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1.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 33(1): 43-48, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-33565399

RESUMO

OBJECTIVE: To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells (iMSC-Exos) on alveolar macrophages (AM) pyroptosis. METHODS: The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells (iMSC) were extracted by rotating ultrafiltration, and the extracted exosomes were identified by transmission electron microscopy, Western blotting and high-resolution adjustable resistance pulse. The rat alveolar macrophage cells (NR8383 cells) were cultured in vitro and the logarithmic growth phase cells were divided into three groups: the control group was added with an equal volume of phosphate buffered saline (PBS) in the AM supernatant; in LPS/ATP group AM cells were stimulated with 500 µg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis; iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP. The cytotoxic activity was detected by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) analysis, the apoptosis and the expression of caspase-1 were observed by immunofluorescence, the levels of inflammatory factors interleukins (IL-1ß and IL-18) released by AM were detected by enzyme linked immunosorbent assay (ELISA), the NOD-like receptor protein 3 (NLRP3) inflammasome pathway and the expression level of pyroptosis related protein gasdermin D (GSDMD) were detected by Western blotting. RESULTS: The extracted exosomes were observed by transmission electron microscopy as round vesicles, expressing exosomal markers CD63 and CD9 showed by Western blotting, high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm, and could be uptaken by AM. Compared with the control group, the cell activity decreased [(0.56±0.05)% vs. (1.06±0.07)%, P < 0.01], the release of necrotic substance LDH increased (U/L: 1 218.86±22.73 vs. 188.30±1.61, P < 0.01), the expression levels of inflammatory factors increased [IL-1ß (ng/L): 958.91±32.78 vs. 194.63±5.14, IL-18 (ng/L): 870.89±21.86 vs. 288.85±24.48, both P < 0.01], and the apoptosis rate [(55.35±6.19)% vs. (12.01±1.32)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 41.06±3.65 vs. 2.80±0.54, P < 0.01) elevated in the AM after LPS/ATP stimulation, suggesting that LPS combined with ATP successfully induced alveolar pyroptosis. Compared with the LPS/ATP group, AM pretreated with iMSC-Exos showed increased cell viability [(0.81±0.05)% vs. (0.56±0.05)%, P < 0.01], decreased LDH secretion (U/L: 535.05±42.55 vs. 1 218.86±22.73, P < 0.01), decreased expression of inflammatory factors [IL-1ß (ng/L): 381.82±19.50 vs. 958.91±32.78, IL-18 (ng/L): 533.77±31.54 vs. 870.89±21.86, both P < 0.01], and decreased apoptosis rate [(19.74±2.96)% vs. (55.35±6.19)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 12.16±1.31 vs. 41.06±3.65, P < 0.01). At the same time, the expression of NLRP3 inflammasome pathway [NLRP3 protein (NLRP3/ß-actin): 0.62±0.06 vs. 1.89±0.11; cleaved caspase-1 protein (cleaved caspase-1/ß-actin): 0.42±0.07 vs. 1.22±0.17, both P < 0.01] and pyrolysis-related protein was significantly inhibited [GSDMD protein (GSDMD/ß-actin): 0.57±0.05 vs. 1.22±0.05, P < 0.01]. CONCLUSIONS: iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP, which may be due to the targeted inhibition of NLRP3 inflammasome pathway, suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.


Assuntos
Exossomos , Células-Tronco Mesenquimais , Animais , Exossomos/metabolismo , Humanos , Inflamassomos , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato , Pirólise , Piroptose , Ratos
2.
Nature ; 590(7847): 635-641, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33429418

RESUMO

Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.


Assuntos
/imunologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Linfócitos T/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Estudos de Coortes , Humanos , Interferon gama/imunologia , Interferons/imunologia , Interferons/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Pneumonia Viral/genética , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T/metabolismo , Fatores de Tempo
3.
Nat Commun ; 12(1): 460, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469029

RESUMO

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação/genética , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Guanosina Trifosfato/metabolismo , Humanos , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Fagocitose , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas rab1 de Ligação ao GTP/genética , Proteínas rab1 de Ligação ao GTP/isolamento & purificação , Proteínas rab1 de Ligação ao GTP/ultraestrutura
4.
Nat Commun ; 12(1): 289, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436554

RESUMO

Mechanical ventilation generates injurious forces that exacerbate lung injury. These forces disrupt lung barrier integrity, trigger proinflammatory mediator release, and differentially regulate genes and non-coding oligonucleotides including microRNAs. In this study, we identify miR-146a as a mechanosensitive microRNA in alveolar macrophages that has therapeutic potential to mitigate lung injury during mechanical ventilation. We use humanized in-vitro systems, mouse models, and biospecimens from patients to elucidate the expression dynamics of miR-146a needed to decrease lung injury during mechanical ventilation. We find that the endogenous increase in miR-146a following injurious ventilation is not sufficient to prevent lung injury. However, when miR-146a is highly overexpressed using a nanoparticle delivery platform it is sufficient to prevent injury. These data indicate that the endogenous increase in microRNA-146a during mechanical ventilation is a compensatory response that partially limits injury and that nanoparticle delivery of miR-146a is an effective strategy for mitigating lung injury during mechanical ventilation.


Assuntos
Técnicas de Transferência de Genes , Lesão Pulmonar/genética , Macrófagos Alveolares/metabolismo , Mecanotransdução Celular , Nanopartículas/química , Respiração Artificial/efeitos adversos , Transferência Adotiva , Animais , Lavagem Broncoalveolar , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-8/metabolismo , Masculino , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Células THP-1 , Regulação para Cima/genética
5.
Life Sci ; 267: 118941, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359748

RESUMO

AIMS: Acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) is a critical clinical syndrome with complex pathology and pathogenesis. Since there is no specific treatment for ALI, it is important to study the mechanism of how ALI develop. Sestrin2 (Sesn2) plays a critical role in the regulation of cellular stress response and oxidant defense. However, the potential function of Sesn2 in ALI/ARDS and the associated mechanism remains unclear. MAIN METHODS: Lipopolysaccharide (LPS) induced ALI model was performed in the wild-type and Sesn2 knockout (Sesn2-/-) mice. The nod-like receptor protein 3 (NLRP3) inflammasome, cell pyroptosis and mitophagy were detected by western blots, immunofluorescent staining, flow cytometry. Lung injury were measured by histopathology and electron microscopy. KEY FINDINGS: Knockout of Sesn2 enhanced LPS-induced ALI. As detailed in Sesn2-/- mice, NLRP3 inflammasome and cell pyroptosis were increased in lungs; IL-1ß and IL-18 in serum and bronchoalveolar lavage fluid (BALF) were further promoted; In the isolated alveolar macrophages from Sesn2-/- mice, mitophagy induced by LPS was markedly inhibited, while reactive oxygen species (ROS), mitochondrial damage and cell pyroptosis were enhanced. Knocking down or overexpressing Sensn2 in J774.A1 cells demonstrated Sesn2 promoted Sequestosome1 (SQSTM1) expression and mitophagy by PTEN-induced putative kinase 1 (Pink1)/Parkin pathway. SIGNIFICANCE: Sesn2 protected ALI by promoting mitophagy that exerts protection of AMs pyroptosis and negative regulation of NLRP3 inflammasomes. These data indicated Sesn2 might be a potential target for ALI treatment.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Macrófagos Alveolares/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Peroxidases/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Inflamassomos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
6.
Mol Med ; 26(1): 98, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33126860

RESUMO

BACKGROUND: Mechanical ventilation, in combination with supraphysiological concentrations of oxygen (i.e., hyperoxia), is routinely used to treat patients with respiratory distress, such as COVID-19. However, prolonged exposure to hyperoxia compromises the clearance of invading pathogens by impairing macrophage phagocytosis. Previously, we have shown that the exposure of mice to hyperoxia induces the release of the nuclear protein high mobility group box-1 (HMGB1) into the pulmonary airways. Furthermore, extracellular HMGB1 impairs macrophage phagocytosis and increases the mortality of mice infected with Pseudomonas aeruginosa (PA). The aim of this study was to determine whether GTS-21 (3-(2,4-dimethoxybenzylidene) anabaseine), an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, could (1) inhibit hyperoxia-induced HMGB1 release into the airways; (2) enhance macrophage phagocytosis and (3) increase bacterial clearance from the lungs in a mouse model of ventilator-associated pneumonia. METHOD: GTS-21 (0.04, 0.4, and 4 mg/kg) or saline were administered by intraperitoneal injection to mice that were exposed to hyperoxia (≥ 99% O2) and subsequently challenged with PA. RESULTS: The systemic administration of 4 mg/kg i.p. of GTS-21 significantly increased bacterial clearance, decreased acute lung injury and decreased accumulation of airway HMGB1 compared to the saline control. To determine the mechanism of action of GTS-21, RAW 264.7 cells, a macrophage-like cell line, were incubated with different concentrations of GTS-21 in the presence of 95% O2. The phagocytic activity of macrophages was significantly increased by GTS-21 in a dose-dependent manner. In addition, GTS-21 significantly inhibited the cytoplasmic translocation and release of HMGB1 from RAW 264.7 cells and attenuated hyperoxia-induced NF-κB activation in macrophages and mouse lungs exposed to hyperoxia and infected with PA. CONCLUSIONS: Our results indicate that GTS-21 is efficacious in improving bacterial clearance and reducing acute lung injury via enhancing macrophage function by inhibiting the release of nuclear HMGB1. Therefore, the α7nAChR represents a possible pharmacological target to improve the clinical outcome of patients on ventilators by augmenting host defense against bacterial infections.


Assuntos
Compostos de Benzilideno/farmacologia , Hiperóxia/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Piridinas/farmacologia , Lesão Pulmonar Induzida por Ventilação Mecânica/tratamento farmacológico , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Proteína HMGB1/metabolismo , Hiperóxia/dietoterapia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/efeitos dos fármacos , Pseudomonas aeruginosa , Células RAW 264.7
7.
Toxicol Lett ; 334: 94-101, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33010382

RESUMO

Silica dust mainly attacks alveolar macrophages (AMs). The apoptosis of AMs is correlated with the progress of silicosis. Our previous study showed that autophagic degradation was blocked in AMs from silicosis patients. However, the effects of nicotine on AM autophagy and apoptosis in silicosis are unknown. In this study, we collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups, according to the tuberous pathological changes observed by X-ray. The AMs from both groups were exposed to nicotine. We found increased levels of LC3, p62, and cleaved caspase-3, decreased levels of LAMP2, and damaged lysosomes after nicotine stimulation of the AMs from both groups. We also found that the autophagy inhibitor 3-methyladenine (3MA) inhibited nicotine-induced apoptosis in the AMs. Furthermore, 3MA reversed both the nicotine-induced decrease in Bcl-2 and the increase in Bax in both groups. These results suggest that nicotine may induce apoptosis by blocking AM autophagic degradation in human silicosis.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Nicotina/toxicidade , Silicose/patologia , Adenina/análogos & derivados , Adenina/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Humanos , Marcação In Situ das Extremidades Cortadas , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Dióxido de Silício/toxicidade , Silicose/metabolismo
8.
PLoS One ; 15(9): e0239289, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32936814

RESUMO

Mycobacterium tuberculosis (Mtb) is the causative agent for tuberculosis, the most extended infectious disease around the world. When Mtb enters inside the pulmonary alveolus it is rapidly phagocytosed by the alveolar macrophage. Although this controls the majority of inhaled microorganisms, in this case, Mtb survives inside the macrophage and multiplies. A posterior chemokine and cytokine cascade generated by the irruption of monocytes, neutrophils and posteriorly, by T-cells, does not necessarily stop the growth of the granuloma. Interestingly, the encapsulation process built by fibroblasts is able to surround the lesion and stop its growing. The success of this last process determines if the host enters in an asymptomatic latent state or continues into a life-threatening and infective active tuberculosis disease (TB). Understanding such dichotomic process is challenging, and computational modeling can bring new ideas. Thus, we have modeled the different stages of the infection, first in a single alveolus (a sac with a radius of 0.15 millimeters) and, second, inside a secondary lobule (a compartment of the lungs of around 3 cm3). We have employed stochastic reaction-diffusion equations to model the interactions among the cells and the diffusive transport to neighboring alveolus. The whole set of equations have successfully described the encapsulation process and determine that the size of the lesions depends on its position on the secondary lobule. We conclude that size and shape of the secondary lobule are the relevant variables to control the lesions, and, therefore, to avoid the evolution towards TB development. As lesions appear near to interlobular connective tissue they are easily controlled and their growth is drastically stopped, in this sense secondary lobules with a more flattened shape could control better the lesion.


Assuntos
Simulação por Computador , Granuloma/patologia , Mycobacterium tuberculosis/patogenicidade , Tuberculose/imunologia , Quimiocinas/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Difusão , Granuloma/imunologia , Granuloma/microbiologia , Humanos , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Tuberculose Latente/patologia , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Mycobacterium tuberculosis/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Tuberculose/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
9.
Nat Commun ; 11(1): 3822, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732898

RESUMO

Alveolar macrophages (AMs) derived from embryonic precursors seed the lung before birth and self-maintain locally throughout adulthood, but are regenerated by bone marrow (BM) under stress conditions. However, the regulation of AM development and maintenance remains poorly understood. Here, we show that histone deacetylase 3 (HDAC3) is a key epigenetic factor required for AM embryonic development, postnatal homeostasis, maturation, and regeneration from BM. Loss of HDAC3 in early embryonic development affects AM development starting at E14.5, while loss of HDAC3 after birth affects AM homeostasis and maturation. Single-cell RNA sequencing analyses reveal four distinct AM sub-clusters and a dysregulated cluster-specific pathway in the HDAC3-deficient AMs. Moreover, HDAC3-deficient AMs exhibit severe mitochondrial oxidative dysfunction and deteriorative cell death. Mechanistically, HDAC3 directly binds to Pparg enhancers, and HDAC3 deficiency impairs Pparg expression and its signaling pathway. Our findings identify HDAC3 as a key epigenetic regulator of lung AM development and homeostasis.


Assuntos
Histona Desacetilases/genética , Homeostase/genética , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Histona Desacetilases/deficiência , Histona Desacetilases/metabolismo , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Macrófagos Alveolares/citologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
10.
Ecotoxicol Environ Saf ; 205: 111186, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853868

RESUMO

Exposure to ambient air particulate matter (PM) is associated with increased cardiorespiratory morbidity and mortality. In this context, alveolar macrophages exhibit proinflammatory and oxidative responses as a result of the clearance of particles, thus contributing to lung injury. However, the mechanisms linking these pathways are not completely clarified. Therefore, the oxinflammation phenomenon was studied in RAW 264.7 macrophages exposed to Residual Oil Fly Ash (ROFA), a PM surrogate rich in transition metals. While cell viability was not compromised under the experimental conditions, a proinflammatory phenotype was observed in cells incubated with ROFA 100 µg/mL, characterized by increased levels of TNF-α and NO production, together with PM uptake. This inflammatory response seems to precede alterations in redox metabolism, characterized by augmented levels of H2O2, diminished GSH/GSSG ratio, and increased SOD activity. This scenario resulted in increased oxidative damage to phospholipids. Moreover, alterations in mitochondrial respiration were observed following ROFA incubation, such as diminished coupling efficiency and spare respiratory capacity, together with augmented proton leak. These findings were accompanied by a decrease in mitochondrial membrane potential. Finally, NADPH oxidase (NOX) and mitochondria were identified as the main sources of superoxide anion () in our model. These results indicate that PM exposure induces direct activation of macrophages, leading to inflammation and increased reactive oxygen species production through NOX and mitochondria, which impairs antioxidant defense and may cause mitochondrial dysfunction.


Assuntos
Macrófagos Alveolares/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Superóxidos/metabolismo , Poluentes Atmosféricos/toxicidade , Animais , Antioxidantes/metabolismo , Cinza de Carvão/toxicidade , Peróxido de Hidrogênio/metabolismo , Inflamação , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo/imunologia , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
11.
Life Sci ; 259: 118285, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32798556

RESUMO

AIMS: Interleukin-1ß (IL-1ß) contributes to the development of bronchopulmonary dysplasia (BPD). Thioredoxin reductase-1 (Txnrd1) inhibition activates nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent responses. Txnrd1 activity is selenium (Se) dependent and Se deficiency is common in prematurity. Auranofin (AFN), a Txnrd1 inhibitor, decreases IL-1ß levels and increases Nrf2 activation in lipopolysaccharide (LPS) treated alveolar macrophages. In lung epithelia, AFN-induced Nrf2 activation is Se dependent. We tested the hypothesis that the effects of Txnrd1 inhibition in alveolar macrophages are Se dependent. MAIN METHODS: To establish Se sufficient (Se+) and deficient (Se-) conditions, alveolar (MH-S) macrophages were cultured in 2.5% fetal bovine serum (FBS) ± 25 nM Na2SeO3. Se- (2.5% FBS) and Se+ (2.5% FBS + 25 nM Na2SeO3) cells were cultured in the presence or absence of 0.05 µg/mL LPS and/or 0.5 µM AFN. Nrf2 activation was determined by measuring NADPH quinone oxidoreductase-1 (Nqo1) and glutathione levels. IL-1ß mRNA (Il1b) and protein levels were measured using qRT-PCR and ELISA. Data were analyzed by ANOVA followed by Tukey's post-hoc. KEY FINDINGS: We detected an independent effect of AFN, but not LPS, on Nqo1 expression and GSH levels in Se+ and Se- cells. LPS significantly increased Il1b and IL-1ß levels in both groups. AFN-mediated attenuation of this effect was not impacted by Se status. SIGNIFICANCE: The beneficial effects of Txnrd1 inhibition in alveolar macrophages are Se-independent and therefore unlikely to be diminished by clinical Se deficiency.


Assuntos
Auranofina/farmacologia , Macrófagos Alveolares/metabolismo , Tiorredoxina Redutase 1/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Auranofina/metabolismo , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/fisiopatologia , Glutationa/metabolismo , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/metabolismo , Macrófagos/metabolismo , Macrófagos Alveolares/fisiologia , Camundongos , Cultura Primária de Células , Selênio/metabolismo , Selênio/farmacologia , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo
12.
Nanotoxicology ; 14(7): 968-984, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32633691

RESUMO

Rich vacancies of semiconductor nanomaterials (NMs) give rise to great enhancement of their physical and chemical properties such as magnetic, catalytic, optical, etc. These NMs possessing extensive applications could inevitably enter into the environment and increase the toxic effects on organisms, so it is imperative to investigate the cytotoxicity of NMs with different types of vacancies. Here, one-dimensional cobalt selenide (CoSe2) NMs with different vacancies were synthesized through the same precursor while calcined at different temperatures (P-CoSe2 which calcined at 200 °C and N-CoSe2 which calcined at 230 °C). According to the positron annihilation spectrum, the VSeSe vacancy associate in P-CoSe2 was endowed with two positive charges, while the VCoCoCoSeSe vacancy associate in N-CoSe2 possessed four negative charges. Cell viability assays revealed that N-CoSe2 had higher toxicity to macrophages than P-CoSe2, which was attributed to higher levels of intracellular reactive oxygen species induced by N-CoSe2. Further investigation showed that N-CoSe2 had higher affinity to the mitochondrion-targeting peptide, leading to its preferential distribution in the mitochondria and consequent induction of mitochondrial superoxide production. In contrast, P-CoSe2 exhibited higher affinity to the endoplasmic reticulum (ER)-targeting peptide, facilitating its preferential distribution in the ER and the nuclei and causing higher damage to both organelles as compared to N-CoSe2. These results demonstrated that type of surface vacancies significantly affected biodistribution of NMs in subcellular organelles, which contributed to differential biological behaviors of the NMs.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Nanoestruturas/toxicidade , Compostos de Selênio/toxicidade , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanoestruturas/química , Coroa de Proteína/química , Ratos , Espécies Reativas de Oxigênio/metabolismo , Compostos de Selênio/química , Solubilidade , Propriedades de Superfície , Distribuição Tecidual
13.
Life Sci ; 257: 118043, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621922

RESUMO

BACKGROUND: Alveolar macrophages (AMs) are the primary targets of silicosis. Blockade of autophagy may aggravate the apoptosis of AMs. Trehalose (Tre), a transcription factor EB (TFEB) activator, may impact the autophagy-lysosomal system in AMs during silicosis. However, the mechanism by which Tre acts upon AMs in silicosis is unknown. METHODS: We collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups. AMs from the two groups were then exposed to Tre. Western blot was used to measure the expression of autophagy-associated proteins. Lysosomal-associated membrane protein 1 (LAMP1) expression was observed using immunofluorescence and western blot. Apoptosis of the AMs was detected by TUNEL assay and western blot. RESULTS: Tre induced localization of TFEB to the nucleus in the AMs of both groups. After Tre exposure, LAMP1 levels increased and LC3 levels decreased in the AMs of both groups, suggesting that Tre may increase the function of the autophagy-lysosomal system. The LC3-II/I ratio in the Tre-exposed AMs was lower than in the AMs not exposed to Tre. The LC3-II/I ratio in AMs subjected to Tre plus Bafilomycin (Baf) was higher than the ratio in cells exposed to Tre or Baf individually. Additionally, p62 levels decreased after Tre stimulation in the AMs of both groups. This indicates that Tre may accelerate the process of autophagic degradation. We also found decreased levels of cleaved caspase-3 after Tre treatment in the AMs of both groups. However, p-mTOR (Ser2448) and p-mTOR (Ser2481) levels did not change significantly after Tre treatment, suggesting that the mTOR signaling pathway was not affected by Tre treatment. CONCLUSION: Our findings suggest that the restoration of autophagy-lysosomal function by Tre may be a potential protective strategy against silicosis.


Assuntos
Silicose/tratamento farmacológico , Trealose/metabolismo , Trealose/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Silicose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição
14.
Expert Opin Pharmacother ; 21(11): 1359-1366, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32511020

RESUMO

INTRODUCTION: Pulmonary alveolar proteinosis (PAP) is a heterogeneous group of rare diseases characterized by the abnormal production and impaired degradation of pulmonary surfactant as a result of malfunctioning of alveolar macrophages. This is due to the downstream dysregulation of the GM-CSF pathway, which can be caused by specific autoantibodies (autoimmune, aPAP formerly known as idiopathic iPAP), direct injury to alveolar macrophages (e.g. by toxic inhaled agents.), or by genetic defects (hereditary or congenital PAP). Few pharmacotherapy options are currently available to treat this disease. AREA COVERED: The authors discuss the exogenous administration of GM-CSF, rituximab, and the potential role of cholesterol lowering medications in this review. The authors, furthermore, provide their opinion on the available pharmacotherapeutic options and give their future perspectives. EXPERT OPINION: Inhaled GM-CSF remains the most commonly used therapy in patients with iPAP but other inhaled therapies such as PPARγ activators should be considered, especially in patients who are partially responsive or unresponsive to traditional treatments.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Proteinose Alveolar Pulmonar/tratamento farmacológico , Rituximab/uso terapêutico , Administração por Inalação , Animais , Autoanticorpos/imunologia , Ensaios Clínicos como Assunto , Terapia Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos adversos , Humanos , Injeções Subcutâneas , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/imunologia , Proteinose Alveolar Pulmonar/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico , Rituximab/administração & dosagem , Rituximab/efeitos adversos
15.
CPT Pharmacometrics Syst Pharmacol ; 9(8): 435-443, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32511867

RESUMO

Azithromycin (AZ), a broad-spectrum macrolide antibiotic, is being investigated in patients with coronavirus disease 2019 (COVID-19). A population pharmacokinetic model was implemented to predict lung, intracellular poly/mononuclear cell (peripheral blood monocyte (PBM)/polymorphonuclear leukocyte (PML)), and alveolar macrophage (AM) concentrations using published data and compared against preclinical effective concentration 90% (EC90 ) for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). The final model described the data reported in eight publications adequately. Consistent with its known properties, concentrations were higher in AM and PBM/PML, followed by lung tissue, and lowest systemically. Simulated PBM/PML concentrations exceeded EC90 following the first dose and for ~ 14 days following 500 mg q.d. for 3 days or 500 mg q.d. for 1 day/250 mg q.d. on days 2-5, 10 days following a single 1,000 mg dose, and for > 20 days with 500 mg q.d. for 10 days. AM concentrations exceeded the 90% inhibitory concentration for > 20 days for all regimens. These data will better inform optimization of dosing regimens for AZ clinical trials.


Assuntos
Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Antibacterianos/farmacocinética , Azitromicina/farmacocinética , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/metabolismo , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Modelos Biológicos , Neutrófilos/metabolismo , Pandemias , Distribuição Tecidual
16.
Cancer Immunol Immunother ; 69(11): 2345-2355, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32514619

RESUMO

The cross-talk between cancer cells and monocyte-derived alveolar macrophages (Mo-AMs) promotes non-small cell lung carcinoma (NSCLC) progression. In this study, we report that both cancer cells and Mo-AMs robustly express beta 3-adrenergic receptor (ADRB3) in NSCLC. ADRB3 supports lung cancer cells proliferation and promotes chronic inflammation. Genetic and pharmacologic inhibition of ADRB3 reverses tumor growth and inflammation in mouse. Furthermore, we demonstrate that M5D1, a novel anti-ADRB3 monoclonal antibody, inhibits human lung cancer cells proliferation and inflammation via affecting the intracellular mTOR pathway and activating p53. In NSCLC patients, we confirmed that upregulation of ADRB3 expression correlates with tumor progression and poor prognosis. Altogether, these results shed light on the role of ADRB3 in NSCLC and suggest that M5D1 could become powerful antitumor weapons.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Macrófagos Alveolares/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Prognóstico
17.
Nanotoxicology ; 14(6): 847-865, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32536243

RESUMO

To determine responses to nanoparticles in a more comprehensive way, current efforts in nanosafety aim at combining the analysis of multiple endpoints and comparing outcomes in different models. To this end, here we used tissue slices from mice as 3D ex vivo models and performed for the first time a comparative study of uptake and impact in liver, lung, and kidney slices exposed under the same conditions to silica, carboxylated and amino-modified polystyrene. In all organs, only exposure to amino-modified polystyrene induced toxicity, with stronger effects in kidneys and lungs. Uptake and distribution studies by confocal microscopy confirmed nanoparticle uptake in all slices, and, in line with what observed in vivo, preferential accumulation in the macrophages. However, uptake levels in kidneys were minimal, despite the strong impact observed when exposed to the amino-modified polystyrene. On the contrary, nanoparticle uptake and accumulation in macrophages were particularly evident in lung slices. Thus, tissue digestion was used to recover all cells from lung slices at different exposure times and to determine by flow cytometry detailed uptake kinetics in lung macrophages and all other cells, confirming higher uptake by the macrophages. Finally, the expression levels of a panel of targets involved in inflammation and macrophage polarization were measured to determine potential effects induced in lung and liver tissue. Overall, this comparative study allowed us to determine uptake and impact of nanoparticles in real tissue and identify important differences in outcomes in the organs in which nanoparticles distribute.


Assuntos
Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas/toxicidade , Poliestirenos/toxicidade , Animais , Feminino , Técnicas In Vitro , Inflamação , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/metabolismo , Especificidade de Órgãos , Poliestirenos/metabolismo , Técnicas de Cultura de Tecidos , Distribuição Tecidual
18.
Nanotoxicology ; 14(7): 929-946, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32538272

RESUMO

Multi-walled carbon nanotubes (MWCNTs) are known to induce pulmonary inflammatory effects through stimulating pro-inflammatory cytokine secretion from alveolar macrophages. Despite extensive studies on MWCNTs' pro-inflammatory reactivity, the understanding of molecular mechanisms involved is still incomplete. In this study, we investigated hemichannel's involvement in MWCNTs-induced macrophage IL-1ß release. Our results showed that the unmodified and COOH MWCNTs could induce ATP release and ATP-P2X7R axis-dependent IL-1ß secretion from THP-1 macrophages. By using various inhibitors, we confirmed that the MWCNTs-induced ATP release was primarily through hemichannels. EtBr dye uptake assay detected significant hemichannels opening in MWCNTs exposed THP-1 macrophages. Inhibition of hemichannels by CBX, 43Gap27, or 10Panx1 pretreatment results in decreased ATP and IL-1ß release. The addition of ATP restored the reduced IL-1ß secretion level from hemichannel inhibition. We also confirmed with five other types of MWCNTs that the induction of hemichannels by MWCNTs strongly correlates with their capacity to induce IL-1ß secretion. Taken together, we conclude that hemichannels-mediated ATP release and subsequent NLRP3 inflammasome activation through P2X7R may be one mechanism by which MWCNTs induce macrophage IL-1ß secretion. Our findings may provide a novel molecular mechanism for MWCNTs induced IL-1ß secretion.


Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Proteínas do Tecido Nervoso/metabolismo , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nanotubos de Carbono/química , Células THP-1
19.
Front Immunol ; 11: 1312, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582222

RESUMO

Respiratory, circulatory, and renal failure are among the gravest features of COVID-19 and are associated with a very high mortality rate. A common denominator of all affected organs is the expression of angiotensin-converting enzyme 2 (ACE2), a protease responsible for the conversion of Angiotensin 1-8 (Ang II) to Angiotensin 1-7 (Ang 1-7). Ang 1-7 acts on these tissues and in other target organs via Mas receptor (MasR), where it exerts beneficial effects, including vasodilation and suppression of inflammation and fibrosis, along an attenuation of cardiac and vascular remodeling. Unfortunately, ACE2 also serves as the binding receptor of SARS viral spike glycoprotein, enabling its attachment to host cells, with subsequent viral internalization and replication. Although numerous reports have linked the devastating organ injuries to viral homing and attachment to organ-specific cells widely expressing ACE2, little attention has been given to ACE-2 expressed by the immune system. Herein we outline potential adverse effects of SARS-CoV2 on macrophages and dendritic cells, key cells of the immune system expressing ACE2. Specifically, we propose a new hypothesis that, while macrophages play an important role in antiviral defense mechanisms, in the case of SARS-CoV, they may also serve as a Trojan horse, enabling viral anchoring specifically within the pulmonary parenchyma. It is tempting to assume that diverse expression of ACE2 in macrophages among individuals might govern the severity of SARS-CoV-2 infection. Moreover, reallocation of viral-containing macrophages migrating out of the lung to other tissues is theoretically plausible in the context of viral spread with the involvement of other organs.


Assuntos
Betacoronavirus/metabolismo , Células Dendríticas/metabolismo , Pulmão/patologia , Macrófagos Alveolares/metabolismo , Peptidil Dipeptidase A/metabolismo , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Pulmão/virologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Pandemias , Tecido Parenquimatoso/patologia , Tecido Parenquimatoso/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
20.
Gene ; 753: 144819, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32485309

RESUMO

Vitamin D is one of the indispensable nutrients of human body. When vitamin D is deficient, it can cause a series of related diseases, such as respiratory tract infection. The regulatory role of vitamin D in inflammatory immune response and defense has attracted more and more attention. However, few studies have shown that vitamin D regulates inflammation and autophagy in Aspergillus fumigatus infected lungs. In this study, we will explain the mechanism of vitamin D regulating inflammation and autophagy in Aspergillus fumigatus infected lungs. METHODS: Different concentrations of Aspergillus fumigatus spores were injected into mice with deficien diets (VitD-) or sufficient vitamin D (VitD + ) , and the survival rates were recorded. Then, the weight changes of rats were measured every other time. At the same time, a gauze was used to filter the lapped lung tissue to get the pulmonary spores and measured the amount of the spores. The mice with the same concentration of Aspergillus fumigatus infected were cut off and the lung tissue for pathological examination in the deficien diets (VitD-) group or sufficient vitamin D (VitD + ) group. Moreover, the expression of inflammation related factors TNF-α, IL-1ß, IL-6 in lung was measured by immunohistochemical method. The expression of TNF-α, IL-1ß, IL-6 in the serum of vitamin D deficiency and sufficient mice were measured by ELISA. In vitro, we obtained macrophages from healthy mice and mixed cultures of Aspergillus fumigatus spores and lung macrophages in medium with or without vitamin D. After the cells were infected with Aspergillus fumigatus spores, the expressions of NF-κB and IL-8 were detected by RT-PCR and Western blot. The RAW264.7 cells transfected with GFP-LC3BII were mixed with Aspergillus fumigatus spores, and the expression of cell fluorescence was observed by the fluorescence microscope with or without chloroquine and rapamycin , and the autophagy flow of the cells was measured by Western blot. In the RAW264.7 cells, Lentivirus transfection and SiRNA technologies were used to enhance or reduce the expression of the NF-κB gene (siNF-κB) for investgating the influence of high or low expression of NF-κB in the autophagic flow of vitamin D + or vitamin D-treated RAW264.7 cells. RESULTS: The survival rate of vitamin D deficient mice infected Aspergillus fumigatus was significantly lower than that of vitamin D sufficient mice, while the number of spores, spore activity, pathological changes of lungs and inflammation in the lungs of vitamin D deficient mice were more severe than that of vitamin D sufficient mice. In vitro cell experiments, when cell was stimulated with vitamin D, the expressions of NF-κB and IL-8 in cells were lower. The autophagic flux and TNF-α, IL-1ß, IL-6 and LC3BII in vitamin D group were significantly lower than those in vitamin D deficiency group. CONCLUSIONS: Vitamin D deficiency can aggravate the inflammatory damage in the lungs of Aspergillus fumigatus. When the body is sufficient in vitamin D, if the lungs infect Aspergillus fumigatus spores, the body may resist the infection of Aspergillus fumigatus by reducing the expression of NF-κB, inflammatory factors and autophagy.


Assuntos
Autofagia/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Vitamina D/metabolismo , Animais , Aspergilose/imunologia , Aspergilose/metabolismo , Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/fisiologia , Citocinas/genética , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Pulmão/metabolismo , Macrófagos/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo , Deficiência de Vitamina D/metabolismo
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