Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2015. / Made available in DSpace on 2016-05-24T17:39:35Z (GMT). No. of bitstreams: 1 339419.pdf: 1426935 bytes, checksum: 0d8c0c0486e49626e60185221fcff86b (MD5) Previous issue date: 2015 / Leflunomida e metotrexato são drogas modificadoras do curso da doença classicamente utilizadas por via sistêmica no tratamento da artrite reumatoide e o sucesso clínico está relacionado com a inibição da proliferação de células imunes e de citocinas na cavidade articular. Baseado nestes potenciais efeitos inibitórios, nossa principal hipótese é que a administração direta dessas drogas na medula é capaz de reduzir a infiltração de leucócitos, edema e incapacitação articular em um modelo de artrite induzida por LPS. Ratos Wistar fêmeas, pesando entre 200 e 220 g, receberam as drogas por via intratecal, 2 horas antes da administração de LPS (30 ng/ 50 µl; i.a.) nas articulações de joelho previamente sensibilizadas com carragenina (300 µg/ 20 µl; i.a.). A incapacitação articular foi avaliada pelo tempo de elevação da pata (TEP; s) durante o deambular de 1 min; o edema pelo aumento do diâmetro articular (DA; cm) e a migração celular pela contagem total de leucócitos (CT; cel/mm3) do fluido sinovial coletado ao final de 5 horas de observação dos parâmetros. As administrações por via intratecal de teriflunomida (metabólito ativo da leflunomida) (0,1 e 20 µg/ 10 µl) e metotrexato (25 µg/ 10 µl) foram capazes de reduzir a migração celular para o fluido sinovial, a incapacitação e o edema articular promovidos pelo LPS, ao contrário de quando administrados por via intraperitoneal, sugerindo que o sítio de interação para os efeitos antiartríticos das drogas foi restrito ao ambiente medular. A coadministração com uridina (10 µg) reverteu apenas os efeitos inibitórios produzidos pela menor dose de teriflunomida, sugerindo que apenas em maiores concentrações o mecanismo de ação da droga independe da inibição da síntese de novo de nucleotídeos. A coadministração por via intratecal de teriflunomida (0,1 µg) e bumetanida (60 µg), um bloqueador do reflexo da raiz dorsal, não potencializou o efeito inibitório da teriflunomida na redução da incapacitação, edema e migração celular, sugerindo que a inibição do reflexo da raiz dorsal e consequentemente da inflamação neurogênica estejam envolvidos na ação medular da teriflunomida. A coadministração de teriflunomida (0,1 µg) e metotrexato (25 µg) por via intratecal promoveu um efeito somatório na inibição da inflamação articular, provavelmente pela adição do mecanismo imunomodulador do metotrexato na inibição da síntese de citocinas pelas células gliais. O presente estudo sugere que o tratamento por via intratecal com teriflunomida e metotrexato modula a inflamação periférica provavelmente por reduzir o aumento da quantidade de citocinas inflamatórias na medula, através da inibição da atividade glial que inibea sensibilização dos terminais neuronais medulares envolvidos na sinalização nociceptiva e na indução da inflamação neurogênica. Esta estratégia pode representar uma nova abordagem terapêutica para o tratamento da artrite reumatoide com menos efeitos colaterais sistêmicos.<br> / Abstract : Leflunomide and methotrexate are disease-modifying antirheumatic drugs classically used systemically in the treatment of rheumatoid arthritis and clinical success is related to inhibition of proliferation of immune cells and cytokines in the joint cavity. Based on these potential inhibitory effects, our central hypothesis is that the direct administration of these drugs on the spinal cord is able to reduce articular incapacitation, edema and synovial leukocyte infiltratio induced in a model of LPS-induced arthritis. Female Wistar rats, weighing between 200 and 220 g were given the drugs intrathecally 2 hours before administration of LPS (30 ng/ 50 µl; i.a.) in the knee joints previously sensitized with carrageenan (300 µg/ 20 µl; i.a.). Inflammatory-induced incapacitation was measured hourly by the paw elevation time (TEP; s) in 1-min period of observation, and edema was evaluated by the articular diameter increase (DA; cm) taken just after each incapacitation measurement. After the 5-h period of observation parameters the synovial fluid was collected from articular capsule for total leukocyte count (CT; cells/ mm3). Intrathecal administration of teriflunomide ( active metabolite of teriflunomide) (0.1 and 20 µg/ 10 µl) and methotrexate (25 g/ 10 µl) were able to reduce cell migration to the synovial fluid, incapacitation and joint edema induced by LPS, unlike when given by intraperitoneal route, suggesting that the interaction site for antiarthritic effects of drugs was restricted to spinal cord microenvironment. Co-administration with uridine (10 µg) just reverses the inhibitory effects produced by the lower dose of intrathecal teriflunomide, suggesting that only in the higher concentrations of the drug mechanism of action independent from inhibition of de novo synthesis of nucleotides. Co-administration of intrathecal teriflunomide (0.1 µg) and bumetanide (60 µg), a blocker of the dorsal root reflex did not potentiate the inhibitory effect of teriflunomide on the reduction of incapacitation, edema and cell migration, suggesting that inhibition of dorsal root reflex and neurogenic inflammation are involved in spinal action of teriflunomide. Co-administration of teriflunomide (0,1 µg) and methotrexate (25 µg) intrathecally caused a summation effect on inhibition of joint inflammation, probably due to the addition of methotrexate immunomodulatory mechanism of inhibition on cytokine synthesis released by glial cells. Thus, this study proposes that the intrathecal methotrexate and leflunomide modulates the peripheral inflammation by inhibiting on increase of cytokines in spinal cord that sensitizes neuronal terminals involved in nociceptive signaling andinduction of neurogenic inflammation. This strategy may represent a novel therapeutic approach for the treatment of rheumatoid arthritis with fewer systemic side effects.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Ciências Médicas, Florianópolis, 2015 / Made available in DSpace on 2016-05-24T17:24:27Z (GMT). No. of bitstreams: 1 338989.pdf: 509773 bytes, checksum: af680f166f1ab4e4301255bed239699f (MD5) Previous issue date: 2015 / Objetivos: a Artrite Reumatoide (AR) classicamente afeta as articulações, porém pode apresentar manifestações extra-articulares, incluindo pulmonares. Neste estudo, busca-se identificar possíveis fatores de risco ou marcadores laboratoriais para o comprometimento pulmonar na AR, especialmente a presença de Fator Reumatoide (FR), anticorpos contra proteínas citrulinadas (ACPA) e marcadores tumorais, relacionando-os às alterações observadas em tomografias computadorizadas de alta resolução (TCAR) do tórax. Método: estudo transversal com portadores de AR entrevistados e examinados por médico especialista e depois submetidos à TCAR do tórax e a coletas sanguíneas para medidas de proteína C reativa (PCR), velocidade de hemossedimentação (VHS), fator reumatoide (FR), ACPA (anti-vimentina e/ou anti-CCP3) e dos marcadores tumorais antígeno carcinoembrionário (CEA), CA 125, CA 15-3 e CA 19-9. Resultados: 96 pacientes realizaram a TCAR do tórax sendo os achados mais frequentes o espessamento brônquico (27/28,1%) e as bronquiectasias (25/26,0%). O FR esteve presente em 63,2% (55/87) e os ACPA (anti-vimentina ou anti-CCP3) em 72,7% (64/88) dos indivíduos. O CEA apresentou-se elevado em 27,5% dos indivíduos não tabagistas e em 67,6% de tabagistas. O CA 19-9 apresentou-se elevado em 6 (7,0%) de 86 pacientes, o CA 15-3 em 3 (3,5%) de 85 pacientes e o CA 125 em 4 (5,3%) de 75 pacientes. Na análise multivariada, foi identificada uma associação estatisticamente significativa entre a elevação do CEA e a presença de alteração em vias aéreas nos pacientes com AR (p=0,014). Conclusões: A elevação do CEA está associada à presença de alteração em vias aéreas em AR. O CEA pode servir como um preditor de doença pulmonar na AR, selecionando os indivíduos que merecem uma maior investigação do aparelho respiratório.<br> / Abstract : Introduction: Rheumatoid arthritis (RA) classically affects the joints but can present extra-articular manifestations, including pulmonary manifestations. The present study aimed to identify possible risk factors or laboratory markers for lung involvement in RA, particularly the presence of rheumatoid factor (RF), anti-citrullinated peptide antibodies (ACPA), and tumor markers, and to correlate them with changes observed on chest high-resolution computerized tomography (HRCT). Method: This cross-sectional study involved RA patients who were examined and questioned by a specialist physician and later subjected to chest HRCT and blood collection for measurement of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), ACPA (anti-vimentin and/or anti-CCP3), and the tumor markers carcinoembryonic antigen (CEA), CA 125, CA 15-3, and CA 19-9. Results: A total of 96 patients underwent chest HRCT. The most frequent findings were bronchial thickening (27/28.1%) and bronchiectasis (25/26.0%). RF was present in 63.2% (55/87) of patients, and ACPA (anti-vimentin or anti-CCP3) were present in 72.7% (64/88) of patients. CEA levels were high in 27.5% of non smokers and 67.6% of smokers. CA-19-9 levels were high in 6 of 86 patients (7.0%), CA 15-3 levels were high in 3 of 85 patients (3.5%), and CA 125 levels were high in 4 of 75 patients (5.3%). Multivariate analysis indicated a statistically significant association between high CEA levels and the presence of airway changes in patients with RA (p=0.014). Conclusions: The elevation of CEA is associated with the presence of airway disease in AR .CEA can serve as a predictor of lung disease in RA and can help identify individuals who require more detailed examination for the presence of respiratory disorders.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2015. / Made available in DSpace on 2016-05-24T17:51:35Z (GMT). No. of bitstreams: 1 337732.pdf: 1919311 bytes, checksum: f784f38a86994b9fd7a8b4fbd0781673 (MD5) Previous issue date: 2015 / A Artrite Reumatoide (AR) é uma doença autoimune caracterizada por inflamação crônica que causa erosões e deformidades nas articulações, podendo ter acometimento de órgãos como pulmão e coração. A molécula HLA-G está relacionada com a inibição de células do sistema imune, como células T e NK, conferindo proteção contra respostas inflamatórias. Entre os polimorfismos ligados à sua expressão, estão os localizados na região 3´UTR do gene HLA-G, que incluem dez sítios polimórficos descritos: um polimorfismo de inserção ou deleção (InDel) de um fragmento de 14pb e nove SNPs (+3001, +3003 T/C, +3010 C/G, +3027 A/C, +3035 C/T, +3142 G/C, +3187 A/G, +3196 G/C e +3227 A/G). A partir disso, o objetivo desse estudo será realizar um estudo caso-controle e verificar a influência desses polimorfismos na manifestação da doença. A família Alu é o elemento móvel mais comum em genomas de primatas, com mais de 1,1 milhões de cópias no Homo sapiens. A inserção AluyHG encontra-se entre os loci HLA-A e HLA-G, podendo ser um marcador da região. A partir disso, o objetivo desse estudo foi genotipar 115 pacientes e 115 controles (classificados por faixa etária) e verificar a influência desses polimorfismos na manifestação da doença. Para isso, foi realizado um levantamento de dados epidemiológicos e clínicos relacionados à manifestação da doença (hábito tabagista, fator reumatoide, proteína C-reativa e velocidade de hemossedimentação) e estes foram testados. O DNA foi extraído de sangue total e a genotipagem feita por PCR e visualização em eletroforese em gel vertical de poliacrilamida em concentração de 7%, corado com nitrato de prata, no caso do polimorfismo de 14pb do HLA-G, e em gel de agarose 1%, corado com GelRedTM, no caso do polimorfismo AluyHG (com 322pb). Os SNPs do HLA-G foram identificados por meio de sequenciamento pelo método de Sanger. Foram realizados cálculos das frequências alélicas e genotípicas, análises de associações e inferência de haplótipos. Após as análises foram encontrados valores significativos de associação dos polimorfismos e a manifestação de AR para os genótipos +3196*G*C (OR=2,387, p=0,026), AluyHG*In (OR=0,524, p=0,006) AluyHG*Del (OR=1,907, p=0,006), AluyHG*In*Del (OR=0,441, p=0,006), AluyHG*Del*Del (OR=2,466, p=0,002). Para os modelos de herança, foram encontradas associações com: +3010*G+GC (OR=0,658, p=0,011), +3010*C+GC (OR=1,519, p=0,011), +3142*G+GC (OR=1,435, p=0,029), +3142*C+GC (OR=0,697, p=0,029), AluyHG*DD+IDxII (OR=0,402, p=0,002), AluyHG*I+ID (OR=0,465, p=0,000), AluyHG*D+ID (OR=2,152, p=0,000). Para os dados clínicos, foram encontradas as seguintes associações: FR e +3010*GG (OR=4,685, p=0,032) e +3196*GC (OR=0,388, p=0,049), CRP e +3196*GC (OR=0,301, p=0,011) e +3196*CC (OR=2,713, p=0,035), VHS e +3003*C (OR=0,296, p=0,042) e +3003*T (OR=3,382, p=0,042). Para os haplótipos e combinações haplotípicas, foram encontrados: UTR5 (OR=2,174, p=0,048), UTR1+UTR5 (OR=5,532, p=0,034); nos modelos de herança UTR5 (OR=2,346, p=0,038), UTR1+AluyHG*Ins (OR=0,556, p=0,047), UTR5+AluyHG*Del (OR=2,247, p=0,050). Em conclusão, este estudo permitiu investigar a possibilidade de associação do gene HLA-G em relação à Artrite Reumatoide, corroborando com dados encontrados na literatura e trazendo alguns dos resultados de associação com doenças autoimunes, que não haviam sido encontrados em nenhuma literatura até o momento. Mais estudos funcionais devem ser realizados para melhor compreender como os polimorfismos genéticos podem agir na patogênese da AR.Palavras chave: Artrite Reumatoide. HLA-G. AluyHG. Doença autoimune. Estudo de associação. Genética epidemiológica. ABSTRACTRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation causing erosions and deformity in the joints, and may be of involvement of organs such as lung and heart. The HLA-G is associated with the inhibition of immune cells, such as NK and T cells, conferring protection against inflammatory responses. Among the polymorphisms linked to its expression are the ones located in the 3'UTR region of the gene that include ten polymorphic sites described: a polymorphism of insertion or deletion (InDel) with a fragment of 14pb and nine SNPs (+3001C/T, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G, +3196G/C and +3227A/G). From this, the objective of this study will be to conduct a case-control study and the influence of these polymorphisms in disease manifestation. Alu family is the most common mobile element in primate genomes, with more than 1.1 million copies in Homo sapiens. The insertion AluyHG located between the loci HLA-A and HLA-G, may be a marker region. From this, the objective of this study was to genotype 115 patients and 115 controls (classified by age group) and the influence of these polymorphisms in the demonstration and development of the disease. For this, a survey was conducted of epidemiological and clinical data related to the development of the disease (smoking habits, rheumatoid factor, C-reactive protein and erythrocyte sedimentation rate) and these were tested. DNA was extracted from whole blood by PCR and genotyping made and viewed in vertical gel electrophoresis in polyacrylamide concentration of 7% stained with silver nitrate in the case of polymorphism of 14bp of the HLA-G, and 1% agarose gel in AluyHG case polymorphism (with 322pb). The HLA-G SNPs were identified through sequencing by the Sanger method. Calculations were made of the allele and genotype frequencies, analyzes of associations and haplotype inference. The analysis found associations of the polymorphisms and the risk of developing RA for genotypes +3196*G*C (OR=2,387, p=0,026), AluyHG*In (OR=0,524, p=0,006) AluyHG*Del (OR=1,907, p=0,006), AluyHG*In*Del (OR=0,441, p=0,006), AluyHG*Del*Del (OR=2,466, p=0,002). For heritage models, associations were found with: +3010*G+GC (OR=0,658, p=0,011), +3010*C+GC (OR=1,519, p=0,011), +3142*G+GC (OR=1,435, p=0,029), +3142*C+GC (OR=0,697, p=0,029), AluyHG*DD+IDxII (OR=0,402, p=0,002), AluyHG*I+ID (OR=0,465, p=0,000), AluyHG*D+ID (OR=2,152, p=0,000). For the clinical data, the following associations were found: RF and +3010*GG (OR=4,685, p=0,032) and +3196*GC (OR=0,388, p=0,049), CRP and +3196*GC (OR=0,301, p=0,011) and +3196*CC (OR=2,713, p=0,035), ESR and +3003*C (OR=0,296, p=0,042) and +3003*T (OR=3,382, p=0,042). For haplotypes and haplotype combinations were found: UTR5 (OR=2,174, p=0,048), UTR1+UTR5 (OR=5,532, p=0,034); in heritage models: UTR5 (OR=2,346, p=0,038), UTR1+AluyHG*Ins (OR=0,556, p=0,047), UTR5+AluyHG*Del (OR=2,247, p=0,050). In conclusion, this study allowed us to investigate the possible association of HLA-G gene in relation to rheumatoid arthritis, corroborating with the data in the literature and bringing some of the association results with autoimmune diseases who had not found any literature to date. More functional studies should be conducted to better understand how genetic polymorphisms can act in the pathogenesis of RA.<br> / Abstract : Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation causing erosions and deformity in the joints, and may be of involvement of organs such as lung and heart. The HLA-G is associated with the inhibition of immune cells, such as NK and T cells, conferring protection against inflammatory responses. Among the polymorphisms linked to its expression are the ones located in the 3'UTR region of the gene that include ten polymorphic sites described: a polymorphism of insertion or deletion (InDel) with a fragment of 14pb and nine SNPs (+3001C/T, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142G/C, +3187A/G, +3196G/C and +3227A/G). From this, the objective of this study will be to conduct a case-control study and the influence of these polymorphisms in disease manifestation. Alu family is the most common mobile element in primate genomes, with more than 1.1 million copies in Homo sapiens. The insertion AluyHG located between the loci HLA-A and HLA-G, may be a marker region. From this, the objective of this study was to genotype 115 patients and 115 controls (classified by age group) and the influence of these polymorphisms in the demonstration and development of the disease. For this, a survey was conducted of epidemiological and clinical data related to the development of the disease (smoking habits, rheumatoid factor, C-reactive protein and erythrocyte sedimentation rate) and these were tested. DNA was extracted from whole blood by PCR and genotyping made and viewed in vertical gel electrophoresis in polyacrylamide concentration of 7% stained with silver nitrate in the case of polymorphism of 14bp of the HLA-G, and 1% agarose gel in AluyHG case polymorphism (with 322pb). The HLA-G SNPs were identified through sequencing by the Sanger method. Calculations were made of the allele and genotype frequencies, analyzes of associations and haplotype inference. The analysis found associations of the polymorphisms and the risk of developing RA for genotypes +3196*G*C (OR=2,387, p=0,026), AluyHG*In (OR=0,524, p=0,006) AluyHG*Del (OR=1,907, p=0,006), AluyHG*In*Del (OR=0,441, p=0,006), AluyHG*Del*Del (OR=2,466, p=0,002). For heritage models, associations were found with: +3010*G+GC (OR=0,658, p=0,011), +3010*C+GC (OR=1,519, p=0,011), +3142*G+GC (OR=1,435, p=0,029), +3142*C+GC (OR=0,697, p=0,029), AluyHG*DD+IDxII (OR=0,402, p=0,002), AluyHG*I+ID (OR=0,465, p=0,000), AluyHG*D+ID (OR=2,152, p=0,000). For the clinical data, the following associations were found: RF and +3010*GG (OR=4,685, p=0,032) and +3196*GC (OR=0,388, p=0,049), CRP and +3196*GC (OR=0,301, p=0,011) and +3196*CC (OR=2,713, p=0,035), ESR and +3003*C (OR=0,296, p=0,042) and +3003*T (OR=3,382, p=0,042). For haplotypes and haplotype combinations were found: UTR5 (OR=2,174, p=0,048), UTR1+UTR5 (OR=5,532, p=0,034); in heritage models: UTR5 (OR=2,346, p=0,038), UTR1+AluyHG*Ins (OR=0,556, p=0,047), UTR5+AluyHG*Del (OR=2,247, p=0,050). In conclusion, this study allowed us to investigate the possible association of HLA-G gene in relation to rheumatoid arthritis, corroborating with the data in the literature and bringing some of the association results with autoimmune diseases who had not found any literature to date. More functional studies should be conducted to better understand how genetic polymorphisms can act in the pathogenesis of RA.