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1.
Chem Biol Interact ; 317: 108962, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31982400

RESUMO

Quaternary ammonium compounds (e.g., benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC)) constitute a group of cationic surfactants are widely used for personal hygiene and medical care despite the potential pulmonary toxicity. To examine whether BAC and CPC aerosols deposited in the alveolar region alter pulmonary function, we studied the effects on pulmonary surfactant using two-step in vitro models; cytotoxicity using A549 alveolar epithelial cell and changes in surface activity of the pulmonary surfactant monolayer using both Surfacten® and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Cell viability was decreased with BAC and CPC dose-dependently. A comparison of cytotoxicity among BAC homologues with different length of alkyl chain showed that C16-BAC, which has the longest alkyl chain, was more cytotoxic than C12- or C14-BAC. Caspase-3/7 activity and cleaved form of caspase-3 and PARP were increased in BAC- and CPC-exposed cells. The elevated caspase-3/7 activity and their cleaved active forms were abolished by caspase-3-inhibitor. Furthermore, we examined the features of the surface pressure/trough area (π-A) isotherm by the Langmuir-Wilhelmy method and atomic force microscopy (AFM) images of lipid monolayers on a subphase containing BAC, CPC, or pyridinium chloride (PC, as a control). The π-A isotherms showed that addition of BAC or CPC yielded dose-dependent increases in surface pressure without compression, indicating that BAC and CPC expand the isotherm to larger areas at lower pressure. The collapse pressure diminished with increasing concentration of CPC. Topographic images indicated that BAC and CPC resulted in smaller condensed lipid domains compared to the control. Conversely, PC without hydrocarbon tail group, showed no cytotoxicity and did not change the isotherms and AFM images. These results indicate that BAC and CPC cause cell death via caspase-3-dependent apoptotic pathway in A549 cells and alter the alveolar surfactant activity. These effects can be attributed to the long alkyl chain of BAC and CPC.


Assuntos
Apoptose/efeitos dos fármacos , Compostos de Benzalcônio/farmacologia , Cetilpiridínio/farmacologia , Células Epiteliais/efeitos dos fármacos , Pulmão/citologia , Mucosa Respiratória/citologia , Células A549 , Compostos de Benzalcônio/química , Sobrevivência Celular/efeitos dos fármacos , Cetilpiridínio/química , Humanos , Tensoativos/metabolismo
2.
Chemosphere ; 241: 125126, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31683444

RESUMO

To study the fate of cyclic volatile methyl siloxanes (cVMS) undergoing photooxidation in the environment and to assess the acute toxicity of inhaled secondary aerosols from cVMS, we used an oxidative flow reactor (OFR) to produce aerosols from oxidation of decamethylcyclopentasiloxane (D5). The aerosols produced from this process were characterized for size, shape, and chemical composition. We found that the OFR produced aerosols composed of silicon and oxygen, arranged in chain agglomerates, with primary particles of approximately 31 nm in diameter. Lung cells were exposed to the secondary organosilicon aerosols at estimated doses of 54-116 ng/cm2 using a Vitrocell air-liquid interface system, and organic gases and ozone exposure was minimized through a series of denuders. Siloxane aerosols were not found to be highly toxic.


Assuntos
Aerossóis/química , Pulmão/efeitos dos fármacos , Siloxanas/química , Células A549 , Aerossóis/toxicidade , Gases/química , Humanos , Pulmão/citologia , Oxirredução , Tamanho da Partícula , Siloxanas/toxicidade
4.
J Surg Res ; 245: 273-280, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421373

RESUMO

BACKGROUND: Transplantation of lungs procured after donation after circulatory death (DCD) is challenging because postmortem metabolic degradation may engender susceptibility to ischemia-reperfusion (IR) injury. Because oxidative mitochondrial DNA (mtDNA) damage has been linked to endothelial barrier disruption in other models of IR injury, here we used a fusion protein construct targeting the DNA repair 8-oxoguanine DNA glycosylase-1 (OGG1) to mitochondria (mtOGG1) to determine if enhanced repair of mtDNA damage attenuates endothelial barrier dysfunction after IR injury in a rat model of lung procurement after DCD. MATERIALS AND METHODS: Lungs excised from donor rats 1 h after cardiac death were cold stored for 2 h after which they were perfused ex vivo in the absence and presence of mt-OGG1 or an inactive mt-OGG1 mutant. Lung endothelial barrier function and mtDNA integrity were determined during and at the end of perfusion, respectively. RESULTS AND CONCLUSIONS: Mitochondria-targeted OGG1 attenuated indices of lung endothelial dysfunction incurred after a 1h post-mortem period. Oxidative lung tissue mtDNA damage as well as accumulation of proinflammatory mtDNA fragments in lung perfusate, but not nuclear DNA fragments, also were reduced by mitochondria-targeted OGG1. A repair-deficient mt-OGG1 mutant failed to protect lungs from the adverse effects of DCD procurement. CONCLUSIONS: These findings suggest that endothelial barrier dysfunction in lungs procured after DCD is driven by mtDNA damage and point to strategies to enhance mtDNA repair in concert with EVLP as a means of alleviating DCD-related lung IR injury.


Assuntos
DNA Glicosilases/administração & dosagem , Endotélio Vascular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Recombinantes de Fusão/administração & dosagem , Traumatismo por Reperfusão/prevenção & controle , Aloenxertos/irrigação sanguínea , Aloenxertos/citologia , Aloenxertos/efeitos dos fármacos , Animais , DNA Glicosilases/genética , Reparo do DNA/efeitos dos fármacos , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/genética , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Humanos , Pulmão/irrigação sanguínea , Pulmão/citologia , Pulmão/efeitos dos fármacos , Transplante de Pulmão , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Perfusão/métodos , Ratos , Proteínas Recombinantes de Fusão/genética , Traumatismo por Reperfusão/patologia , Coleta de Tecidos e Órgãos/métodos
5.
Life Sci ; 241: 117166, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31843527

RESUMO

AIMS: Congenital diaphragmatic hernia (CDH) is a lethal birth defect characterized by congenital lung malformation, and the severity of pulmonary hypoplasia directly affects the prognosis of infants with CDH. Using a nitrofen-induced CDH rat model, we previously reported that Foxa2 expression was downregulated in CDH lungs by proteomics analysis. Here, we investigate the role of miR-130a-5p/Foxa2 axis in lung development of the nitrofen-induced CDH and evaluate its potential role in vivo prenatal therapy. MAIN METHODS: Nitrofen was orally administrated on embryonic day (E) 8.5 to establish a rat CDH model, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5 and E21.5. The binding sites of miR-130a-5p on Foxa2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. The expression levels of miR-130a-5p and Foxa2 were detected using qRT-PCR, ISH, IHC and western blotting. The role of miR-130a-5p/Foxa2 axis in CDH-associated lung development was investigated in ex vivo lung explants. KEY FINDINGS: We found that Foxa2 was downregulated in CDH lung tissues, and Foxa2 upregulating improved CDH branching morphogenesis in ex vivo lung explants. Meanwhile, we also showed that miR-130a-5p was significantly upregulated in CDH lungs and thus inversely correlated with Foxa2. Increasing miR-130a-5p abundance with mimics decreases Foxa2-driven Shh/Gli1 signaling and inhibits branching morphogenesis in ex vivo lung explants. SIGNIFICANCE: This study was the first to show that the miR-130a-5p/Foxa2 axis played a crucial role in CDH-associated pulmonary hypoplasia. These findings may provide relevant insights into the prenatal diagnosis and prenatal therapy of CDH.


Assuntos
Desenvolvimento Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator 3-beta Nuclear de Hepatócito/metabolismo , Hérnias Diafragmáticas Congênitas/patologia , Pulmão/citologia , MicroRNAs/genética , Organogênese , Animais , Proliferação de Células , Células Cultivadas , Feminino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Herbicidas/toxicidade , Hérnias Diafragmáticas Congênitas/induzido quimicamente , Hérnias Diafragmáticas Congênitas/metabolismo , Pulmão/fisiologia , Masculino , Éteres Fenílicos/toxicidade , Gravidez , Ratos , Ratos Sprague-Dawley , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
6.
Nat Protoc ; 14(12): 3303-3332, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31732721

RESUMO

Alveolar epithelial type II cells (AEC2s) are the facultative progenitors of lung alveoli and serve as the surfactant-producing cells of air-breathing organisms. Although primary human AEC2s are difficult to maintain stably in cell cultures, recent advances have facilitated the derivation of AEC2-like cells from human pluripotent stem cells (hPSCs) in vitro. Here, we provide a detailed protocol for the directed differentiation of hPSCs into self-renewing AEC2-like cells that can be maintained for up to 1 year in culture as epithelial-only spheres without the need for supporting mesenchymal feeder cells. The month-long protocol requires recapitulation of the sequence of milestones associated with in vivo development of the distal lung, beginning with differentiation of cells into anterior foregut endoderm, which is followed by their lineage specification into NKX2-1+ lung progenitors and then distal/alveolar differentiation to produce progeny that express transcripts and possess functional properties associated with AEC2s.


Assuntos
Células Epiteliais Alveolares/citologia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Células Epiteliais Alveolares/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Autorrenovação Celular/genética , Autorrenovação Celular/fisiologia , Células Epiteliais/citologia , Células Alimentadoras , Humanos , Pulmão/citologia , Células-Tronco Pluripotentes/fisiologia
8.
Clin Exp Rheumatol ; 37 Suppl 119(4): 115-124, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31573469

RESUMO

OBJECTIVES: Nintedanib is approved for the treatment of idiopathic pulmonary fibrosis (IPF) and was demonstrated to slow disease progression in patients with IPF by reducing decline in forced vital capacity by 50%. Recently, nintedanib has been reported to exert anti-fibrotic activity on systemic sclerosis (scleroderma, SSc) skin fibroblasts and to diminish skin and lung fibrosis in mouse models. The goal of the present study was to determine the effects of nintedanib on a cellular model of SSc-associated interstitial lung disease (ILD). METHODS: Study was performed using lung fibroblasts (LF) isolated from five patients with SSc-ILD and from three control subjects. RESULTS: Nintedanib inhibited LF proliferation and migration in a concentration- and time-dependent manner. The proliferation rate of LF stimulated with PDGF in the presence of nintedanib was reduced 1.9-fold within 24 h as compared to cells stimulated with PDGF alone. Migration of SSc-ILD LF incubated with 100 nM nintedanib was reduced from 62.8±12.5% to 39.1±9.0% in the presence of PDGF and from 38.2±7.9% to 26.6±7.2% in serum-free medium. Nintedanib attenuated PDGF-induced Ca2+ efflux, reduced α-SMA promoter activity and α-SMA protein expression. Furthermore, nintedanib blocked PDGF-induced differentiation of normal LF to myofibroblasts, reduced production of collagen and fibronectin, and decreased contractility of SSc-ILD LF in both floating and fixed collagen gels. CONCLUSIONS: Our data demonstrate significant antifibrotic efficacy of nintedanib in SSc-ILD LF suggesting that nintedanib has the potential not only to prevent but also to reverse the increased activity of LF consequently attenuating excessive lung fibrosis observed in SSc-ILD.


Assuntos
Fibrose Pulmonar Idiopática , Indóis/uso terapêutico , Doenças Pulmonares Intersticiais , Inibidores de Proteínas Quinases/uso terapêutico , Escleroderma Sistêmico , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/etiologia , Pulmão/citologia , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/etiologia , Escleroderma Sistêmico/complicações
9.
Chem Commun (Camb) ; 55(88): 13223-13226, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31595909

RESUMO

There are a limited number of near-infrared (NIR) emitting (λem = 700-900 nm) molecular probes for imaging applications. A NIR-emitting probe that exhibits emission at ∼800 nm with a large Stokes shift was synthesized and found to exhibit excellent selectivity towards mitochondria for live-cell imaging. The photophysical properties were attributed to an excited "cyanine structure" via intramolecular charge transfer (ICT) involving a phenol group.


Assuntos
Carbocianinas/química , Fibroblastos/química , Corantes Fluorescentes/química , Oligodendroglia/química , Imagem Óptica , Fenóis/química , Linhagem Celular , Humanos , Raios Infravermelhos , Pulmão/citologia , Estrutura Molecular , Espectrometria de Fluorescência
10.
Sheng Li Xue Bao ; 71(5): 689-697, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31646322

RESUMO

The aim of the present study was to investigate the role of ferroptosis in acute lung injury (ALI) mouse model induced by oleic acid (OA). ALI was induced in the mice via the lateral tail vein injection of pure OA. The histopathological score of lung, lung wet-dry weight ratio and the protein content of bronchoalveolar lavage fluid (BALF) were used as the evaluation indexes of ALI. Iron concentration, glutathione (GSH) and malondialdehyde (MDA) contents in the lung tissues were measured using corresponding assay kits. The ultrastructure of pulmonary cells was observed by transmission electron microscope (TEM), and the expression level of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA was detected by quantitative polymerase chain reaction (q-PCR). Protein expression levels of glutathione peroxidase 4 (GPX4), ferritin and transferrin receptor 1 (TfR1) in lung tissues were determined by Western blot. The results showed that histopathological scores of lung tissues, lung wet-dry weight ratio and protein in BALF in the OA group were higher than those of the control group. In the OA group, the mitochondria of pulmonary cells were shrunken, and the mitochondrial membrane was ruptured. The expression level of PTGS2 mRNA in the OA group was seven folds over that in the control group. Iron overload, GSH depletion and accumulation of MDA were observed in the OA group. Compared with the control group, the protein expression levels of GPX4 and ferritin in lung tissue were down-regulated in the OA group. These results suggest that ferroptosis plays a potential role in the pathogenesis of ALI in our mouse model, which may provide new insights for development of new drugs for ALI.


Assuntos
Lesão Pulmonar Aguda/patologia , Apoptose , Ácido Oleico , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Líquido da Lavagem Broncoalveolar/química , Ciclo-Oxigenase 2/metabolismo , Ferritinas/metabolismo , Glutationa/análise , Glutationa Peroxidase/metabolismo , Ferro/análise , Sobrecarga de Ferro/fisiopatologia , Pulmão/citologia , Pulmão/patologia , Malondialdeído/análise , Camundongos , Microscopia Eletrônica de Transmissão , Membranas Mitocondriais/ultraestrutura
11.
Vet Immunol Immunopathol ; 217: 109955, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31639586

RESUMO

The purpose of this work was to characterize the cellular phenotype in inflammatory infiltrates of fetal tissues from pregnant heifers immunized and experimentally challenged with Neospora caninum. Fetuses from 20 heifers separated into 5 groups were obtained. The experiment was designed as follow: Group A, heifers inoculated intravenously with live tachyzoites of Argentine strain NC-6 (n = 4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (n = 4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (n = 4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (n = 4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (n = 4). Experimental challenge was performed at 70 days of gestation and all heifers were euthanized 34 days later. Fetal tissues were taken for histological studies. Inflammatory lesions were observed in brain and lung, and immunhistochemistry was used to identify CD3+, CD20+ and MHC II+ cells. The majority of the cells that infiltrate and circumscribe the lesions in the brain and lung tissue expressed MHC II antigen; varying between 70-90% of the total cellular infiltrate. CD3+ cells were also present within the lesions, contributing to up to 30% of the inflammatory cells. CD20+ cells appeared as a marginal group, in some cases, with a range between 10 and 25%. As expected, the immunolabeling of MHC II + and CD3 + cells in fetal tissues was associated with fetal infection with N. caninum. There were statistically significant differences in the distribution and population of the inflammatory infiltrate in relation to the immunogenic treatment and the type of tissue, with inflammatory cells being markedly less extensive fetuses from group A (dams previously exposed to N. caninum) and in brain tissue. This work showed that Neospora-infection induced MHC II+ and CD3+ cells in bovine fetuses from dams receiving experimental vaccines.


Assuntos
Coccidiose/imunologia , Feto/imunologia , Imunização/veterinária , Neospora/imunologia , Vacinas Protozoárias/imunologia , Animais , Encéfalo/citologia , Encéfalo/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Feminino , Feto/citologia , Imuno-Histoquímica , Pulmão/citologia , Pulmão/imunologia , Gravidez
12.
J Biol Regul Homeost Agents ; 33(5): 1415-1424, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31556264

RESUMO

Transforming growth factor-beta (TGF-ß) functions in fibrogenesis as a profibrotic mediator, regulating cell proliferation, migration, apoptosis and collagen production of fibroblasts. microRNA-155 (miR-155), the expression of which has been related to bleomycin-induced idiopathic pulmonary fibrosis, has been involved in TGF-ß induced epithelial-mesenchymal transition. Here, we found that miR-155 expression was decreased in human pulmonary fibroblasts by TGF-ß treatment. We overexpressed miR-155 in fibroblasts to investigate the functional impact of miR-155 on TGF-ß-induced fibrotic phenotype of fibroblasts. It is suggested that miR-155 overexpression attenuated the stimulatory effect of TGF-ß on fibroblast proliferation, migration and collagen synthesis, by evidence from assessment of cell cycle, viability, apoptosis, migration and collagen content. Furthermore, quantitative measurement showed that SMAD1 gene expression was decreased following miR-155 inhibition, thereby demonstrating an indirect miRNA-SMAD interaction that links miR-155 to TGF-ß signaling. Our work helped uncover an miRNA-mediated mechanism of fibroblast response to TGF-ß. Moreover, it will help to achieve a better understanding of the regulatory roles of miR-155 in fibrogenesis.


Assuntos
Fibroblastos/efeitos dos fármacos , MicroRNAs/genética , Fator de Crescimento Transformador beta/farmacologia , Células Cultivadas , Fibrose , Humanos , Pulmão/citologia
13.
Nat Commun ; 10(1): 3964, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481690

RESUMO

Resident tissue macrophages (RTM) can fulfill various tasks during development, homeostasis, inflammation and repair. In the lung, non-alveolar RTM, called interstitial macrophages (IM), importantly contribute to tissue homeostasis but remain little characterized. Here we show, using single-cell RNA-sequencing (scRNA-seq), two phenotypically distinct subpopulations of long-lived monocyte-derived IM, i.e. CD206+ and CD206-IM, as well as a discrete population of extravasating CD64+CD16.2+ monocytes. CD206+ IM are peribronchial self-maintaining RTM that constitutively produce high levels of chemokines and immunosuppressive cytokines. Conversely, CD206-IM preferentially populate the alveolar interstitium and exhibit features of antigen-presenting cells. In addition, our data support that CD64+CD16.2+ monocytes arise from intravascular Ly-6Clo patrolling monocytes that enter the tissue at steady-state to become putative precursors of CD206-IM. This study expands our knowledge about the complexity of lung IM and reveals an ontogenic pathway for one IM subset, an important step for elaborating future macrophage-targeted therapies.


Assuntos
Pulmão/citologia , Macrófagos Alveolares/citologia , Monócitos/citologia , Animais , Citometria de Fluxo , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Fenótipo , Análise de Sequência de RNA , Análise de Célula Única/métodos
14.
Adv Exp Med Biol ; 1169: 95-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487021

RESUMO

Epithelial stem cells reside within multiple regions of the lung where they renew various region-specific cells. In addition, there are multiple routes of regeneration after injury through built-in heterogeneity within stem cell populations and through a capacity for cellular plasticity among differentiated cells. These processes are important facets of respiratory tissue resiliency and organism survival. However, this regenerative capacity is not limitless, and repetitive or chronic injuries, environmental stresses, or underlying factors of disease may ultimately lead to or contribute to tissue remodeling and end-stage lung disease. This chapter will review stem cell heterogeneity among pulmonary epithelia in the lower respiratory system, discuss recent findings that may challenge long-held scientific paradigms, and identify several clinically relevant research opportunities for regenerative medicine.


Assuntos
Pulmão , Células-Tronco , Animais , Diferenciação Celular , Humanos , Pulmão/citologia , Células-Tronco/citologia
15.
Respir Res ; 20(1): 172, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370853

RESUMO

Genome wide association (GWA) studies have reproducibly identified signals on chromosome 4q24 associated with lung function and COPD. GSTCD (Glutathione S-transferase C-terminal domain containing) represents a candidate causal gene in this locus, however little is currently known about the function of this protein. We set out to further our understanding of the role of GSTCD in cell functions and homeostasis using multiple molecular and cellular approaches in airway relevant cells. Recombinant expression of human GSTCD in conjunction with a GST activity assay did not identify any enzymatic activity for two GSTCD isoforms questioning the assignment of this protein to this family of enzymes. Protein structure analyses identified a potential methyltransferase domain contained within GSTCD, with these enzymes linked to cell viability and apoptosis. Targeted knockdown (siRNA) of GSTCD in bronchial epithelial cells identified a role for GSTCD in cell viability as proliferation rates were not altered. To provide greater insight we completed transcriptomic analyses on cells with GSTCD expression knocked down and identified several differentially expressed genes including those implicated in airway biology; fibrosis e.g. TGFBR1 and inflammation e.g. IL6R. Pathway based transcriptomic analyses identified an over-representation of genes related to adipogenesis which may suggest additional functions for GSTCD. These findings identify potential additional functions for GSTCD in the context of airway biology beyond the hypothesised GST activity and warrant further investigation.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Homeostase/fisiologia , Pulmão/fisiologia , Miócitos de Músculo Liso/fisiologia , Proteínas/genética , Mucosa Respiratória/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Pulmão/citologia , Proteínas/metabolismo , Mucosa Respiratória/citologia
16.
Int J Pharm ; 569: 118616, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31415873

RESUMO

Respiratory tract infections caused by multidrug-resistant Gram-negative bacteria are serious burdens to the public. Our previous findings indicated that co-loading of colistin and ciprofloxacin via liposomes improved in vitro antimicrobial activities against multidrug resistant Pseudomonas aeruginosa as compared to the monotherapies. The current study aims to investigate the transport behavior of colistin and ciprofloxacin in liposomes using the in vitro Calu-3 cell monolayer, which is a lung epithelial model cultured under the air-interfaced condition. The cell viability results demonstrated that there was no obvious toxicity of cells exposed to single or co-administered drugs at the concentration ≤500 µg/mL. Transport of ciprofloxacin into the cells was easier than that of colistin, which reached a plateau rapidly. Colistin was less trapped in the mucus or adhered to the apical cell membrane, and less transported across the cell monolayer than ciprofloxacin. The deposition of ciprofloxacin on the apical side increased over time (from 1 to 4 h). There was no drug-drug interaction observed during the transport of ciprofloxacin and colistin across the cell monolayer, when they were dosed together in the solution form. The amount of drug transported across the cell monolayer was decreased in both agents when loaded in liposomes. Both drugs were more trapped in the mucus or more adhered to the apical side cell membrane of the cell monolayer when they were in liposomes. This study demonstrated that co-delivery of colistin and ciprofloxacin in a single liposome can reduce transport capacity of both drugs across the lung epithelial cell monolayer and enhance drug retention on the lung epithelial surfaces; therefore, it is a promising approach to treat the respiratory infections caused by multidrug resistant Pseudomonas aeruginosa.


Assuntos
Antibacterianos/administração & dosagem , Ciprofloxacino/administração & dosagem , Colistina/administração & dosagem , Células Epiteliais/metabolismo , Pulmão/citologia , Linhagem Celular , Humanos , Lipossomos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa
17.
Int J Mol Med ; 44(4): 1399-1413, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432143

RESUMO

At present, thousands of circular RNAs (circRNAs) have been found in cancer and various tissues from different species. However, the expression of circRNAs during rat lung development remains largely unknown. In the present study, circRNA expression profiles were screened in three mixed rat lung tissues at 3 time­points [embryonic day (E) 19, E21 and post­natal (P) day 3] during fetal rat development with circRNA high­throughput sequencing. Preliminary results were verified by reverse transcription­PCR (RT­PCR) at 4 time­points (E16, E19, E21 and P3). A total of 375 circRNAs were differently expressed in E19 vs. E21 (fold change ≥1.5; P<0.05). At the same time, a total of 358 circRNAs were differently expressed in E21 vs. P3 (fold change ≥1.5; P<0.05). A total of 3 circRNAs (rno_circ:chr7:24777879­24784993, rno_circ:chr14:14620910­14624933 and rno_circ:chr3:1988750­â€‹1998592) were characterized by having consistent fold changes (≥1.5) between 3 time­points (E19, E21 and P3) and were selected for RT­PCR at 4 time­points (E16, E19, E21 and P3). Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of parent genes of the differentially expressed circRNAs revealed that these circRNAs may serve important roles in lung development. The present results support that these new found circRNAs participate in lung development. Furthermore, these findings may help to clarify the physiopathological mechanism of normal rat lung development, and may further provide a physiopathological basis of lung developmental diseases.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pulmão/embriologia , Pulmão/metabolismo , Organogênese/genética , /genética , Animais , Biologia Computacional/métodos , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Pulmão/citologia , MicroRNAs/genética , Gravidez , Ratos
18.
Acta Histochem ; 121(7): 852-865, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31445760

RESUMO

Anatolian ground squirrel (Spermophilus xanthoprymnus) is a true hibernator. This animal transiently reduces pulmonary function during hibernation. Continuance of pulmonary function is very important to survive ground squirrels during the hibernation. Natriuretic peptides may be key players in the modulation of pulmonary hemostasis. However, NPs' role in pulmonary function during hibernation remains unclear. We aimed to investigate the localization and distribution of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) in squirrel lungs during pre-hibernation and hibernation periods using immunohistochemistry. Our immunohistochemical data indicate that ANP, BNP, and CNP were produced by the mucosal epithelium of terminal and respiratory bronchioles, smooth muscle cells in the lamina propria of terminal bronchioles and vascular smooth muscle cells, alveolar type II cells, and macrophages. ANP immunoreactivity was weaker than BNP and CNP immunoreactivities in these cells. The results also demonstrate that the number of ANP, BNP and CNP positive alveolar type II cells tended to increase, although statistically non-significant, during the hibernation period, but the expression of NPs in other pulmonary cells is unaffected by hibernation. This study firstly investigates ANP, BNP and CNP distribution in the Anatolian ground squirrel lung. However, further studies are required to dissect their functional roles during the hibernation.


Assuntos
Regulação da Expressão Gênica/fisiologia , Hibernação/fisiologia , Pulmão , Peptídeos Natriuréticos/biossíntese , Mucosa Respiratória , Sciuridae/metabolismo , Animais , Imuno-Histoquímica , Pulmão/citologia , Pulmão/metabolismo , Masculino , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo
19.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31451621

RESUMO

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia worldwide, and interleukin-22 (IL-22) helps contain pneumococcal burden in lungs and extrapulmonary tissues. Administration of IL-22 increases hepatic complement 3 and complement deposition on bacteria and improves phagocytosis by neutrophils. The effects of IL-22 can be tempered by a secreted natural antagonist, known as IL-22 binding protein (IL-22BP), encoded by Il22ra2 To date, the degree to which IL-22BP controls IL-22 in pulmonary infection is not well defined. Here, we show that Il22ra2 inhibits IL-22 during S. pneumoniae lung infection and that Il22ra2 deficiency favors downregulation of oxidative phosphorylation (OXPHOS) genes in an IL-22-dependent manner. Il22ra2-/- mice are more resistant to S. pneumoniae infection, have increased IL-22 in lung tissues, and sustain longer survival upon infection than control mice. Transcriptome sequencing (RNA-seq) analysis of infected Il22ra2-/- mouse lungs revealed downregulation of genes involved in OXPHOS. Downregulation of this metabolic process is necessary for increased glycolysis, a crucial step for transitioning to a proinflammatory phenotype, in particular macrophages and dendritic cells (DCs). Accordingly, we saw that macrophages from Il22ra2-/- mice displayed reduced OXPHOS gene expression upon infection with S. pneumoniae, changes that were IL-22 dependent. Furthermore, we showed that macrophages express IL-22 receptor subunit alpha-1 (IL-22Ra1) during pneumococcal infection and that Il22ra2-/- macrophages rely more on the glycolytic pathway than wild-type (WT) controls. Together, these data indicate that IL-22BP deficiency enhances IL-22 signaling in the lung, thus contributing to resistance to pneumococcal pneumonia by downregulating OXPHOS genes and increasing glycolysis in macrophages.


Assuntos
Interleucinas/metabolismo , Pneumonia Pneumocócica/metabolismo , Receptores de Interleucina/metabolismo , Animais , Linhagem Celular , Suscetibilidade a Doenças , Células Epiteliais/fisiologia , Regulação da Expressão Gênica , Interleucinas/genética , Antígenos Comuns de Leucócito , Pulmão/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Oxirredução , Fosforilação , Pneumonia Pneumocócica/imunologia , Receptores de Interleucina/genética , Streptococcus pneumoniae
20.
Nat Commun ; 10(1): 3841, 2019 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-31451696

RESUMO

Human lung tissue-resident NK cells (trNK cells) are likely to play an important role in host responses towards viral infections, inflammatory conditions and cancer. However, detailed insights into these cells are still largely lacking. Here we show, using RNA sequencing and flow cytometry-based analyses, that subsets of human lung CD69+CD16- NK cells display hallmarks of tissue-residency, including high expression of CD49a, CD103, and ZNF683, and reduced expression of SELL, S1PR5, and KLF2/3. CD49a+CD16- NK cells are functionally competent, and produce IFN-γ, TNF, MIP-1ß, and GM-CSF. After stimulation with IL-15, they upregulate perforin, granzyme B, and Ki67 to a similar degree as CD49a-CD16- NK cells. Comparing datasets from trNK cells in human lung and bone marrow with tissue-resident memory CD8+ T cells identifies core genes co-regulated either by tissue-residency, cell-type or location. Together, our data indicate that human lung trNK cells have distinct features, likely regulating their function in barrier immunity.


Assuntos
Imunidade nas Mucosas , Células Matadoras Naturais/metabolismo , Pneumopatias/imunologia , Pulmão/citologia , Transcriptoma/imunologia , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Conjuntos de Dados como Assunto , Feminino , Humanos , Células Matadoras Naturais/imunologia , Pulmão/imunologia , Pulmão/cirurgia , Pneumopatias/patologia , Pneumopatias/cirurgia , Masculino , Pessoa de Meia-Idade , Pneumonectomia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo
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