Histomorphometric analysis of cutaneous remodeling in the early stage of the scleroderma model

INTRODUCTION/OBJECTIVES: Systemic sclerosis, or scleroderma, is a rheumatic disease characterized by autoimmunity, vasculopathy, and fibrosis of the skin and several internal organs. In the present study, our aim was to assess the skin alterations in animals with scleroderma during the first stages of disease induction. METHODS: To induce scleroderma, female New Zealand rabbits (n = 12) were subcutaneously immunized with 1 mg/ml of collagen V (Col V) in complete Freund's adjuvant, twice with a thirty-day interval. Fifteen days later, the animals received an intramuscular booster with type V collagen in incomplete Freund's adjuvant, twice with a fifteen-day interval. The control group was inoculated with 1 ml of 10 mM acetic acid solution diluted with an equal amount of Freund's adjuvant. Serial dorsal skin biopsies were performed at 7, 15, and 30 days and stained with H&E, Masson's trichrome and Picrosírius for morphological and morphometric analyses. RESULTS: Immunized rabbits presented a significant increase in collagen in skin collected seven days after the first immunization (p=0.05). CONCLUSION: The results from this experimental model may be very important to a better understanding of the pathogenic mechanisms involved in the beginning of human SSc. Therapeutic protocols to avoid early remodeling of the skin may lead to promising treatments for SSc in the future

Aerobic Exercise Attenuated Bleomycin-Induced Lung Fibrosis in Th2-Dominant Mice

Introduction The aim of this study was to investigate the effect of aerobic exercise (AE) in reducing bleomycin- induced fibrosis in mice of a Th2-dominant immune background (BALB/c). Methods BALB/c mice were distributed into: sedentary, control (CON), Exercise-only (EX), sedentary, bleomycin-treated (BLEO) and bleomycin-treated+exercised (BLEO+EX);(n = 8/group). Following treadmill adaptation, 15 days following a single, oro-tracheal administration of bleomycin (1.5U/kg), AE was performed 5 days/week, 60min/day for 4 weeks at moderate intensity (60% of maximum velocity reached during a physical test) and assessed for pulmonary inflammation and remodeling, and cytokine levels in bronchoalveolar lavage (BAL). Results At 45 days post injury, compared to BLEO, BLEO+EX demonstrated reduced collagen deposition in the airways (p<0.001) and also in the lung parenchyma (p<0.001). In BAL, a decreased number of total leukocytes (p<0.01), eosinophils (p<0.001), lymphocytes (p<0.01), macrophages (p<0.01), and neutrophils (p<0.01), as well as reduced pro-inflammatory cytokines (CXCL-1;p<0.01), (IL-1 beta;p<0.001), (IL-5;p<0.01), (IL-6;p<0.001), (IL-13;p<0.01) and pro-fibrotic growth factor IGF-1 (p<0.001) were observed. Anti-inflammatory cytokine IL-10 was increased (p<0.001). Conclusion AE attenuated bleomycin-induced collagen deposition, inflammation and cytokines accumulation in the lungs of mice with a predominately Th2-background suggesting that therapeutic AE (15-44 days post injury) attenuates the pro-inflammatory, Th2 immune response and fibrosis in the bleomycin model

Tradução e validação da Clinician Administered PTSD Scale (CAPS-CA-5) em crianças e adolescentes portugueses

Introduction: The aim of this study was to translate and validate into European Portuguese the CAPS-CA-5 (Clinician Administered PTSD Scale for Children and Adolescents), a semi-structured scale for the diagnosis of post-traumatic stress disorder in children and adolescents, according to the DSM-5 criteria. Material and Methods: This study was developed in three stages. In the first stage, the translation and back-translation of CAPS-CA-5 into European Portuguese was carried out. In the second stage, the version obtained in the previous step was subjected to a pre-test. In the third stage, the final version of CAPS-CA-5, the KIDCOPE questionnaires and the Depression, Anxiety and Stress Scale-Children were applied to 101 children who had experienced at least one potentially traumatic event. The children included in this study were between seven and 18 years old and had a follow-up period in a Child Psychiatry or Pediatrics Clinic in one of the three hospitals involved in this project of at least one month. Results: Regarding the confirmatory factor analysis, our results show that the CAPS-CA-5 is a suitable psychometric instrument to assess the diagnosis and symptoms severity of post-traumatic stress disorder according to DSM-5. Convergent validity was comparable to its original version. Although there were negative relationships with almost all of its clusters, these were not statistically significant when applied with the positive coping strategies of the KIDCOPE. The European Portuguese version of the CAPS-CA-5 showed a good internal consistency (Cronbach’s α for the total scale was 0.89). Conclusion: The European Portuguese version of CAPS-CA-5 has similar psychometric properties to its original versionIntrodução: O objetivo deste estudo foi traduzir e validar para português europeu a CAPS-CA-5 (Clinician Administered PTSD Scale for Children and Adolescents), uma escala semiestruturada para o diagnóstico de perturbação de stress pós-traumático em crianças e adolescentes, de acordo com os critérios do DSM-5. Material e Métodos: Este estudo foi desenvolvido em três etapas. Na primeira, foi realizada a tradução e contra-tradução da CAPS- -CA-5 para português europeu. Na segunda etapa, a versão obtida anteriormente foi submetida a um pré-teste. Na terceira etapa, a versão final da CAPS-CA-5, os questionários KIDCOPE e a Escala de Depressão, Ansiedade e Stresse - Crianças foram aplicados em 101 crianças que experienciaram pelo menos um evento potencialmente traumático. As crianças incluídas neste estudo tinham entre sete e 18 anos e tinham um período de acompanhamento em consulta de Psiquiatria Infantil ou Pediatria de pelo menos um mês, num dos três hospitais envolvidos neste projeto. Resultados: Em relação à análise fatorial confirmatória, os nossos resultados mostram que a CAPS-CA-5 é um instrumento psico-métrico adequado para avaliar o diagnóstico e a gravidade dos sintomas de perturbação de stresse pós-traumático de acordo com o DSM-5. A validade convergente foi comparável à versão original. Embora tenha havido relações negativas com quase todos os seus clusters, estas não foram estatisticamente significativas quando aplicadas com as estratégias de coping positivo do KIDCOPE. A versão em português europeu da CAPS-CA-5 apresentou boa consistência interna (α de Cronbach para a escala total foi de 0,89). Conclusão: A versão em português europeu do CAPS-CA-5 possui propriedades psicométricas semelhantes à sua versão originalinfo:eu-repo/semantics/publishedVersio

Collagen V- induced nasal tolerance in scleroderma experimental model

Objetivo: Verificar o remodelamento da pele e produção de anticorpos em modelo experimental de esclerodermia em coelhos, após indução de tolerância nasal com colágeno tipo V. Métodos: Coelhas Nova Zelândia (N=12) foram imunizadas com 1mg/ml de colágeno V (Col V) em adjuvante completo de Freund e dois reforços com adjuvante incompleto de Freund. Seis coelhas imunizadas receberam uma dose diária de 25ug de Col V, iniciado via nasal (grupo tolerado) 150 dias do começo das imunizações e seis animais foram somente imunizadas (grupo imunizado). Um grupo imunizado com adjuvante de Freund serviu como controle. Biopsias de pele foram coletadas em 0, 75, 120, 150 e 210 dias e coradas pelo H&E, tricrômico de Masson e Sírius red para analise morfológica e morfométrica. Os colágenos I, III e V, além de TGFbeta e PDGF foram imunomarcados por imunofluorescência. Os soros dos animais foram coletados em 0, 150 e 210 dias para determinar anticorpos anti-colágenos I, III, IV e V e anti-nucleares. Resultados: Os animais imunizados mostraram progressivo decréscimo da derme papilar, atrofia de anexos, aumento no depósito dos colágenos I, III e V e aumento da expressão de TGFbeta e PDGF. Os tolerados apresentaram aumento dos anexos cutâneos e significante diminuição no depósito dos colágenos I, III e V, TGFbeta e PDGF. O grupo de imunizados e de tolerados apresentaram anticorpos anti-colágenos III e IV e antinucleares. Conclusões: A indução de tolerância nasal com Col V diminuiu o remodelamento da pele observado no modelo experimental de esclerodermia e inibiu a síntese de citocinas fibrogênicas. Portanto, a tolerância nasal com Col V pode ser uma opção terapêutica promissora para o controle do remodelamento cutâneo em pacientes com esclerodermia.Objective: Our aim was to verify the skin remodeling and antibody production in experimental model of scleroderma in rabbits, after induction of tolerance by daily nasal administration of human type V collagen (Col V). Methods: Female New Zealand rabbits (N=12) were immunized with 1mg/ml of Col V in complete Freund\'s adjuvant, followed by more two boosters in incomplete Freund\'s adjuvant. Six immunized rabbits received daily nasal administrating of 25ug of Col V (tolerated group), started 150 days after the first immunization, and the others animals (N=6) were only immunized (immunized group). Finally a group of rabbits immunized with Freund\'s adjuvant served as control. Skin biopsies were collected at 0, 75, 120, 150 and 210 days, and stained with H&E, Masson\'s trichrome and Sirius red for morphological and morphometric analysis. Types I, III and V collagen, TGFbeta and PDGF were immunostained by immunofluorescence. The sera of animals were colleted at 0, 150 and 210 days to determine anti types I, III, IV and V collagen and antinuclear antibodies. Results: The immunized animals showed progressive decrease of papillary dermis, appendages atrophy, increase of types I, III and V collagen deposition and increased expression of TGF-beta and PDGF. The tolerated rabbits presented increase of cutaneous appendages and significant decrease of types I, III and V and TGF-beta and PDGF. Both immunized and tolerated rabbits presented anti types III and IV antibodies and antinuclear antibodies. Conclusions: Col V nasal tolerance reduced skin remodeling in experimental model of scleroderma and inhibited synthesis of fibrotic cytokines. Therefore, the nasal tolerance with type V collagen can be a promising therapeutic option to control the skin remodeling in patients with scleroderma

Auto-imunidade e colágeno V Autoimmunity and collagen V

As proteínas da matriz extracelular (MEC) e seus componentes estão sendo amplamente estudados na literatura médica, assim como sua relação com o remodelamento tecidual presente nas doenças reumáticas. Neste artigo, mostramos a importância do estudo do colágeno do tipo V no entendimento da etiologia da esclerodermia, no que se refere ao desencadeamento da auto-imunidade nesta enfermidade. Estudos em nosso laboratório demonstram que a sensibilização com colágeno do tipo V em coelhos pode resultar em um modelo animal de esclerodermia. Diante destes fatos, sugerimos que pesquisas neste campo podem ser de grande valia no desenvolvimento de novas condutas terapêuticas.<br>The extracellular matrix (ECM) proteins and their components have been widely studied in medical literature, as well its relationship with the tecidual remodeling present in the rheumatic disease. In this paper we show the importance of understanding the role of type V collagen as an important trigger of rheumatic autoimmune diseases. Studies in our laboratory demonstrate that type V collagen sensibilization in rabbits, could result in an animal scleroderma model. In this way we suggested that researches in this field can be worthy in development of new therapeutic procedures

Manifestations in the esophagus in the scleroderma model induced by type V collagen in rabbits

Introdução: a esclerose sistêmica progressiva acomete diversas vísceras, das quais o esôfago é uma das mais freqüentemente lesadas. No entanto, o mecanismo de autoimunidade nesta enfermidade é ainda desconhecido. Objetivo: avaliar morfologicamente esôfagos de animais após imunização com colágeno tipo V, correlacionando-os com as alterações observadas em humanos. Materiais e métodos: foram analisados esôfagos de coelhos do modelode esclerodermia induzido por colágeno V. As amostras foram analisadas morfologicamente pelas técnicas de H&amp;E, Tricrômico de Masson, Picro Sirius e Imunofluorescência. Resultados: observou-se aumento progressivo das fibras colágenas, caracterizado por alterações no epitélio, submucosa, camada muscular e adventícia. A análise da imunofluorescência revelou a existência de remodelamento dos diferentes tipos de colágeno da matriz. Discussão: o estudo de espécimes esofágicos em humanos é difícil. Na maioria das vezes só é possível analisá-lo em fases avançadas da doença. No modelo estudado acreditamos que as alterações observadas estão relacionadas a um processo inicial da doença e que o estudo de animais sacrificados após um tempo maior irá revelar uma fase mais avançada da doença.Backgrounds: Sclerosis systemic that usually affects the esophagus. However its patologic mechanism is still unknown. Objective: Evaluate the manifestation of the esophagus in a experimental model of scleroderma induced by type V collagen in rabbits. Matherial and Methods: 39 rabbits submited to the model immunization were morfologicaly analised. Results: A progressive deposit of collagen fibers was observed in H&amp;E and Tricomico of Masson. Which was confirmed by the imunofluorescence. Conclusion: We concluded that the characteristics observeds are similar to what happens in the esophagus of human, but in an earlier period

Aerobic Exercise Attenuated Bleomycin-Induced Lung Fibrosis in Th2-Dominant Mice

Introduction The aim of this study was to investigate the effect of aerobic exercise (AE) in reducing bleomycin- induced fibrosis in mice of a Th2-dominant immune background (BALB/c). Methods BALB/c mice were distributed into: sedentary, control (CON), Exercise-only (EX), sedentary, bleomycin-treated (BLEO) and bleomycin-treated+exercised (BLEO+EX);(n = 8/group). Following treadmill adaptation, 15 days following a single, oro-tracheal administration of bleomycin (1.5U/kg), AE was performed 5 days/week, 60min/day for 4 weeks at moderate intensity (60% of maximum velocity reached during a physical test) and assessed for pulmonary inflammation and remodeling, and cytokine levels in bronchoalveolar lavage (BAL). Results At 45 days post injury, compared to BLEO, BLEO+EX demonstrated reduced collagen deposition in the airways (p<0.001) and also in the lung parenchyma (p<0.001). In BAL, a decreased number of total leukocytes (p<0.01), eosinophils (p<0.001), lymphocytes (p<0.01), macrophages (p<0.01), and neutrophils (p<0.01), as well as reduced pro-inflammatory cytokines (CXCL-1;p<0.01), (IL-1 beta;p<0.001), (IL-5;p<0.01), (IL-6;p<0.001), (IL-13;p<0.01) and pro-fibrotic growth factor IGF-1 (p<0.001) were observed. Anti-inflammatory cytokine IL-10 was increased (p<0.001). Conclusion AE attenuated bleomycin-induced collagen deposition, inflammation and cytokines accumulation in the lungs of mice with a predominately Th2-background suggesting that therapeutic AE (15-44 days post injury) attenuates the pro-inflammatory, Th2 immune response and fibrosis in the bleomycin model

Butylated hydroxytoluene induces type-V collagen and overexpression of remodeling genes/proteins in experimental lung fibrosis

Anomalous histoarchitecture with increased levels of type-V collagen (Col V) in lungs of human idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM) airway-centered interstitial fibrosis suggest that this collagen can be a possible trigger involved in the pathogenesis of these diseases. Butylated hydroxytoluene (BHT) injury model revealed a distal involvement of lung parenchyma with significant endothelial injury and fibrotic response, contrasting with the BLM airway-centered insult. We undertook this study to analyze whether BHT alters distal airway/alveolar epithelial cells (AECs) and extracellular matrix (ECM) signaling involved in the initiation and progression of pulmonary fibrosis in a different pathway concerning overexpression of Col V. Female mice C57BL/6 (n=6) were instilled intraperitoneally with 400mg/kg of BHT dissolved in 1 mL of corn oil and euthanized at day 14 or 21 after BHT administration. Morphometry, immunohistochemistry and transmission electron microscopy were performed to characterize microscopic and submicroscopic changes of AECs and endothelial cells through transforming growth factor beta (TGF-β) basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) expression. Immunofluorescence and immunogold electron microscopy were performed to characterize Col V. Quantitative polymerase chain reaction (qPCR) was used to confirm differential levels of RNA messenger. BHT lungs showed marked fibrotic areas and hyperplastic AECs. The alveolar damage caused destruction of elastic fibers and a critical increase of Col V in ECM of distal lung parenchyma. Fibrogenesis-promoting markers TGF-β, bFGF and VEGF were also overexpressed in situ, coinciding with up-regulation in remodeling enzymes, growth factors, cytokines, transduction and transcription genes. BHT alters distal lung parenchyma signaling involved in pulmonary fibrosis highlighted similarities to human IPF in a pathway involving Col V arising as a promissory model to identify effective therapeutic targets

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N=10) were immunized with collagen V plus Freund’s adjuvant and compared with animals inoculated with adjuvant only (N=10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69±80.31; 24.46±2.58; 70.51±7.66, respectively) in immunized rabbits when compared with animals from control group (164.91±15.67; 12.89±1.05; 32±3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients