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Master's Dissertation
DOI
https://doi.org/10.11606/D.17.2020.tde-07042021-144946
Document
Author
Full name
Djúlio César Zanin da Silva
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2020
Supervisor
Committee
Rodrigues, Maria Carolina de Oliveira (President)
Frade, Marco Andrey Cipriani
Oliveira, Carolina Caliári
Title in Portuguese
Expressão de marcadores de fibrose em pacientes com esclerose sistêmica submetidos a transplante autólogo de células-tronco hematopoéticas
Keywords in Portuguese
Doenças autoimunes
Esclerose sistêmica
Fibrose
Tecido conjuntivo
Terapia celular
Transplante de células-tronco hematopoéticas
Abstract in Portuguese
A esclerose sistêmica (ES) é uma doença autoimune marcada por desregulação da imunidade, vasculopatia, fibrose da pele e de órgãos internos. O Transplante Autólogo de Células-Tronco Hematopoéticas (TACTH) tem sido investigado como opção de tratamento para pacientes com formas graves e progressivas de ES. Estudos clínicos mostram que o TACTH promove melhora clínica, mas os mecanismos terapêuticos básicos associados ao procedimento não estão completamente esclarecidos. O presente estudo teve como objetivo avaliar se o TACTH promove mudanças na expressão de moléculas associadas ao processo fibrótico e/ou fisiologia do tecido conjuntivo em pacientes com ES, correlacionando-os com medidas clínicas de fibrose cutânea. Avaliamos, retrospectivamente, dados clínicos, amostras de soro e de pele de 44 pacientes com ES submetidos ao TACTH. Biópsias de pele (obtidas do antebraço) foram avaliadas por imunohistoquímica com anticorpos anti-MMP-1, -MMP-2, -MMP-3, -MMP-9, -TIMP-1, -α-SMA e -TGF-β e, coradas com Hematoxilina e Eosina (H&E) e Picrosirius Red. A análise das imagens obtidas foi realizada pelo software ImageJ. Amostras de soro disponíveis de 27 pacientes, foram avaliadas por Ensaio Multiplex, para COL1A1, COL4A1, FGF-1, MMP-1, MMP-3, MMP-12, MMP-13, PDGF-AA, PDGF-BB, S100A9 e TIMP-1. A idade média e o desvio padrão (DP) dos pacientes foi de 36 (9,9) anos, sendo 82% pertencentes ao sexo feminino. O tempo médio entre o diagnóstico e a realização do transplante foi de 3,3 (±3,1) anos. Após o TACTH, houve melhora clínica da fibrose cutânea, com redução do escore cutâneo de Rodnan modificado aos 6 e 12 meses pós-TACTH. Histologicamente, após o TACTH, observamos diminuição significativa da densidade de colágeno (avaliada por Picrosirius Red) e da espessura da pele (avaliada por H&E). As análises imunohistoquímicas demonstraram aumento da expressão de metaloproteinases (MMP-1, 2, 3 e 9) e de seu inibidor tecidual (TIMP-1), pós-TACTH. Embora a expressão cutânea de α-SMA e de TGF-β não tenham se alterado significativamente pós-transplante, houve correlação positiva entre a expressão de TGF-β e a densidade de colágeno na pele no período pós-TACTH. No soro, houve redução nos níveis de MMP-1, S100A9, PDGF-AA, PDGF-BB, TIMP-1 e COL4A1 após o TACTH, quando comparados aos níveis pré-TACTH. Os níveis séricos de COL1A1 aumentaram depois do TACTH. As concentrações séricas de MMP-3 aumentaram em 6 meses, mas decaíram para os níveis basais em 12 meses após o TACTH. Os níveis séricos de MMP-12, MMP-13 e FGF-1 não se alteraram após o transplante. Assim, o TACTH diminuiu a fibrose da pele e modificou a expressão de moléculas relacionadas à manutenção do tecido conjuntivo, inflamação e dano endotelial, em pacientes com ES. Acreditamos, portanto, que além do controle da autorreatividade imunológica, outros mecanismos terapêuticos contribuam para os desfechos clínicos dos pacientes com ES submetidos ao TACTH.
Title in English
Expression of fibrosis markers in systemic sclerosis patients treated with autologous hematopoietic stem cell transplantation
Keywords in English
Autoimmune diseases
Cell-based therapy
Connective tissue
Fibrosis
Hematopoietic stem cell transplantation
Systemic sclerosis
Abstract in English
Systemic sclerosis (SSc) is an autoimmune disease marked by immunological dysregulation, vasculopathy, and fibrosis of the skin and internal organs. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) has been investigated as a treatment option for patients with severe and progressive SSc. Clinical trials have shown that AHSCT promotes clinical improvement. However, the basic therapeutic mechanisms associated with the procedure are not completely understood. The present study aimed to evaluate if AHSCT modifies the expression of molecules associated with the fibrotic process and/or connective tissue physiology in patients with SSc, and how these findings correlate with clinical skin fibrosis scores. We retrospectively evaluated clinical data, serum and skin samples from 44 patients with SSc who underwent AHSCT. Skin biopsies (obtained from the forearm) were marked, through immunohistochemistry, with anti-MMP-1, -MMP-2, -MMP-3, -MMP-9, -TIMP-1, -α-SMA and -TGF-β antibodies, and stained with Hematoxylin and Eosin (H&E) and Picrosirius Red. Microscopy images were analyzed by ImageJ software. Serum samples available from 27 patients were evaluated by Multiplex Assay for COL1A1, COL4A1, FGF-1, MMP-1, MMP-3, MMP-12, MMP-13, PDGF-AA, PDGF-BB, S100A9 and TIMP-1. Patients' mean age (standard deviation, SD) was 36 (9.9) years and 82% were female. Average time between diagnosis and transplantation was 3.3 (3.1) years. After AHSCT, there was clinical improvement of skin fibrosis, with reduction in the modified Rodnan's skin score (mRSS) at 6 and 12 months after AHSCT. Histologically, there was significant decrease in collagen density (assessed by Picrosirius Red) and in skin thickness (assessed by H&E) after AHSCT. Immunohistochemical analyses showed increased expression of metalloproteinases (MMP-1, 2, 3 and 9) and its tissue inhibitor (TIMP-1), post-AHSCT. Although there are no significant changes in the cutaneous expression of α-SMA and TGF-β after transplantation, there was positive correlation between the expression of TGF-β and the collagen density in skin in the post-AHSCT period. In the serum samples, there was reduction of MMP-1, S100A9, PDGF-AA, PDGF-BB, TIMP-1 and COL4A1 levels after AHSTC. Serum COL1A1 levels increased after AHSCT. Serum MMP-3 concentrations increased at 6 months but declined to baseline levels at 12 months after AHSCT. Serum levels of MMP-12, MMP-13 and FGF-1 did not change after transplantation. In conclusion, AHSCT decreases skin fibrosis and modifies the expression of molecules related to the maintenance of connective tissue, inflammation, and endothelial damage, in patients with SSc. We believe, therefore, that in addition to the control of immunological autoreactivity, other therapeutic mechanisms contribute to the clinical outcomes of patients with SSc undergoing TACTH.
 
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Publishing Date
2021-04-19
 
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