Introduction: Systemic sclerosis (SSc) is a generalized connective tissue disease affecting the skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. To date, none of the tested drugs have demonstrated convincing efficacy in the treatment of SSc. S100A4 is involved in the regulation of cell motility, proliferation, apoptosis, angiogenesis and remodeling of the extracellular matrix. It was originally described as a promoter of metastasis in tumors, however, its pro-inflammatory properties have recently been demonstrated in inflammatory rheumatic diseases. The aim of this study was to assess the role of S100A4 in pathological activation of fibroblasts in SSc and in experimental models of dermal fibrosis. Results: The expression of S100A4 was increased in the skin of SSc patients, in SSc fibroblasts and in experimental fibrosis in a TGF-β / Smad dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, inhibition of S100A4 or its complete deficit abrogated the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4 knock-out mice (S100A4-/- ) were protected from bleomycin-induced skin fibrosis with reduced dermal thickening,...

Introduction: Systemic sclerosis (SSc) is a generalized connective tissue disease affecting the skin and internal organs. The pathogenesis of SSc is characterized by inflammation, vasculopathy and fibrosis. To date, none of the tested drugs have demonstrated convincing efficacy in the treatment of SSc. S100A4 is involved in the regulation of cell motility, proliferation, apoptosis, angiogenesis and remodeling of the extracellular matrix. It was originally described as a promoter of metastasis in tumors, however, its pro-inflammatory properties have recently been demonstrated in inflammatory rheumatic diseases. The aim of this study was to assess the role of S100A4 in pathological activation of fibroblasts in SSc and in experimental models of dermal fibrosis. Results: The expression of S100A4 was increased in the skin of SSc patients, in SSc fibroblasts and in experimental fibrosis in a TGF-β / Smad dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, inhibition of S100A4 or its complete deficit abrogated the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4 knock-out mice (S100A4-/- ) were protected from bleomycin-induced skin fibrosis with reduced dermal thickening,...

Systemic sclerosis (SSc), or scleroderma, is a complex, rare disease of unknown etiology. Macrophages constitute a large portion of the immune cell infiltrate in the skin of patients with SSc, and are an important target of study. Particularly, the M2 macrophage has been implicated scleroderma and other fibrotic diseases as a key contributor to fibrotic processes. However, the definition of an M2 macrophage appears to change with context, and is poorly elucidated in different species. With varying characterizations between species and disease models, there is a need to establish some consensus on how to identify this macrophage in an uniform manner across species. We used a bioinformatic approach to identify a unique gene signature for the M2 macrophage phenotype, which is shared between human and mouse systems. We were able to confirm a 7-gene subset of this theorized signature using human and mouse in vitro systems. In addition, we selected one of the identified genes, Clec7a, and characterized its expression at the protein level on different macrophage phenotypes, across several human and mouse models. Our data show that Clec7a is a more selective marker of murine M2 macrophages than current reference markers, and is useful in human models as well. Using our M2-specific gene signature, we also identified a potential inhibitor of the signature and showed its effects on M2 marker expression. Finally, we showed some preliminary work into Clec7a expression in skin tissue from patients with scleroderma. Overall, our data suggest that Clec7a may be a valuable addition to the panel of markers used to characterize M2 macrophages and distinguish between macrophage phenotypes, and perhaps provide clarity into the development and function of the M2 macrophage. Better understanding of the M2 macrophage would ultimately be useful to the study of fibrotic diseases such as scleroderma, wherein this macrophage phenotype may be a viable target for antifibrotic therapy. / Thesis / Master of Science (MSc)

Made available in DSpace on 2015-03-26T13:42:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3332103 bytes, checksum: 7f48d2c607fe012e89c268995b92737e (MD5) Previous issue date: 2011-07-19 / Scleroderma laeve is a basidiomycete fungus capable of forming ectomycorrhizas with Eucalyptus grandis. Significant increases in lateral root production were observed in E. grandis plants inoculated with S. leave during the presymbiotic phase. This was also observed during the colonization, differentiation, and functioning stages of the symbiosis. On the third day of contact of the fungus with the host, the partial elongation of epidermal cells and mantle formation were observed, and, on the fifteenth day, the beginning of Hartig net formation was evident. On the 30th day, lateral root numbers in the plants in contact with the fungus was higher than in the control plants. The elongation of the epidermal cell and the thickening of the mantle could also be observed. The emerging lateral roots colonized by the fungus did not present root hairs, were completely enveloped by the fungal mantle, and had a fully developed Hartig net between the epidermal cells. The periodical sampling of lateral roots and ectomycorrhizas at different developmental stages in the in vitro system allowed following the morphological development of ectomycorrhizas and the corresponding changes in the expression of the gene coding for ATP synthase subunit VI in S. leave. The partial sequence of the ATP synthase subunit VI gene obtained possesses 503 bp and no intron. This sequences showed an identity to the sequences of the genes that code for e ATP synthase subunit VI in Scleroderma hypogaeum, Pisolithus arhizus, and Gyroporus cyanescens, among others, confirming the close relationship among these organisms. The expression analysis of the ATP synthase subunit VI gene in S. laeve by RT-PCR, at the different developmental stages of the symbiotic association, showed that the gene is expressed in all the phases of mycorrhization. / Scleroderma laeve é um fungo Basidiomiceto capaz de formar ectomicorrizas com Eucalyptus grandis. Foi observado aumento significativo do número de raízes laterais em plântulas de E. grandis em contato com S. laeve durante a fase de préinfecção em relação às plântulas não inoculadas. A inoculação com S. laeve também aumentou o número de raízes laterais em plântulas de E. grandis durante as etapas de colonização, diferenciação e funcionamento em relação às plântulas não inoculadas. No terceiro dia de contato entre o fungo e a planta, foi observado alongamento parcial das células da epiderme e formação do manto fúngico e no 15º dia foi observada a iniciação da formação da rede de Hartig. No trigésimo dia, o número de raízes laterais nas plântulas em contato com o fungo foi quatro vezes maior do que nas plântulas controle. Ocorreu, ainda, alongamento das células da epiderme e engrossamento do manto. As raízes laterais emergentes que formavam as ectomicorrizas não apresentavam pêlos radiculares, estavam totalmente envolvidas pelo manto fúngico e possuíam a rede de Hartig completamente formada entre as células alongadas da epiderme. Utilizando o sistema in vitro foi possível acompanhar, retirando-se raízes laterais e ectomicorrizas em diferentes estádios, as mudanças morfológicas da simbiose e de expressão do gene que codifica a subunidade seis de ATP sintase em S. laeve. A seqüência parcial do gene obtida possui 503 pb, na qual não foi observada a presença de introns. Essa sequência apresentou identidade com as sequências dos genes que codificam a subunidade seis de ATP sintase em S. hypogaeum, Pisolithus arhizus, Gyroporus cyanescens, entre outros, confirmando o parentesco entre esses organismos. A análise da expressão do gene que codifica a subunidade seis de ATP sintase em S. laeve por RT-PCR, nas diferentes etapas da associação, mostrou que esse gene é expresso em todas as etapas da micorrização.

Introdução: Os afro-brasileiros compreendem um grupo de negros e pardos com uma ancestralidade diversa. Como as diferenças raciais podem estar associadas a apresentações distintas, torna-se relevante realizar uma análise de associações clínicas e sorológicas de pacientes afro-brasileiros com esclerose sistêmica (ES). Objetivos: Analisar a apresentação clínica e laboratorial, características demográficas e sobrevida dos afro-brasileiros com ES. Métodos: Foram avaliados soros de 260 pacientes adultos com ES (57 afro-brasileiros e 203 brancos). Pacientes com síndromes de sobreposição foram excluídos. Dados clínicos e demográficos foram obtidos de um banco de dados de registros eletrônicos. A análise laboratorial incluiu anti-CENP-A/ CENP-B, Scl70, RNA polimerase III, Ku, fibrilarina, Th/To, PM/Scl75 e PM/Scl100 por line imunoblotting e anticorpos antinucleares por imunofluorescência indireta em células HEp-2. Resultados: Em relação aos brancos, os pacientes afro-brasileiros com ES apresentaram menor tempo de doença (9,0±5,4 anos vs. 11,3±7,5 anos, P=0,001), maior frequência do padrão nucleolar (28% vs. 13%, P=0,008) e menores frequências do padrão centromérico (14% vs. 29%, P=0,026) e CENP-B (18% vs. 34%, P=0,017). Comparações adicionais entre grupos étnicos de acordo com a forma clínica revelaram que pacientes afro-brasileiros com ES difusa apresentaram maior frequência de hipertensão pulmonar (P=0,017), envolvimento cardíaco (P=0,037), padrão nucleolar (P=0,036) e anticorpos antifibrilarina (P=0,037). De maneira diferente foi observado para a forma limitada apenas uma menor frequência de envolvimento esofágico (P=0,050) e padrão centromérico (P=0,049). A análise de sobrevida mostrou que os afro-brasileiros apresentaram maior mortalidade quando ajustados para sexo e forma clínica (risco relativo 3,65; intervalo de confiança de 95% 1,05-12,62, P=0,041). Conclusão: Os pacientes afro-brasileiros com ES, apesar de sua forte impressão de ancestralidade europeia, apresentam características distintas de acordo com a forma clínica e uma doença mais grave do que os brancos, com um padrão muito semelhante ao descrito para negros de outros países / Introduction: African-Brazilians comprise a group of blacks and \"pardos\" with a diverse ancestry. As racial differences can be associated with distinct presentations, it becomes relevant to assess the clinical and serological associations of African-Brazilians with systemic sclerosis (SSc). Objectives: To analyze clinical and laboratory presentation, demographic features and survival of Afro-Brazilians with SSc. Methods: Sera from 260 adult SSc patients (57 African-Brazilians and 203 whites) were evaluated. Patients with overlap syndromes were excluded. Clinical and demographic data were obtained from an electronic register database. Laboratory analysis included: anti-CENPA/CENP-B, Scl70, RNA polymerase III, Ku, fibrillarin, Th/To, PM/Scl75 and PM/Scl100 by line immunoassay and antinuclear antibodies by indirect immunofluorescence on HEp-2 cells. Results: In relation to whites, African-Brazilian patients with SSc presented shorter disease duration (9.0±5.4 years vs. 11.3±7.5 years, P=0.001), higher frequency of nucleolar pattern (28% vs. 13%, P=0.008) and lower frequencies of centromeric pattern (14% vs. 29%, P=0.026) and CENP-B (18% vs. 34%, P=0.017). Further comparison of ethnic groups according to subsets revealed that African-Brazilian patients with diffuse SSc presented higher frequency of pulmonary hypertension (P=0.017), heart involvement (P=0.037), nucleolar pattern (P=0.036) and anti-fibrillarin antibodies (P=0.037). A different pattern was observed for the limited subset with solely a lower frequency of esophageal involvement (P=0.050) and centromeric pattern (P=0.049). Survival analysis showed that African-Brazilians had a higher mortality, when adjusted for gender and clinical subset (relative risk 3.65, confidence interval 95% 1.05-12.62, P=0.041). Conclusion: African-Brazilian patients with SSc, despite their strong imprint of European ancestry, have distinct characteristics according to clinical subset and an overall more severe disease than whites, with a pattern very similar to the described for blacks from other countries

A lesão pulmonar é a principal causa de morte da Esclerose Sistêmica (ES), e as alterações principais são: o acometimento intersticial e o vascular. No presente estudo analisamos através do microscópio confocal a laser 40 artérias pulmonares de pequeno e médio calibre de pacientes com ES e 16 controles. Medimos a área do lúmen, a área total do vaso e fizemos a subtração da área total do vaso menos a do lúmen, e a porcentagem da área do lúmen em relação à área total do vaso. Observou-se que a área do lúmen e a porcentagem da área do lúmen em relação a área total do vaso são significativamente menores na ES em relação ao controle, e que a diferença entre a área total do vaso e a área do lúmen foi maior no grupo ES. Os achados confirmaram a hipótese inicial de acometimento das artérias pulmonares na ES, que se encontram espessadas devido à inflamação, infiltração celular em suas camadas e ativação endotelial / Lung injury is the leading cause of death in Systemic Sclerosis (SSc), and the main changes are: the vascular and interstitial involvement. In this study we analyzed through the confocal laser microscope 40 lung arteries of small and medium-sized of patients with SSc and 16 arteries of control group. We measured the lumen area, the total vessel area, made the subtracting the total vessel area minus the lumen area and the percentage between the lumen area and total vessel area. It was observed that the lumen area and the percentage between the lumen area and total vessel area were significantly lower in SSc group compared to control group, and the difference between the total vessel and the lumen area was higher in SSc. The findings confirmed the initial hypothesis of pulmonary arterial injury in SSc, wich are thickened due to inflammation, cellular infiltration into its layers and endothelial activation

A lesão pulmonar é a principal causa de morte da Esclerose Sistêmica (ES), e as alterações principais são: o acometimento intersticial e o vascular. No presente estudo analisamos através do microscópio confocal a laser 40 artérias pulmonares de pequeno e médio calibre de pacientes com ES e 16 controles. Medimos a área do lúmen, a área total do vaso e fizemos a subtração da área total do vaso menos a do lúmen, e a porcentagem da área do lúmen em relação à área total do vaso. Observou-se que a área do lúmen e a porcentagem da área do lúmen em relação a área total do vaso são significativamente menores na ES em relação ao controle, e que a diferença entre a área total do vaso e a área do lúmen foi maior no grupo ES. Os achados confirmaram a hipótese inicial de acometimento das artérias pulmonares na ES, que se encontram espessadas devido à inflamação, infiltração celular em suas camadas e ativação endotelial / Lung injury is the leading cause of death in Systemic Sclerosis (SSc), and the main changes are: the vascular and interstitial involvement. In this study we analyzed through the confocal laser microscope 40 lung arteries of small and medium-sized of patients with SSc and 16 arteries of control group. We measured the lumen area, the total vessel area, made the subtracting the total vessel area minus the lumen area and the percentage between the lumen area and total vessel area. It was observed that the lumen area and the percentage between the lumen area and total vessel area were significantly lower in SSc group compared to control group, and the difference between the total vessel and the lumen area was higher in SSc. The findings confirmed the initial hypothesis of pulmonary arterial injury in SSc, wich are thickened due to inflammation, cellular infiltration into its layers and endothelial activation

The overall objective of this thesis work was to develop new insights into the etiology of scleroderma, a human systemic autoimmune disease, by analyzing the autoantibodies to centrosome antigens that develop during the disease. Centrosomes are perinuclear organelles that form microtubule arrays, including mitotic spindles that ensure the faithful segregation of chromosomes during mitosis. These studies used a novel methodology to determine the prevalence of anti-centrosome autoantibodies in patients with scleroderma. Recombinant centrosome antigens were used to determine the antigenic specificity of anti-centrosome antibody subsets by immunoblotting. Centrosome marker antibodies were used in indirect immunofluorescence assays to distinguish centrosomes within the polymorphic staining pattern frequently given by scleroderma sera. We found that 43% of patients are autoreactive to centrosomes, a prevalence higher than has been reported for any other scleroderma autoantigen. Half of the centrosome-positive patients also had autoantibodies against other antigens used in scleroderma diagnosis. However, in the remaining half of these patients, anti-centrosome antibodies represented the sole class of autoantibodies that was detectable. Anti-centrosome antibodies were detected in only a small percentage of normal individuals and patients with other connective tissue diseases. These data suggest that anti-centrosome autoantibodies may represent a new diagnostic tool in scleroderma. Upon examination of anti-centrosome autoantibody development in an animal model, it appeared that this autoantibody specificity may develop in mice as a consequence of an infection. An infectious agent was isolated by plaque-formation from carrier mice. Further characterization of the infectious agent was undertaken to obtain information on its physical, morphological and cytopathological properties. The infectious agent was identified by sequence and unique antigenic properties to be homologous to the pig pathogen Mycoplasma hyorhinis. When reintroduced into naive mice, the murine mycoplasma triggered anti-centrosome autoantibody development. While anti-centrosome autoantibodies of IgM isotype are part of the repertoire of naive unimmunized mice, mycoplasma infection specifically triggered the development of anti-centrosome IgG. Moreover, centrosome autoreactivity was prevented by antibiotic treatment. The autoantibody response evolved to recruit additional specificities, having IgM isotypes, reactive to endoplasmic reticulum-associated autoantigens.

Made available in DSpace on 2015-03-26T13:51:53Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4350402 bytes, checksum: bd6bf9393be337ef287514591c95188f (MD5) Previous issue date: 2011-07-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The ectomycorrhizal association is a mutualistic interaction between plant roots and soil fungi that causes morphophysiological changes in the plant root system. The nutritional benefits result from the capacity of the fungus to increase the uptake of mineral nutrients by the host plant in exchange for photoassimilates. For the ectomycorrhizal association between Scleroderma laeve and Eucalyptus grandis, there is no information on which genes are decisive for the symbiosis and how they relate to the formation of ectomycorrhizas. Transcripts of the genes ras and ef1α were dentified as differentially expressed in many symbiotic associations and are related to signal transduction pathways and protein synthesis, respectively. Thus, the objectives of this work were to establish the ectomycorrhizal association between S. laeve and E. grandis in vitro, to isolate partial sequences of the genes ras and ef1α of S. laeve, and to evaluate the functional expression of these genes during ectomycorrhizal formation. Our works demonstrates the ectomycorrhizal association between S. laeve and E. grandis in vitro for the first time. The typical structures of ectomycorrhizas, such as the mantle and the Hartig net, were observed. At three days of contact between S. laeve and E. grandis roots the beginning of mantle formation could be observed. At 15 days, the mantle was completely formed, the epidermal cells were elongated, and the Hartig net formation was in progress. At 30 days, the ectomycorrhizas presented all the typical morphological structures fully developed. To evaluate gene expression during the association, partial sequences of ras and ef1α were isolated and the transcripts evaluated at the pre-symbiotic phase and at 3, 15 and 30 days after physical contact of the fungus with the plant roots. The transcripts of the gene ef1α were expressed at all evaluated times. Transcripts of ras were only detected in the ectomycorrhizas after three, 15, and 30 days of contact. These results are fundamental for a better understanding of the ectomycorrhizal association between S. laeve and E. grandis and suggest that ras-mediated signal transduction pathways may be functional during the establishment of the symbiosis between the fungus and the host roots. / A associação ectomicorrízica é uma interação mutualista entre raízes de plantas e fungos do solo, resultando em mudanças morfofisiológicas do sistema radicular das plantas. Os benefícios nutricionais advêm da capacidade do fungo em aumentar a absorção de nutrientes minerais pelas plantas, recebendo em troca os fotoassimilados. Na associação entre Scleroderma laeve e Eucalyptus grandis ainda não se tem informações de quais genes são decisivos e estão relacionados a este processo. Transcritos dos genes ras e ef1α foram identificados durante a formação da simbiose e sendo diferencialmente expressos na associação ectomicorrízica, e estão relacionados a vias de transdução de sinal e atuando na síntese protéica, respectivamente. Assim, os objetivos deste trabalho foram estabelecer a associação ectomicorrízica in vitro entre S. laeve e E. grandis, isolar sequências parciais dos genes ras e ef1α do fungo ectomicorrízico S. laeve, e avaliar a expressão funcional destes genes durante as fases de formação das ectomicorrizas. Este trabalho comprova a associação in vitro entre S. laeve e E. grandis, sendo registrada pela primeira vez. As estruturas típicas das ectomicorrizas, como a formação do manto fúngico e da rede de Hartig foram observadas. Nos tempos avaliados, em três dias de contato já havia a formação do manto fúngico. Aos 15 dias, o manto fúngico estava completamente formado, as células da epiderme alongadas e a rede de Hartig, em formação. Aos 30 dias, as ectomicorrizas apresentavam todas as estruturas típicas desenvolvidas. Para avaliar a expressão dos genes durante a associação, sequências parciais dos genes ras e ef1α foram isolados, e os transcritos destes genes foram avaliados na fase pré-simbiótica e aos três, 15 e 30 dias após o contato físico. Os transcritos do gene ef1α foram expressos durante todos os tempos avaliados. Os transcritos do gene ras foram detectados nas ectomicorrizas após três, 15 e 30 dias. Estes resultados são fundamentais para uma melhor compreensão da associação ectomicorrízica entre S. laeve e E. grandis e sugerem que as vias de transdução de sinais mediada por ras podem ser funcionais durante o estabelecimento da simbiose entre os fungos e as raízes de plantas.

INTRODUÇÃO: Já é sabido que a capacidade funcional é reduzida nos pacientes com doenças reumatológicas. Entretanto, há poucos estudos que avaliam a capacidade funcional em pacientes com esclerodermia, uma doença rara com manifestações vasculares, músculo-esqueléticas, e viscerais. OBJETIVO: Avaliar a capacidade funcional de pacientes com esclerodermia do sexo feminino. MÉTODO: Treze pacientes sem limitação pulmonar (matriculadas no ambulatório de esclerodermia da Disciplina de Reumatologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo) e 13 controles (funcionárias do mesmo Hospital), todas sedentárias e pareadas por idade e índice de massa corporal, foram submetidas à um teste de esforço máximo em esteira. RESULTADOS: O consumo máximo de oxigênio das pacientes foi estatisticamente mais baixo do que o apresentado pelo grupo controle (p=0,0395), assim como a porcentagem atingida do consumo máximo de oxigênio (p=0,0383) e a intensidade máxima de esforço alcançada (p=0,0395). O tempo entre os limiares ventilatórios também foi mais baixo no grupo esclerodermia (p=0,0271), porém não houve diferença estatística entre os grupos em relação ao consumo de oxigênio medido nos limiares (p=0,6021 para o limiar anaeróbio e p=0,3387 para o ponto de compensação respiratório). CONCLUSÃO: As pacientes com esclerodermia, mesmo sem comprometimento pulmonar, apresentam uma capacidade mais baixa de desempenhar um esforço físico de moderado a intenso quando comparadas à mulheres sedentárias da mesma idade e índice de massa corporal. / INTRODUCTION: It is already known that funcional capacity of patients with rheumatic diseases is reduced. However, there are few studies that evaluate functional capacity in patients with scleroderma, a rare disease with vascular, skeletal muscle and visceral manifestations. OBJECTIVE: To evaluate functional capacity of scleroderma female patients. METHOD: Thirteen patients without pulmonary impairment (registered at Scleroderma Service from Rheumatology Discipline from Clinicas Hospital from University of São Paulo School of Medicine) and 13 controls (employees from the same Hospital), all sedentary and paired by age and body mass index, undertook a treadmill exercise test. RESULTS: Maximal oxygen consumption of patients was statistically lower than control group (p=0,0395), as well as the percentage of predicted VO2 max (p=0,0383) and maximal attained exercise intensity (p=0,0395). Time between ventilatory thresholds was also lower in scleroderma group (p=0,0271), but there was no statistical difference between groups in oxygen consumption measured in thresholds (p=0,6021 for anaerobic threshold and e p=0,3387 for respiratory compensation point). CONCLUSION: Scleroderma patients, even without pulmonary disease, present a lower ability for a moderate to intense physical effort when compared with sedentary women of the same age and body mass index.