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BACKGROUND: Upon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. In this study we investigated the potential role of myeloperoxidase in the endothelial cell injury caused by neutrophil-derived microparticles. RESULTS: Microparticles were produced by activating human neutrophils with a calcium ionophore and characterized by flow cytometry and transmission and scanning electron microscopy. Myeloperoxidase activity was measured by luminol-dependent chemiluminescence. Neutrophil microparticles-induced injuries and morphological alterations in human umbilical vein endothelial cells (HUVECs) were evaluated by microscopy and flow cytometry. Neutrophil microparticles were characterized as structures bounded by lipid bilayers and were less than 1 µm in diameter. The microparticles also expressed CD66b, CD62L and myeloperoxidase, which are all commonly expressed on the surface of neutrophils, as well as exposition of phosphatidylserine. The activity of the myeloperoxidase present on the microparticles was confirmed by hypochlorous acid detection. This compound is only catalyzed by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. The addition of sodium azide or taurine inhibited and reduced enzymatic activity, respectively. Exposure of HUVEC to neutrophil microparticles induced a loss of cell membrane integrity and morphological changes. The addition of sodium azide or myeloperoxidase-specific inhibitor-I consistently reduced the injury to the endothelial cells. Taurine addition reduced HUVEC morphological changes. CONCLUSIONS: We have demonstrated the presence of active myeloperoxidase in neutrophil microparticles and that the microparticle-associated myeloperoxidase cause injury to endothelial cells. Hence, the microparticle-associated myeloperoxidase-hydrogen peroxide-chloride system may contribute to widespread endothelial cell damage in conditions of neutrophil activation as observed in vasculitis and sepsis.
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Micropartículas Derivadas de Células/enzimología , Células Endoteliales/patología , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patología , Membrana Celular/ultraestructura , Micropartículas Derivadas de Células/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Células Endoteliales de la Vena Umbilical Humana , Humanos , Peróxido de Hidrógeno/metabolismo , Estrés OxidativoRESUMEN
This corrects the article on p. 406 in vol. 10, PMID: 29949837.
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PURPOSE: The use of tolerogenic dendritic cells (TolDCs) to control exacerbated immune responses may be a prophylactic and therapeutic option for application in autoimmune and allergic conditions. The objective of this work was to evaluate the effects of TolDC administration in a mouse model of allergic airway inflammation caused by mite extract. METHODS: Mouse bone marrow-derived TolDCs were induced by incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and dexamethasone, and then characterized by flow cytometry and cytokine production by enzyme-linked immunosorbent assay (ELISA). For the in vivo model of Blomia tropicalis-induced allergy, mice transplanted with antigen-pulsed TolDCs were sensitized intraperitoneally with B. tropicalis mite extract (BtE) adsorbed to aluminium hydroxide. After challenge by nasal administration of BtE, bronchoalveolar lavage fluid (BALF), lungs, spleen and serum were collected for analysis. RESULTS: Induction of TolDCs was efficiently achieved as shown by low expression of major histocompatibility complex (MHC) II, programmed death-ligand (PD-L) 2 and pro-inflammatory cytokine production, and up-regulation of interleukin (IL)-10, upon LPS stimulation in vitro. Transplantation of 1 or 2 doses of BtE-pulsed TolDCs reduced the number of inflammatory cells in BALF and lungs as well as mucus deposition. Moreover, compared to saline-injected controls, TolDC-treated mice showed lower serum levels of anti-BtE immunoglobulin E (IgE) antibodies as well as reduced Gata3 and IL-4 gene expression in the lungs and decreased IFN-γ levels in the supernatant of splenocyte cultures Transplantation of TolDCs increased the percentage of the regulatory T cells in the spleen and the lungs. CONCLUSIONS: Preventive treatment with TolDCs protects against dust mite-induced allergy in a mouse model, reinforcing the use of tolerogenic dendritic cells for the management of allergic conditions.
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PURPOSE: Doxorubicin (DOX) is a chemotherapeutic that is widely used for the treatment of many human tumors. However, the development of cardiotoxicity has limited its use. The aim of the present study was to evaluate the possible efficacy of mito-TEMPO (mito-T) as a protective agent against DOX-induced cardiotoxicity in mice. METHODS: C57BL/6 mice were treated twice with mito-T at low (5 mg/kg body weight) or high (20 mg/kg body weight) dose and once with DOX (24 mg/kg body weight) or saline (0.1 mL/20 g body weight) by means of intraperitoneal injections. The levels of malondialdehyde (MLDA), a marker of lipid peroxidation, and serum levels of creatine kinase were evaluated 48 h after the injection of DOX. RESULTS: DOX induced lipid peroxidation in heart mitochondria (p < 0.001), and DOX-treated mice receiving mito-T at low dose had levels of MLDA significantly lower than the mice that received only DOX (p < 0.01). Furthermore, administration of mito-T alone did not cause any significant changes from control values. Additionally, DOX-treated mice treated with mito-T at high dose showed decrease in serum levels of total CK compared to mice treated with DOX alone (p < 0.05). CONCLUSION: Our results indicate that mito-T protects mice against DOX-induced cardiotoxicity.
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Antibióticos Antineoplásicos/toxicidad , Cardiotónicos/farmacología , Cardiotoxicidad/prevención & control , Doxorrubicina/toxicidad , Compuestos Organofosforados/farmacología , Piperidinas/farmacología , Animales , Antibióticos Antineoplásicos/administración & dosificación , Cardiotónicos/administración & dosificación , Cardiotoxicidad/etiología , Creatina Quinasa/sangre , Relación Dosis-Respuesta a Droga , Doxorrubicina/administración & dosificación , Femenino , Inyecciones Intraperitoneales , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/patología , Compuestos Organofosforados/administración & dosificación , Piperidinas/administración & dosificaciónRESUMEN
Introdução: A doxorrubicina (DOX) é um quimioterápico antracíclico amplamente usado para o tratamento de diversos tumores humanos, entretanto, o desenvolvimento de reações adversas à droga, em particular, cardiotoxicidade, tem limitado seu uso. Embora a toxicidade cardíaca induzida pela DOX pareça ser multifatorial, a hipótese mais investigada tem sido a formação de espécies reativas de oxigênio (ROS) e há evidências apontando para as mitocôndrias cardíacas como alvos primários da toxicidade da DOX. Esse dano oxidativo pode iniciar peroxidação lipídica e pode ser potencialmente limitado pelo uso de antioxidantes. Objetivo: O objetivo do presente estudo foi avaliar a possível eficácia do ácido lipoico (AL) e do Mito-TEMPO (Mito-T) como agentes protetores contra a cardiotoxicidade induzida pela DOX in vitro e in vivo e investigar se essa proteção pode afetar a atividade antitumoral da DOX. Método e Resultados: A capacidade do AL e Mito-T eliminar radicais livres foi avaliada usando o teste do 2,2-difenil-1-picril-hidrazila (DPPH). Menor atividade antioxidante do AL (29%) comparada ao Mito-T (63%) foi observada. DOX reduziu a viabilidade de células H9c2 (CI50 = 40,83 M, IC 95% = 28,64 58,21 M) e aumentou a concentração de malondialdeído (MLDA), um marcador de peroxidação lipídica, confirmando a citotoxicidade induzida pela DOX in vitro. O pré-tratamento com AL ou Mito-T não promoveu proteção contra o dano induzido pela DOX in vitro. Uma única injeção intraperitoneal (i.p.) de DOX (24 mg/kg de peso corpóreo) induziu redução significante no peso corpóreo (p<0,001), elevação da atividade sérica total de creatina quinase (p<0,05) e creatina quinase-MB (p<0,05), aumento na concentração de malondialdeído em mitocôndrias (p<0,05) e tecido cardíaco (p<0,01) em camundongos da linhagem C57BL/6 após 48 horas. O pré-tratamento dos animais com Mito-T (5 mg/kg de peso corpóreo, i.p., por dois dias, 48 e 24 horas antes da DOX) reduziu significativamente a peroxidação lipídica de mitocôndrias cardíacas (p<0,01) indicando o direcionamento do antioxidante para a mitocôndria. O tratamento com Mito-T ou AL, duas vezes, 24 e uma hora antes do tratamento com DOX, inibiu a atividade sérica de creatina quinase total (p<0,05). Além disso, o tratamento de camundongos apresentando tumor B16F10 com AL não interferiu na eficácia antitumoral da DOX. Conclusão: Os dados sugerem que a combinação de AL com DOX pode ser benéfica para o tratamento de câncer, entretanto, são necessárias novas investigações para confirmar essa suposição.
Introduction: Doxorubicin (DOX) is an anthracycline chemotherapeutic that is widely used for the treatment of many human tumours, however, the development of adverse drug reactions in particular cardiotoxicity has limited its use. Although doxorubicin-induced cardiac toxicity appears to be multifactorial, the most thoroughly investigated hypothesis has been the formation of reactive oxygen species (ROS) and there is evidence pointing to cardiac mitochondria as primary targets of the toxicity of DOX. This oxidative injury can initiate lipidic peroxidation and may be potentially limited by the use of antioxidants. Aim: The aim of the present study was to evaluate the possible efficacy of lipoic acid (LA) and Mito-TEMPO (Mito-T) as a protective agent against DOX-induced cardiotoxicity in vitro and in vivo and to investigate whether this protection may affect the antitumor activity of DOX. Method and Results: Free radical scavenging capacity of LA and Mito-T was assayed using 1,1-diphenyl-2-picrylhydrazy (DPPH) assay. Lower antioxidant activity for LA (29%) compared to Mito-T (63%) were observed. DOX reduced H9c2 viability (IC50 = 40.83 M, 95% CI = 28.64 58.21 M) and increased the levels of malondialdehyde (MLDA), a marker of lipid peroxidation, confirming DOX-induced cytotoxicity in vitro. Pretreatment with LA or Mito-T did not provide protection against DOX-induced damage in vitro. A single intraperitoneal (i.p.) injection of DOX (24 mg/kg body weight) induced a significant reduction in body weight (p<0.001), elevation of serum activity of total creatine kinase (p<0.05) and creatine kinase-MB (p<0.05), increase in malondialdehyde levels in cardiac mitochondria (p<0.05) and cardiac tissue (p<0.01) in C57BL/6 mice after 48 hours...