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1.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652988

RESUMO

In this Review, we briefly describe the basic virology and pathogenesis of SARS-CoV-2, highlighting how stem cell technology and organoids can contribute to the understanding of SARS-CoV-2 cell tropisms and the mechanism of disease in the human host, supporting and clarifying findings from clinical studies in infected individuals. We summarize here the results of studies, which used these technologies to investigate SARS-CoV-2 pathogenesis in different organs. Studies with in vitro models of lung epithelia showed that alveolar epithelial type II cells, but not differentiated lung alveolar epithelial type I cells, are key targets of SARS-CoV-2, which triggers cell apoptosis and inflammation, while impairing surfactant production. Experiments with human small intestinal organoids and colonic organoids showed that the gastrointestinal tract is another relevant target for SARS-CoV-2. The virus can infect and replicate in enterocytes and cholangiocytes, inducing cell damage and inflammation. Direct viral damage was also demonstrated in in vitro models of human cardiomyocytes and choroid plexus epithelial cells. At variance, endothelial cells and neurons are poorly susceptible to viral infection, thus supporting the hypothesis that neurological symptoms and vascular damage result from the indirect effects of systemic inflammatory and immunological hyper-responses to SARS-CoV-2 infection.


Assuntos
/patologia , Organoides/virologia , Células-Tronco/virologia , Animais , Apoptose , Sistema Cardiovascular/citologia , Sistema Cardiovascular/patologia , Sistema Cardiovascular/virologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Trato Gastrointestinal/citologia , Trato Gastrointestinal/patologia , Trato Gastrointestinal/virologia , Humanos , Inflamação/patologia , Inflamação/virologia , Pulmão/citologia , Pulmão/patologia , Pulmão/virologia , Organoides/patologia , Células-Tronco/patologia , Tropismo Viral , Internalização do Vírus
2.
Nat Commun ; 12(1): 1446, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664261

RESUMO

Invariant natural killer T cells (iNKT cells) differentiate into thymic and peripheral NKT1, NKT2 and NKT17 subsets. Here we use RNA-seq and ATAC-seq analyses and show iNKT subsets are similar, regardless of tissue location. Lung iNKT cell subsets possess the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. Following antigenic stimulation, iNKT cells undergo chromatin and transcriptional changes delineating two populations: one similar to follicular helper T cells and the other NK or effector like. Phenotypic analysis indicates these changes are observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to additional subsets.


Assuntos
Pulmão/citologia , Células T Matadoras Naturais/citologia , Subpopulações de Linfócitos T/citologia , Timo/citologia , Animais , Diferenciação Celular/imunologia , Cromatina/genética , Feminino , Pulmão/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Transcriptoma/genética
3.
Nat Commun ; 12(1): 1029, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589635

RESUMO

A primary challenge in single-cell RNA sequencing (scRNA-seq) studies comes from the massive amount of data and the excess noise level. To address this challenge, we introduce an analysis framework, named single-cell Decomposition using Hierarchical Autoencoder (scDHA), that reliably extracts representative information of each cell. The scDHA pipeline consists of two core modules. The first module is a non-negative kernel autoencoder able to remove genes or components that have insignificant contributions to the part-based representation of the data. The second module is a stacked Bayesian autoencoder that projects the data onto a low-dimensional space (compressed). To diminish the tendency to overfit of neural networks, we repeatedly perturb the compressed space to learn a more generalized representation of the data. In an extensive analysis, we demonstrate that scDHA outperforms state-of-the-art techniques in many research sub-fields of scRNA-seq analysis, including cell segregation through unsupervised learning, visualization of transcriptome landscape, cell classification, and pseudo-time inference.


Assuntos
Redes Neurais de Computação , Análise de Sequência de RNA/estatística & dados numéricos , Análise de Célula Única/estatística & dados numéricos , Aprendizado de Máquina não Supervisionado/estatística & dados numéricos , Animais , Teorema de Bayes , Benchmarking , Separação Celular/métodos , Cerebelo/química , Cerebelo/citologia , Embrião de Mamíferos , Humanos , Fígado/química , Fígado/citologia , Pulmão/química , Pulmão/citologia , Camundongos , Células-Tronco Embrionárias Murinas/química , Células-Tronco Embrionárias Murinas/citologia , Pâncreas/química , Pâncreas/citologia , Retina/química , Retina/citologia , Análise de Célula Única/métodos , Córtex Visual/química , Córtex Visual/citologia , Zigoto/química , Zigoto/citologia
4.
Ecotoxicol Environ Saf ; 210: 111892, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33429317

RESUMO

Human activities have generated air pollution, with extremely small particles (PM 2.5, particulate matter less than 2.5 µm in diameter) and liquid droplets, which become a menace to human health. Among the pollutants, polycyclic aromatic hydrocarbons (PAHs), which enhance the risks of pulmonary dysfunction and cancer development, have been extensively studied. Numerous studies have addressed the effects of PAHs on the respiratory system, whereas the effects on lung stem/progenitor cells remain unknown. Here, we provide evidence that benzo[a]pyrene (BaP), a major toxic PAH, induces fibrotic changes with a loss of α-1,6-fucosylation in CD54+CD157+CD45- cells (lung stem cells). In studies with aryl hydrocarbon receptor (AHR) antagonist, we found that these effects by BaP are independent of the canonical AHR pathway. In addition, these BaP-induced fibrotic changes are reduced by TGF-ß antagonist, suggesting an alternative pathway of BaP toxicity is different from other PAH/AHR signaling pathways. Finally, it was observed that BaP impairs the spheroid formation and the podoplanin expression of CD54+CD157+CD45- cells, indicating that BaP suppresses the differentiation of lung stem cells. Taken together, our findings reveal specific BaP-induced injuries in CD54+CD157+CD45- cells.


Assuntos
Poluentes Atmosféricos/toxicidade , Benzo(a)pireno/toxicidade , Pulmão/citologia , Células-Tronco/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fibrose , Camundongos , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Células-Tronco/patologia , Fator de Crescimento Transformador beta/antagonistas & inibidores
5.
Toxicol Lett ; 341: 83-93, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33508333

RESUMO

Proliferation and migration of lung epithelial cells following the injury to the epithelial lining of alveoli and airways in the lung are pivotal for remodeling and repair of the wound to restore normal lung function. In the present study, we examined the modulatory effect of carboxylated nanodiamonds (cNDs) on the cell division, migration, and adhesion of epithelial cells in the well-established in vitro model of wound repair and cell migration. Flow cytometry and confocal microscopy results indicated that both LA4 and A549 cells effectively internalized fluorescent carboxylated nanodiamonds (cFNDs) and the internalized nanodiamonds were essentially localized in the cytoplasmic region. Treatment with cNDs blocked the division and migration of cells to fill the scratch wound. Live cell imaging and time-lapse videography of the wound healing process indicated a significant inhibition of cell proliferation activity in cND-treated cells and blocked the wound repair process. Trans-well cell-migration assay results further support the inhibitory effect of cNDs on the cell migration process. Western blotting and immunofluorescence staining indicated that the crucial proteins involved in epithelial-mesenchymal transition (EMT) and cell migration i.e. ß-catenin, Vimentin, NM-myosin, and Focal Adhesion Kinase (FAK) were downregulated after treatment with cNDs, while the expression of E-cadherin and Claudin-1, major cell adhesion markers remained unaltered. Taken together, our results indicate that the decline in cell proliferation activity, downregulation in the expression of various crucial protein like ß-Catenin, NM-myosin, FAK, and Vimentin involved in the cell migration and unaltered expression of cell adhesion molecules E-cadherin and Claudin-1, may be the factors that contribute to the cND-mediated inhibition of EMT during the wound repair process in the monolayers of lung epithelial cells.


Assuntos
Movimento Celular , Proliferação de Células , Células Epiteliais/efeitos dos fármacos , Pulmão/citologia , Nanodiamantes , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
6.
Life Sci ; 269: 119085, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33482190

RESUMO

Pulmonary fibrosis (PF), which is characterized by excessive matrix formation, may ultimately lead to irreversible lung damage and thus death. Fibroblast activation has been regarded as a central event during PF pathogenesis. In our previous study, we confirmed that the miR-627/high-mobility group box protein 1 (HMGB1)/Nuclear factor kappa beta (NF-κB) axis modulates transforming growth factor beta 1 (TGFß1)-induced pulmonary fibrosis. In the present study, we investigated the upstream factors leading to miR-627 dysregulation in the process of pulmonary fibroblast activation and PF. The lncRNA MIR155 host gene (MIR155HG) was found to be abnormally upregulated in pulmonary fibrosis tissues and TGFß1-stimulated normal human primary lung fibroblasts (NHLFs). By directly binding to miR-627, MIR155HG inhibited miR-627 expression. MIR155HG overexpression enhanced TGFß1-induced increases in HMGB1 protein expression and p65 phosphorylation, NHLF proliferation, and extracellular matrix (ECM) deposition. In contrast, miR-627 overexpression attenuated the TGFß1-induced changes in NHLFs and significantly reversed the effects of MIR155HG overexpression. Under TGFß1 stimulation, miR-627 inhibition promoted, whereas JSH-23 treatment inhibited NF-κB activation; in NHLFs, NF-κB overexpression upregulated, whereas JSH-23 treatment downregulated MIR155HG expression. In tissue samples, HMGB1 protein levels and p65 phosphorylation were increased; MIR155HG was negatively correlated with miR-627 and positively correlated with HMGB1. In conclusion, we validated that the MIR155HG/miR-627/HMGB1/NF-κB axis formed a regulatory loop that modulates TGFß1-induced NHLF activation. Considering the critical role of NHLF activation in PF pathogenesis, the NF-κB/MIR155HG/miR-627/HMGB1 regulatory loop could exert a vital effect on PF pathogenesis. Further in vivo and clinical investigations are required to confirm this model.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/citologia , Proteína HMGB1/metabolismo , MicroRNAs/genética , NF-kappa B/metabolismo , Fibrose Pulmonar/patologia , RNA Longo não Codificante/genética , Estudos de Casos e Controles , Proliferação de Células , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteína HMGB1/genética , Humanos , Pulmão/citologia , Pulmão/metabolismo , NF-kappa B/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
7.
Cytokine ; 140: 155430, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33508651

RESUMO

In vitro interferon (IFN)α treatment of primary human upper airway basal cells has been shown to drive ACE2 expression, the receptor of SARS-CoV-2. The protease furin is also involved in mediating SARS-CoV-2 and other viral infections, although its association with early IFN response has not been evaluated yet. In order to assess the in vivo relationship between ACE2 and furin expression and the IFN response in nasopharyngeal cells, we first examined ACE2 and furin levels and their correlation with the well-known marker of IFNs' activation, ISG15, in children (n = 59) and adults (n = 48), during respiratory diseases not caused by SARS-CoV-2. A strong positive correlation was found between ACE2 expression, but not of furin, and ISG15 in all patients analyzed. In addition, type I and III IFN stimulation experiments were performed to examine the IFN-mediated activation of ACE2 isoforms (full-length and truncated) and furin in epithelial cell lines. Following all the IFNs treatments, only the truncated ACE2 levels, were upregulated significantly in the A549 and Calu3 cells, in particular by type I IFNs. If confirmed in vivo following IFNs' activation, the induction of the truncated ACE2 isoform only would not enhance the risk of SARS-CoV-2 infection in the respiratory tract.


Assuntos
/genética , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Interferons/farmacologia , /efeitos dos fármacos , Células A549 , Adulto , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular Tumoral , Criança , Citocinas/genética , Células Epiteliais/metabolismo , Humanos , Interferons/metabolismo , Pulmão/citologia , Pessoa de Meia-Idade , Ubiquitinas/genética
8.
Cell Res ; 31(2): 126-140, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33420426

RESUMO

The current coronavirus disease 2019 (COVID-19) pandemic presents a global public health challenge. The viral pathogen responsible, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), binds to the host receptor ACE2 through its spike (S) glycoprotein, which mediates membrane fusion and viral entry. Although the role of ACE2 as a receptor for SARS-CoV-2 is clear, studies have shown that ACE2 expression is extremely low in various human tissues, especially in the respiratory tract. Thus, other host receptors and/or co-receptors that promote the entry of SARS-CoV-2 into cells of the respiratory system may exist. In this study, we found that the tyrosine-protein kinase receptor UFO (AXL) specifically interacts with the N-terminal domain of SARS-CoV-2 S. Using both a SARS-CoV-2 virus pseudotype and authentic SARS-CoV-2, we found that overexpression of AXL in HEK293T cells promotes SARS-CoV-2 entry as efficiently as overexpression of ACE2, while knocking out AXL significantly reduces SARS-CoV-2 infection in H1299 pulmonary cells and in human primary lung epithelial cells. Soluble human recombinant AXL blocks SARS-CoV-2 infection in cells expressing high levels of AXL. The AXL expression level is well correlated with SARS-CoV-2 S level in bronchoalveolar lavage fluid cells from COVID-19 patients. Taken together, our findings suggest that AXL is a novel candidate receptor for SARS-CoV-2 which may play an important role in promoting viral infection of the human respiratory system and indicate that it is a potential target for future clinical intervention strategies.


Assuntos
/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Mucosa Respiratória/citologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Brônquios/citologia , Brônquios/metabolismo , Linhagem Celular , Humanos , Pulmão/citologia , Pulmão/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas/análise , Receptores Proteína Tirosina Quinases/análise , Mucosa Respiratória/metabolismo , Glicoproteína da Espícula de Coronavírus/análise , Internalização do Vírus
9.
Theranostics ; 11(5): 2170-2181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33500718

RESUMO

Introduction: An increasing number of children with severe coronavirus disease 2019 (COVID-19) is being reported, yet the spectrum of disease severity and expression patterns of angiotensin-converting enzyme 2 (ACE2) in children at different developmental stages are largely unknow. Methods: We analysed clinical features in a cohort of 173 children with COVID-19 (0-15 yrs.-old) between January 22, 2020 and March 15, 2020. We systematically examined the expression and distribution of ACE2 in different developmental stages of children by using a combination of children's lung biopsies, pluripotent stem cell-derived lung cells, RNA-sequencing profiles, and ex vivo SARS-CoV-2 pseudoviral infections. Results: It revealed that infants (< 1yrs.-old), with a weaker potency of immune response, are more vulnerable to develop pneumonia whereas older children (> 1 yrs.-old) are more resistant to lung injury. The expression levels of ACE2 however do not vary by age in children's lung. ACE2 is notably expressed not only in Alveolar Type II (AT II) cells, but also in SOX9 positive lung progenitor cells detected in both pluripotent stem cell derivatives and infants' lungs. The ACE2+SOX9+ cells are readily infected by SARS-CoV-2 pseudovirus and the numbers of the double positive cells are significantly decreased in older children. Conclusions: Infants (< 1 yrs.-old) with SARS-CoV-2 infection are more vulnerable to lung injuries. ACE2 expression in multiple types of lung cells including SOX9 positive progenitor cells, in cooperation with an unestablished immune system, could be risk factors contributing to vulnerability of infants with COVID-19. There is a need to continue monitoring lung development in young children who have recovered from SARS-CoV-2 infection.


Assuntos
/metabolismo , Pulmão/citologia , Células-Tronco/metabolismo , Adolescente , Biópsia , Criança , Pré-Escolar , Feminino , Humanos , Sistema Imunitário , Lactente , Recém-Nascido , Pulmão/virologia , Masculino , RNA-Seq , Fatores de Risco , Fatores de Transcrição SOX9/metabolismo , Análise de Célula Única , Células-Tronco/virologia
10.
Environ Toxicol Pharmacol ; 83: 103576, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33385576

RESUMO

Establishing accurate dosimetry is important for assessing the toxicity of xenobiotics as well as for comparing responses between different test systems. In this study, we used acrolein as a model toxicant and defined the concentration-response relationships of the key adverse responses in normal human bronchial epithelial (NHBE) cells and human mucoepidermoid pulmonary carcinoma (NCI-H292) cells. Direct trace analysis of intracellular free acrolein is extremely challenging, if not impossible. Therefore, we developed a new method for indirectly estimating the intracellular uptake of acrolein. A 10-min treatment was employed to capture the rapid occurrence of the key alkylation reactions of acrolein. Responses, including protein carbonylation, GSH depletion, and GSH-acrolein (GSH-ACR) adduct formation, were all linearly correlated with acrolein uptake in both cell types. Compared to the NCI-H292 mucoepidermoid carcinoma cells, NHBE cells were more sensitive to acrolein exposure. Furthermore, results from the time-course studies demonstrated that depletion and conjugation of GSH were the primary adverse events and directly associated with the cytotoxicity induced by acrolein. In summary, these data suggest that cell susceptibility to acrolein exposure is closely associated with acrolein uptake and formation of GSH-ACR adducts. The dosimetric analysis presented in this study may provide useful information for computational modeling and risk assessment of acrolein using different test systems.


Assuntos
Acroleína/toxicidade , Células Epiteliais/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutationa/metabolismo , Humanos , Pulmão/citologia , Carbonilação Proteica
11.
Methods Mol Biol ; 2241: 49-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486727

RESUMO

Flow cytometry is a critical tool that can be employed to detect unique cells and to isolate cells from tissues based on their antigen profiles. While mouse eosinophils can be readily detected by one or more distinct antigen profiles, many of these strategies do not result in accurate eosinophil counts. We present here our basic protocol, which permits quantitative detection of eosinophils and isolation of eosinophils from bone marrow, spleen, and lung tissue of allergen-challenged wild-type and unchallenged IL5 transgenic mice. With small protocol variations, eosinophils can be isolated from small intestines and muscle tissue, the latter from infiltrates characteristic of muscular dystrophy (mdx) mice.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Citometria de Fluxo/métodos , Alérgenos/imunologia , Animais , Sangue/metabolismo , Células Sanguíneas/citologia , Medula Óssea/imunologia , Células da Medula Óssea/citologia , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Feminino , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Receptores de IgG/imunologia , Baço/citologia , Baço/imunologia
12.
Methods Mol Biol ; 2241: 75-87, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486729

RESUMO

Murine models of asthma are developed to better understand the mechanisms of asthma including eosinophil recruitment in the airways with the aim of evaluating new therapeutic strategies. They are intended to model the typical features of human disease, in particular airway inflammation, hyperresponsiveness (AHR), and remodeling. The phenotype of inflammatory cells recovered from the bronchoalveolar lavage fluid (BAL) is studied with innovative flow cytometry techniques while airway obstruction is measured using the forced oscillation technique, and airway responsiveness approached by barometric plethysmography in awake and unconstrained animals. We here describe models of asthma of house dust mite (HDM) as a clinically relevant allergen: a short study design (8 days) model of hypereosinophilic asthma and a chronic (31 days) asthma model, both suitable to evaluate the potential of new drug candidates to prevent allergic asthma.


Assuntos
Desenvolvimento de Medicamentos/métodos , Eosinófilos/citologia , Pyroglyphidae/imunologia , Alérgenos/imunologia , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas , Modelos Animais de Doenças , Eosinófilos/metabolismo , Hipersensibilidade/imunologia , Contagem de Leucócitos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos/imunologia , Camundongos Transgênicos , Transtornos Respiratórios/imunologia , Hipersensibilidade Respiratória/imunologia , Células Th2/imunologia
13.
Methods Mol Biol ; 2147: 149-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32840818

RESUMO

The fabrication of functional biomaterials for organ replacement and tissue repair remains a major goal of biomedical engineering. Advances in additive manufacturing (AM) technologies and computer-aided design (CAD) are advancing the tools available for the production of these devices. Ideally, these constructs should be matched to the geometry and mechanical properties of the tissue at the needed implant site. To generate geometrically defined and structurally supported multicomponent and cell-laden biomaterials, we have developed a method to integrate hydrogels with 3D-printed lattice scaffolds leveraging surface tension-assisted AM.


Assuntos
Materiais Biocompatíveis/síntese química , Microtecnologia/métodos , Impressão Tridimensional , Engenharia Tecidual/instrumentação , Tecidos Suporte/química , Materiais Biocompatíveis/química , Engenharia Biomédica/instrumentação , Engenharia Biomédica/métodos , Células Cultivadas , Desenho Assistido por Computador , Fibroblastos/citologia , Regeneração Tecidual Guiada/instrumentação , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Pulmão/citologia , Medicina Regenerativa/instrumentação , Tensão Superficial
14.
Methods Mol Biol ; 2223: 183-200, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226596

RESUMO

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Imunofenotipagem/métodos , Pulmão/metabolismo , Coloração e Rotulagem/métodos , Imunidade Adaptativa , Animais , Anticorpos/química , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos B/classificação , Linfócitos B/citologia , Linfócitos B/metabolismo , Biomarcadores/análise , Carbocianinas/química , Contagem de Células , Sobrevivência Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Eosinófilos/citologia , Eosinófilos/metabolismo , Citometria de Fluxo/instrumentação , Humanos , Imunidade Inata , Pulmão/citologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/metabolismo , Camundongos , Monócitos/citologia , Monócitos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Cultura Primária de Células , Linfócitos T/classificação , Linfócitos T/citologia , Linfócitos T/metabolismo
15.
Methods Mol Biol ; 2223: 201-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226597

RESUMO

Bronchoalveolar lavage (BAL) is a technique used to collect the contents of the airways. The fluid recovered, called BAL fluid (BALF), serves as a dynamic tool to identify various disease pathologies ranging from asthma to infectious diseases to cancer in the lungs. A wide array of tests can be performed with BALF, including total and differential leukocyte counts (DLC), enzyme-linked immunosorbent assays (ELISA) or flow-cytometric quantitation of inflammatory mediators, such as cytokines, chemokines and adhesion molecules, and assessment of nitrate and nitrite content for estimation of nitric oxide synthase (NOS) activity. Here, we describe a detailed procedure for the collection of BALF for a variety of downstream usages, including DLC by cytological and flow-cytometry-based methods, multiplex cytokine analysis by flow cytometry, and NOS activity analysis by determining nitrate and nitrite levels.


Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Citometria de Fluxo/métodos , Pulmão/citologia , Macrófagos Alveolares/citologia , Neutrófilos/citologia , Animais , Basófilos/citologia , Basófilos/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Eosinófilos/citologia , Eosinófilos/metabolismo , Humanos , Contagem de Leucócitos , Pulmão/imunologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Camundongos , Monócitos/citologia , Monócitos/metabolismo , Neutrófilos/metabolismo , Óxido Nítrico Sintase/imunologia , Óxido Nítrico Sintase/metabolismo , Traqueostomia/métodos
16.
Life Sci ; 267: 118927, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33358908

RESUMO

AIMS: Gender disparities exist in smoking-related lung cancer epidemiology, but the molecular basis has not been explored so far. We aimed at identifying genes with gender-bias expression pattern in smoking lung cancer patients for understanding the molecular basis of gender bias in smokers using meta-analysis of microarray gene expression data. MATERIALS AND METHODS: Transcriptome of around 1100 samples from 13 studies were used in the meta-analysis to identify 'Lung Cancer genes specific to Female-Smokers' (LCFS) and 'Lung Cancer genes specific to Male-Smokers' (LCMS). The expression profiles of these genes were validated with an independent microarray report and TCGA-RNA-sequencing data. The molecular interactions, pathway, and other functional annotations were portrayed for the key genes identified. KEY FINDINGS: We identified 1159 gender-biased genes in smoking lung cancer patients. Of these, 400 and 474 genes showed differential expression in cancerous compared to normal lung of women (LCFS) and men (LCMS), respectively. While many up-regulated LCFS were involved in 'immune responses' including T-cell activation, leukocyte cell-cell adhesion, the LCMS were mainly involved in 'positive regulation of gene expression', signaling pathways including RAS, VEGF, insulin-receptor signaling, and 'cell cycle'. SIGNIFICANCE: The strategic-method identified genes, particularly, SNX20, GIMAP6, MTMR2, FAM171B, IDH1, MOBP, FBXO17, LPXN and WIPF1, which were consistently differentially expressed in at least 4 studies, and in agreement with RNA-Seq data. Exploring their functions could be beneficial to the gender-based diagnosis, prognosis, and treatment of lung cancer in smokers. The current meta-analysis supports existing knowledge of sexual-dimorphism of immune responses in cancer.


Assuntos
Fumar Cigarros/genética , Neoplasias Pulmonares/metabolismo , Moléculas de Adesão Celular/genética , Fumar Cigarros/efeitos adversos , Proteínas do Citoesqueleto/genética , Bases de Dados Genéticas , Feminino , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/citologia , Pulmão/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosfoproteínas/genética , Prognóstico , Proteínas Tirosina Fosfatases não Receptoras/genética , RNA/genética , Fatores Sexuais , Nexinas de Classificação/genética , Transcriptoma
17.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33234678

RESUMO

Viruses rely on their host for reproduction. Here, we made use of genomic and structural information to create a biomass function capturing the amino and nucleic acid requirements of SARS-CoV-2. Incorporating this biomass function into a stoichiometric metabolic model of the human lung cell and applying metabolic flux balance analysis, we identified host-based metabolic perturbations inhibiting SARS-CoV-2 reproduction. Our results highlight reactions in the central metabolism, as well as amino acid and nucleotide biosynthesis pathways. By incorporating host cellular maintenance into the model based on available protein expression data from human lung cells, we find that only few of these metabolic perturbations are able to selectively inhibit virus reproduction. Some of the catalysing enzymes of such reactions have demonstrated interactions with existing drugs, which can be used for experimental testing of the presented predictions using gene knockouts and RNA interference techniques. In summary, the developed computational approach offers a platform for rapid, experimentally testable generation of drug predictions against existing and emerging viruses based on their biomass requirements.


Assuntos
Interações Hospedeiro-Patógeno , Pulmão , Replicação Viral , Antivirais/farmacologia , Biomassa , /virologia , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/metabolismo , Glicólise/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Pulmão/citologia , Pulmão/metabolismo , Análise do Fluxo Metabólico , Modelos Biológicos , /metabolismo , Biologia de Sistemas , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
18.
Nucleic Acids Res ; 49(1): 322-339, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33330905

RESUMO

Many APOBEC cytidine deaminase members are known to induce 'off-target' cytidine deaminations in 5'TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.


Assuntos
Desaminase APOBEC-1/antagonistas & inibidores , DNA de Cadeia Simples/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína de Replicação A/metabolismo , Desaminase APOBEC-1/metabolismo , Ligação Competitiva , Linhagem Celular , Linhagem Celular Tumoral , Citidina/metabolismo , Dano ao DNA , Replicação do DNA , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/química , Desaminação , Difusão Facilitada , Histonas/análise , Humanos , Pulmão/citologia , Pulmão/embriologia , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/genética , Neoplasias/patologia , Especificidade de Órgãos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína de Replicação A/genética
19.
Artigo em Inglês | MEDLINE | ID: mdl-33375152

RESUMO

Although nanoparticles (NPs) have been used as simplified atmospheric particulate matter (PM) models, little experimental evidence is available to support such simulations. In this study, we comparatively assessed the toxic effects of PM and typical NPs (four carbonaceous NPs with different morphologies, metal NPs of Fe, Al, and Ti, as well as SiO2 NPs) on human lung epithelial A549 cells. The EC50 value of PM evaluated by cell viability assay was 148.7 µg/mL, closest to that of SiO2 NPs, between the values of carbonaceous NPs and metal NPs. All particles caused varying degrees of reactive oxygen species (ROS) generation and adenosine triphosphate (ATP) suppression. TiO2 NPs showed similar performance with PM in inducing ROS production (p < 0.05). Small variations between two carbonaceous NPs (graphene oxides and graphenes) and PM were also observed at 50 µg/mL. Similarly, there was no significant difference in ATP inhibition between carbonaceous NPs and PM, while markedly different effects were caused by SiO2 NP and TiO2 NP exposure. Our results indicated that carbonaceous NPs could be served as potential surrogates for urban PM. The identification of PM model may help us further explore the specific roles and mechanisms of various components in PM.


Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais/efeitos dos fármacos , Nanopartículas Metálicas , Nanopartículas , Material Particulado/toxicidade , Células A549 , Humanos , Pulmão/citologia , Nanopartículas Metálicas/toxicidade , Nanopartículas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/toxicidade
20.
PLoS Biol ; 18(12): e3000879, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33382684

RESUMO

Correlative light, electron, and ion microscopy (CLEIM) offers huge potential to track the intracellular fate of antibiotics, with organelle-level resolution. However, a correlative approach that enables subcellular antibiotic visualisation in pathogen-infected tissue is lacking. Here, we developed correlative light, electron, and ion microscopy in tissue (CLEIMiT) and used it to identify the cell type-specific accumulation of an antibiotic in lung lesions of mice infected with Mycobacterium tuberculosis. Using CLEIMiT, we found that the anti-tuberculosis (TB) drug bedaquiline (BDQ) is localised not only in foamy macrophages in the lungs during infection but also accumulate in polymorphonuclear (PMN) cells.


Assuntos
Pulmão/diagnóstico por imagem , Microscopia/métodos , Tuberculose/diagnóstico por imagem , Animais , Antituberculosos , Diarilquinolinas/metabolismo , Diarilquinolinas/farmacologia , Feminino , Pulmão/citologia , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Testes de Sensibilidade Microbiana , Microscopia Eletrônica/métodos , Mycobacterium tuberculosis/patogenicidade
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