Development of different degrees of elastase-induced emphysema in mice: a randomized controlled experimental study

Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity.Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined.Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase.Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes.Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity. Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined. Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase. Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes

Development of different degrees of elastaseinduced emphysema in mice : a randomized controlled experimental study

Introduction: Mouse models of emphysema are important tools for testing different therapeutic strategies. The aim of this study was to develop a mouse model of emphysema induced by different doses of elastase in order to produce different degrees of severity. Methods: Thirty female mice (C57BL/6) were used in this study. Different doses of porcine pancreatic elastase were administered intratracheally once a week for four weeks, as follows: 0.1 U (n=8), 0.15 U (n=7), and 0.2 U (n=7). Control mice (n=8) received 50 microL of sterile saline solution intratracheally. Lung mechanics were analyzed by plethysmography. Mean linear intercept and volume fraction occupied by collagen and elastic fibers were determined. Results: An increase in lung resistance was observed with 0.2 U of elastase [median (P-25-P75): 2.02 (1.67; 2.34) cmH2O.s/mL], as well as a decrease in tidal volume and minute ventilation. Peak expiratory flow increased significantly in the groups treated with 0.15 U and 0.2 U of elastase. Mean linear intercept was higher with 0.15 U and 0.2 U of elastase, with destruction of alveolar walls [median (P-25-P75): 30.31 (26.65-43.13) microm and 49.49 (31.67-57.71) microm respectively]. The volume fraction occupied by collagen and elastic fibers was lower in the group receiving 0.2 U of elastase. Conclusion: Four intratracheal instillations of 0.2 U of elastase once a week induced changes in lung function and histology, producing an experimental model of severe pulmonary emphysema, whereas 0.15 U resulted in only histological changes

Caracterization of autoantibody-autoantigen systems associated with cytoplasm discrete speckled immunofluorescence pattern

Objetivo - Identificacao e caracterizacao de auto-anticorpos e auto-antigenos associados ao padrao de imunofluorescencia indireta (IFI) de Pontos Citoplasmaticos Isolados (PU). Materiais e metodos - Foram selecionados soros que apresentavam o padrao de PCI a IFI. Esses soros foram analisados por IFI dupla em microscopia confocal, imunomicroscopia eletronica, immunoblotting, imunoprecipitacao de antigenos proteicos sintetizados in vitro, HPTLC de lipideos seguida de imunocoloracao e analise de espectrometria de massas para antigenos lipidicos. Os dados clinicos foram obtidos por revisao de prontuarios e entrevistas com os medicos assistentes. Resultados Dois sistemas de auto-antigeno-auto-anticorpo associados com o padrao de IFI de pontos citoplasmaticos isolados (PCI) foram identificados e denominados de padrao PCI-I e PCI-II. O padrao PCI-I foi caracterizado por 3 a 20 pontos brilhantes bem delimitados e homogeneamente distribuidos pelo citoplasma. O padrao PCI-11 foi caracterizado por apresentar mais de 30 pontos por todo o citoplasma, com predominancia perinuclear, grande heterogeneidade de tamanho e tendencia a aglomeracao. De acordo com esta classificacao foram obtidos 27 soros PCI-I e 6 soros PCI-II. Por IFI dupla em microscopia confocal e imuno-microscopia eletronica os soros PCI-11 mostraram co-localizacao com os anticorpos monoclonais anti-LAMP-2 (proteina-2 associada a membrana do lisossomo) e anti-EEA1 (antigeno-1 do endossomo primario) e com receptor de transferrina, marcadores da via endociticaexocitica. Os dominios citoplasmaticos reconhecidos pelos auto-anticorpos PCI-11 parecem ser delimitados por membrana. Por immunoblotting soros PCI-11 reconheceram uma banda com mobilidade relativa de 180kDa e imunoprecipitaram a proteina EEA1. Em contraste, os anticorpos PCI-I nao apresentaram co-localizacao com anti-EEA1 e anti-LAMP-2, porem apresentaram co-localizacao parcial com o anticorpo monoclonal anti-GW182, que reconhece uma proteina de 182 kDa (GW182), presente em aglomerados citoplasmaticos de mRNA. Os soros PCI-I nao demonstraram qualquer reatividade ao immunoblotting com diversos extratos celulares e nao imunoprecipitaram as proteinas GW182 e EEA1 sintetizadas in vitro. IFI apos extracao lipidica previa do substrato mostrou abolicao drastica do padrao PCI-I mas nao do PCI-II. Por HPTLC/imunocoloracao dos 27 soros PCI-I 24 reagiram com uma banda do extrato lipidico de celulas HEp-2, que foi identificada comoa(au)BV UNIFESP: Teses e dissertaçõe

Experience of an university hospital on the implementation of I and II brazilian consensuses for standardization of ANA in HEp-2 Cells

Objetivo: Analisar a prevalência, padrões e títulos do Fator Antinuclear (FAN) por imunofluorescência indireta (IFI) em células HEp-2 em um hospital universitário após a adoção do I e II Consensos Nacional para Padronização dos Laudos de FAN em Células HEp-2. Método: Foi realizado um estudo transversal, em que foram revisados os laudos de FAN por IFI originários de solicitações encaminhadas ao Serviço de Patologia Clínica do Hospital de Clínicas de Porto Alegre (SPC/HCPA) entre 2002 e 2005. Resultados: Foram analisados 12.095 testes de FAN no período entre 2002 e 2005. As solicitações com resultado reagente foram de 2.577 (21,30%), com média anual de 644±233). Houve um aumento significativo na proporção de resultados reagentes posterior à adoção dos Consensos (p < 0,001). A Reumatologia foi a especialidade que mais solicitou exames por paciente atendido, mas houve um declínio nesse número nos anos posteriores à adoção do Consenso, ocorrido em 2004 (p < 0,001). O padrão de imunofluorescência de FAN mais frequentemente encontrado foi o padrão nuclear pontilhado fino, presente em 52,3% dos resultados reagentes (453/866), e os títulos mais encontrados foram 1/80 e 1/160 (27,8% e 29,4%, respectivamente). Conclusão: Após a adoção do Consenso Nacional de Padronização de Laudos de FAN, observou-se um aumento de exames com resultados reagentes, na sua maioria, com títulos baixos e padrão nuclear pontilhado fino. Na Reumatologia, observou-se uma diminuição no número de solicitações desse exame. As potenciais causas para essas observações são discutidas, mas seu real impacto sobre a situação clínica do paciente e seu tratamento merece ser mais bem estudado.Objective: To evaluate the prevalence of patterns and titers of antinuclear antibodies (ANA) detected by indirect immunofluorescence (IIF) technique on HEp-2 cells in a university hospital following the introduction of I and II Brazilian Consensuses for Standardization of ANA in HEp-2 Cells. Methods: A transversal study was performed between 2002 and 2005 during which all ANA orders to Serviço de Hospital de Clínicas de Porto Alegre (SPC/HCPA) and cognate results were reviewed. Results: 12.095 tests of ANA were revised. The number of positive results during this period was 2.577 (21.30%), annual mean 644 (SD: 233). A marked increase in the number of positive results was observed following the introduction of the Consensuses (p < 0.001). Rheumatology was the medical specialty which requested the highest number of ANA testing per patient although a significant decrease of these numbers was observed after the introduction of the Consensus in 2004 (p < 0,001). Nuclear fine speckled immunofluorescence labeling was the most frequently ANA pattern observed, 52.3% (453/866), and low ANA titers (1/80 and 1/160) more commonly detected (27.8% and 29.4%, respectively). Conclusion: Following the introduction of the Brazilian Consensus for standardization of ANA in HEp-2 cells an increased number of positive results was observed, mostly in low titers and with nuclear fine speckled immunofluorescence pattern. Moreover, there were decreasing numbers of ANA orders by rheumatologists in the same period. Potential causes for these observations are discussed but the real impact in the clinical condition of the patient and therapy deserves to be better studied