur citrullination. Pour tester ce modèle, nous avons injecté des PAD à des souris et suivi les LT et les Ac anti-PAD ainsi que les ACPA. Nous avons montré que l’immunisation par PAD induit des LT anti-PAD, des Ac anti-PAD ainsi que des Ac anti-peptides de fibrinogène citrulliné.B-Raf est une ser/thr kinase de la voie des MAPK impliquée dans l’inflammation et l’activation des LT. Les LT de patients PR ont une sur-activation de la voie des MAPK, induisant une rupture de tolérance envers les auto-antigènes. Notre hypothèse est que des mutations du gène BRAF pourraient être à l’origine des AAc anti-B-Raf. Nous avons identifié la présence d’une mutation du gène BRAF entrainant la substitution de la valine en alanine en position 600 (V600A) qui est trouvée en plus grande quantité dans les cellules du sang périphérique et les LT des patients PR. V600A n’est pas corrélée à la présence d’AAc anti-B-Raf, mais augmente l’activité kinase de B-Raf qui pourrait activer la voie des MAPK dans les LT et une rupture de tolérance envers les auto-antigènes. / Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune joint disease. RA most specific autoantibodies (AAb) recognize citrulline proteins (ACPA). 30% of patients with RA have anti-B-Raf AAb. ACPA are IgG that arise in the absence of associated T cell responses. PAD enzymes, responsible for the citrullination, are also targets of AAb in RA. We thus propose a mechanism to explain the emergence of ACPA. We hypothesize that anti-citrullinated protein immunization arises because, at first, PAD is recognized by T cells, which, in turn help the production of AAb to neighbor proteins citrullinated by PAD. To test this model, we primed mice with PAD proteins and looked for immune response to PAD and citrullinated proteins. We found that PAD immunization leads to T cell response, Ab anti-PAD as well as anti-citrullinated fibrinogen peptides Ab production. Anti-PAD immunization could induce anti-citrullinated protein immunization.B-Raf, a ser-thr kinase of the MAPK signalling pathway, is involved in inflammation and in T cell activation. An overexpression of B-Raf is observed in T cells from RA patients increasing T cell activation to autoantigens. Our hypothesis is that BRAF gene mutations could trigger a rupture of tolerance and AAb production in RA. We have identified a BRAF mutation, a valine residue at position 600 is substituted by an alanine (V600A). V600A is found more often and at greater quantities in patients with RA, noteworthy in their T cells. This mutation does not correlate with Anti-B-Raf AAb presence but this is a strong enhancer of B-Raf kinase activity. This could lead to abnormal T cells activation and explain tolerance rupture in RA.

Objetivo: este estudo objetivou avaliar a presença do anticorpo anti-alfaenolase (AAAE) IgM na doença de Behçet (DB) e suas possíveis associações com as manifestações clínicas e atividade da doença. Métodos: noventa e sete pacientes com DB foram comparados a 36 pacientes com enteroartrite (EA) [24 com doença de Crohn (DC) e 12 com retocolite ulcerativa (RCU)], além de 87 controles saudáveis. Os testes para detecção do AAAE IgM foram realizados por Immunoblotting. A atividade de doença foi avaliada por índices padronizados, como o Formulário de Atividade Atual da Doença de Behçet (BR-BDCAF) para os pacientes com DB e o Índice de Harvey-Bradshaw (HBI) para os pacientes com DC e RCU. Uma segunda avaliação foi realizada somente nos pacientes com DB (n=56) para a detecção do AAAE IgM, avaliação de atividade de doença e dosagem de proteína-C-reativa (PCR). Resultados: maior prevalência do AAAE IgM foi encontrada na DB (17,7%) comparativamente à EA (2,8%) e aos controles saudáveis (2,3%), p < 0,001. A frequência do AAAE IgM foi maior na DB ativa quando comparada à DB inativa (30,2% vs. 7,4%, p=0,006). Este achado foi confirmado em uma segunda avaliação de 56 pacientes do grupo com DB (45,5% vs. 13,3%, p=0,02). A média do BR-BDCAF foi maior no grupo com AAAE IgM positivo, em ambas avaliações (9,1 ± 5,4 vs. 4,9 ± 4,9, p=0,002; 5,0 ± 4,9 vs. 2,2 ± 2,9, p=0,01, respectivamente). Os pacientes com DB em atividade mucocutânea e articular apresentaram maior incidência do AAAE IgM, tanto na primeira quanto na segunda avaliação (64,7% vs. 27,5%, p=0,005; 36,4% vs. 7,1%, p=0,039, respectivamente). Conclusões: os presentes dados corroboram que a alfa-enolase é um antígeno alvo na DB, particularmente associada à atividade mucocutânea e articular da doença. Além disso, o AAAE IgM pode distinguir a DB da EA, especialmente em pacientes com alta atividade de doença / Objective: this study aimed to assess IgM AAEA in systemic Behçet\'s disease (BD) and its possible association with clinical manifestations and disease activity. Methods: ninety-seven consecutively selected BD patients were compared to 36 enteropathic spondyloarthritis (ESpA) [24 Crohn\'s disease (CD) and 12 ulcerative colitis (UC)] patients and 87 healthy controls. IgM AAEA was detected by Immunoblotting. Disease activity was assessed by standardized indexes, Brazilian BD Current Activity Form (BR-BDCAF) for BD and Harvey-Bradshaw Index (HBI) for CD and UC patients. A second evaluation was performed in BD patients (n=56), regarding IgM AAEA presence, disease activity scores and C-reactive protein (CRP). Results: higher IgM AAEA prevalence was found in BD (17.7%) compared to ESpA (2.8%) and healthy controls (2.3%), p < 0.001. IgM AAEA frequency was higher in active BD compared to inactive BD (30.2% vs. 7.4%, p=0.006), a finding confirmed in the second cross-sectional evaluation of 56 of these BD patients (45.5% vs. 13.3%, p=0.02). Mean BR-BDCAF scores were higher in IgM AAEA positive group on both evaluations (9.1 ± 5.4 vs. 4.9 ± 4.9, p=0.002; 5.0 ± 4.9 vs. 2.2 ± 2.9, p=0.01, respectively). BD patients with mucocutaneous and articular symptoms presented higher IgM AAEA positivity in the first and second evaluations (64.7% vs. 27.5%, p=0.005; 36.4% vs. 7.1%, p=0.039 respectively). Conclusions: these data support the notion that alpha-enolase is a target antigen in BD, particularly associated with disease activity, mucocutaneous and articular involvement. In addition, IgM AAEA may distinguish BD from ESpA, especially in patients with high disease activity

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22144号 / 医博第4535号 / 新制||医||1039(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一, 教授 生田 宏一, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...

Vor zirka 45 Jahren wurde das La Protein (SS-B) erstmals als Autoantigen von Patienten mit Systemischen Lupus Erythematodes (SLE) oder Primären Sjögren’s Syndrom beschrieben. Jahrzehntelange Forschungen befassten sich mit seiner Struktur- und Funktionsaufklärung, sowie der Untersuchung von monoklonalen anti-La Antikörpern (anti-La mAK). Doch noch immer werfen nukleäre Antigene wie das La Protein und die gegen sie gerichteten Autoantikörper verschiedene Fragen auf. Diese betreffen besonders deren Entstehung und die pathophysiologische Bedeutung. So war es die Zielsetzung dieser Arbeit, die pathophysiologische Bedeutung des La Proteins in der Autoimmunkrankheit und der Krankheitsentstehung bei SLE-Patienten weiter aufzuklären. Im Zentrum der Untersuchungen stand dabei die Redoxsensitivität des La Proteins und deren Einfluss auf die räumliche Proteinstruktur, die zelluläre Lokalisation, die Funktion der Nukleinsäurebindung, sowie die Antigenität. Dabei konnte erstmals mit Hilfe von CD-Spektroskopieanalysen deutlich gezeigt werden, dass die drei Cysteine (C18, C232 und C245) des La Proteins für die Struktur eine zentrale Rolle spielen. Es konnte demonstriert werden, dass die Fähigkeit zur redoxabhängigen Strukturumfaltung zum Verlust der protektiven Wirkung des La Proteins auf gebundene Nukleinsäure führt, wodurch diese für Nukleasen zugänglich gemacht wird und abgebaut werden kann. Dies ließ sich in der vorliegenden Arbeit mit Hilfe verschiedener Experimente verifizieren, beispielsweise durch die Anwesenheit von Kupferionen (Cu2+), oder auch durch die Änderung des pH-Wertes von 7,0 auf 4,5. Parallel hierzu wurden neben Wildtyp La Protein auch verschiedene Cysteinmutanten getestet, um die Redoxabhängigkeit auch durch den Austausch der Cysteine C18, C232 und C245 zu zeigen. Die Änderungen des Redoxzustands beeinflussten jedoch nicht nur Sekundär- und Tertiär-struktur des La Proteins, sondern auch sein Di- und Oligomerisationsverhalten, sowie die Antigenerkennung durch bestimmte anti-La mAK (mAK der 312B Gruppe). Erstmals konnte in dieser Arbeit auch gezeigt werden, dass Patientenautoantikörper nicht nur gegen die nor-malerweise nukleär vorliegende, reduzierte Form des La Proteins existieren. Es waren eben-so Patientenautoantikörper gegen die zytoplasmatische, oxidierte Form nachweisbar. Auch auf zellulärer Ebene wurde die Wirkung einer redoxabhängigen Umfaltung des La Proteins deutlich. Durch den Einfluss von reaktiven Sauerstoffspezies (ROS) konnte eine zytoplasmatische Anreicherung beobachtet werden. Die dazu bereits bekannten Daten aus der Literatur konnten mit dieser Arbeit nochmals belegt und darüber hinaus ergänzt werden. Die zytoplasmatische Anreicherung wurde in der vorliegenden Arbeit für verschiedene ROS Stimuli gezeigt, darunter H2O2, Cu(II)SO4, Fe(II)Cl2 und darüber hinaus auch für NO-Glutathion (Stimulus reaktiver Stickstoffspezies NO). Des Weiteren konnte erstmals eine zytoplasmatische La Anreicherung durch eine Veränderung des intrazellulären ROS Levels nach Rezeptorstimulation gezeigt werden. Spannender Weise gelang dies für dendritische Zellen, wie moDCs und slanDCs, als auch für Endothelzellen (HUVECs). Die Induktion erfolgte dabei über Toll-like Rezeptoren (TLR), wie TLR4 (LPS-abhängiger Toll-like Rezeptor) und auch über TLR7/8, zwei Toll-like Rezeptoren, die für die Bindung von ssRNA, beispielsweise nach Virusinfektion zuständig sind. Unter dem Einfluss dieser Stimuli, kommt es zur Umfaltung des La Proteins, zum Loslösen von seinem potentiellen, indirekt über den anti-La mAK 7B6 nachgewiesenen Bindepartner und zum Verlust der nukleären Lokalisation. Im Zytoplasma kann die oxidierte Proteinvariante ihre protektive Aufgabe bezüglich der gebundenen Nukleinsäure nicht weiter ausführen. Es kommt unter normalen Umständen zu Dissoziation der gebundenen Nukleinsäuren und einem potentiellen Abbau dieser. Da bei Autoimmunpatienten jedoch sehr häufig eine Reduktion der Nukleaseaktivität nachweisbar ist, könnte der Nukleinsäureabbau in diesen Patienten gestört sein, was zu einem oxidierten La Protein mit noch gebundener Nukleinsäure führen würde. Neben der zytoplasmatischen Lokalisation wurde für das La Protein auch eine Lokalisation an der Zelloberfläche diskutiert. Die hier durchgeführten Studien mit den verschiedenen Sauerstoff- oder Stickstoffstressstimuli zeigten jedoch unter den gewählten Bedingungen keine Exposition des La Proteins auf die Zelloberfläche. Daher wurden apoptotische und nekrotische Zellen als mögliche La Proteinquelle für die Dekoration lebender Nachbarzellen untersucht. War in der Literatur noch über „apototic bodies“ als Quelle des La Proteins als Autoantigen spekuliert wurden, so konnte hier gezeigt werden, dass das La Protein aus humanen, apoptotischem Zellmaterial zu keiner nachweislichen Oberflächendekoration lebender Zellen führte. Anders verhielt es sich bei nekrotischem Zellmaterial. Hier konnte zum Beispiel humanes oxidiertes La Protein auf den murinen Zellen nachgewiesen werden. Im Rahmen dieser Analysen zeigte sich darüber hinaus, dass sowohl oxidiertes als auch reduziertes La Protein auf die Oberfläche verschiedener Zelltypen binden kann. Insgesamt konnte jedoch mehr oxidiertes La Protein auf den verschiedensten Zellentypen nachge-wiesen werden, als reduziertes La Protein unter gleichen Bedingungen. Auch ergaben sich Unterschiede bezüglich der Proteinbindung zwischen einzelnen Zelltypen. Vertreter von Anti-gen präsentierenden Zellen, wie Monozyten oder B-Zellen (Radji), sowie Endothelzellen (HUVECs), aber auch murine A9 Fibroblasten konnten im Vergleich zu T-Zellen und NK-Zellen, mehr La Protein auf ihrer Oberfläche binden. Diese Resultate lassen eine pathophysiologische Bedeutung des La Proteins bei SLE Patienten erkennen. Im Zuge von Zellschä-digungen, wie beispielsweise nach UV-Stress, kommt es zu einem nekrotischen Zellzerfall. Dieser führt zur Freisetzung von oxidiertem La Protein, welches auf benachbarte Zellen bindet. Dadurch können sie zum Ziel einer komplementsystemvermittelten Immunreaktion, sowie einer antikörpervermittelten, NK-Zellen gestützten Zelllyse (ADCC) werden. Für die Initiation einer Immunantwort, und im Besonderen für die Reifung autoreaktiver B-Zellen zu autoantikörpernproduzierenden Plasmazellen, bedarf es jedoch zunächst einer Aktivierung von dendritischen Zellen. Im Rahmen dieser Arbeit konnte gezeigt werden, dass lösliches La Protein dendritische Zellen aktivieren kann, belegt über die dokumentierte, finale TNFα Sekretion. Dabei war jedoch nicht entscheidend, ob La in einem reduzierten oder oxidierten Zustand vorlag, sondern das es assoziierte Nukleinsäuren aufwies. Experimentell führten an La Protein gebundene Nukleinsäuren zur Aktivierung von dendritischen Zellen (slanDCs). Wurde die Nukleinsäuren jedoch zuvor durch RNaseA Behandlung oder die Inkubation in Serum eines gesunden Spenders abgebaut, so lag keine oder eine nur sehr geringe Aktivierung von slanDCs vor. Eine Inkubation von La Protein in SLE Patientenserum hatte keine solche verminderte Aktivierung dendritischer Zellen zur Folge. Unter Berücksich-tigung der oben geschilderten Versuchsergebnisse ließ sich dieses Resultat mit der geringeren Nukleaseaktivität bei SLE Patienten begründen, was bereits aus der Literatur bekannt ist. Diese reduzierte Nukleaseaktivität stellt somit offensichtlich einen entscheidenden Faktor bei der Aktivierung einer Autoimmunantwort in Zusammenhang mit dem La Protein und anti-La Autoantikörpern dar. Mit dieser Arbeit konnte also eindeutig gezeigt werden, dass das La Protein unter sich wechselnden Redoxbedingungen seine Struktur ändert. Dabei spielen die drei enthaltenden Cysteine eine bedeutende Rolle. Derartige Strukturveränderungen beeinflussen die Nukleinsäureschutzfunktion und die Erkennung durch Antikörper. Darüber hinaus bestätigte sich eine ROS induzierte Anreicherung von La im Zytoplasma. Auch die Fähigkeit des La Proteins, aus nekrotischem Zellmaterial auf die Oberflächen von lebenden Zellen zu binden, wurde gezeigt. Dadurch wäre es für Autoantikörper bei SLE Patienten zugänglich und somit pathophysiologisch relevant. Außerdem konnte auch die Eigenschaft von nukleinsäurege-koppelten La Proteins zur Aktivierung dendritischer Zellen belegt werden, was zur krankheitsauslösenden Aktivierung von autoreaktiven T- und im weiteren Verlauf von B-Zellen führen kann. Dadurch konnten die in dieser Arbeit erhaltenen Resultate deutliche Hinweise auf die pathophysiologische Bedeutung des La Proteins, nicht nur während der Autoimmunerkrankung, sondern auch für die Frühphase der Krankheitsentstehung, geben.

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...