Carboxyhaemoglobin and pulmonary epithelial permeability in man.

PubMed Central

Jones, J G; Minty, B D; Royston, D; Royston, J P

1983-01-01

The effect of cigarette smoke exposure on pulmonary epithelial permeability was studied in 45 smokers and 22 non-smokers. An index of cigarette smoke exposure was obtained from the carboxyhaemoglobin concentration (HbCO%). Pulmonary epithelial permeability was proportional to the half-time clearance rate of technetium-99m-labelled diethylene triamine pentacetate (99mTc DTPA) from lung to blood (T1/2LB). The relationship between T1/2LB and HbCO% was hyperbolic in form and the data could be fitted to the quadratic formula (formula; see text) where the parameters a0, a1, and a2 represent respectively the asymptotic T1/2LB value at large carboxyhaemoglobin values and the slope and shape of the curve. The values of these parameters were a0 4.4 (2.6), a1 = 77.8 (15.5), and a2 -25.5 (9.7) (SE). This is the first demonstration of a dose-response relationship between carboxyhaemoglobin and an increased permeability of the lungs in man and provides a technique for identifying the roles of carbon monoxide and other cigarette smoke constituents in causing increased pulmonary epithelial permeability. PMID:6344310

Mineral fiber-mediated activation of phosphoinositide-specific phospholipase c in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells.

PubMed

Loreto, Carla; Carnazza, Maria Luisa; Cardile, Venera; Libra, Massimo; Lombardo, Laura; Malaponte, Grazia; Martinez, Giuseppina; Musumeci, Giuseppe; Papa, Veronica; Cocco, Lucio

2009-02-01

Given the role of phosphoinositide-specific phospholipase C (PLC) isozymes in the control of cell growth and differentiation we were prompted to analyze the expression of some of these PLC in human bronchoalveolar carcinoma-derived alveolar epithelial A549 cells. The effects of several fluoro-edenite fibers were compared with those of tremolite, a member of the calcic amphibole group of asbestos that originates from Calabria (Italy), and crocidolite, that, due to its high toxicity, is one of the most studied asbestos amphiboles. Our data show an increased expression of both PLC beta1 and PLC gamma1 in A549 cells treated with asbestos-like fibers, hinting at a role of PLC signalling in those cancerous cells.

Mesenchymal stem cells promote cell invasion and migration and autophagy-induced epithelial-mesenchymal transition in A549 lung adenocarcinoma cells.

PubMed

Luo, Dan; Hu, Shiyuan; Tang, Chunlan; Liu, Guoxiang

2018-03-01

Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment-induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial-mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E-cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture-mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture-mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell-containing microenvironments and MSC-induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion. Copyright © 2018 John Wiley & Sons, Ltd.

In vitro cytotoxicity effect and antibacterial performance of human lung epithelial cells A549 activity of Zinc oxide doped TiO2 nanocrystals: Investigation of bio-medical application by chemical method.

PubMed

Kaviyarasu, K; Geetha, N; Kanimozhi, K; Maria Magdalane, C; Sivaranjani, S; Ayeshamariam, A; Kennedy, J; Maaza, M

2017-05-01

We report the synthesis of high quality ZnO doped TiO 2 nanocrystals by chemical method at room temperature (RT), it can cause serious oxidative stress and DNA damage to human lung epithelial cells (A549) lines. Our aim in this study, to reduce the cytotoxicity effect of ZnO doped TiO 2 nanocrystals are widely in biological fields. Several studies have been performed to understand the influence of ZnO doped titanium dioxide (TiO 2 -NPs) on cell function; however the effects of nanoparticle against to exposure on the cell membrane have been duly addressed fascinatingly so far. However, In this interaction, which may alter cell metabolism and integrity, it is one of the importance to understand the modifications of the cell membrane, mechanisms of pulmonary A549 cell lines nanoparticles were uptake and the molecular pathway during the initial cell responses are still unclear and much more investigative efforts are need to properly characterize the ZnO doped titanium dioxide nanoparticles were reported successfully. In particular of the epithelial cells, upon particles are exposed human pulmonary epithelial cells (A549) to various concentrations of composition, structure and morphology of the nanocrystals were analyzed by X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). XRD assessed the crystal structure of the nanocrystals which identified peaks associated with (002), (100) and (101) planes of hexagonal wurtzite-type ZnO with lattice constants of a=b=3.249Å and c=5.219Å. The IR results showed high purity of products and indicated that the nanocrystals are made up of TiO and ZnO bonds. The Photoluminescence (PL) spectra are dominated by a strong narrow band edge emission tunable in the blue region of the visible spectra indicating a narrow size distribution of ZnO/TiO 2 nanocrystals which exhibits antibacterial activity over a broad range of bacterial species and in particular against Stre. Mut where it out competes four other

Preprocessing with Photoshop Software on Microscopic Images of A549 Cells in Epithelial-Mesenchymal Transition.

PubMed

Ren, Zhou-Xin; Yu, Hai-Bin; Shen, Jun-Ling; Li, Ya; Li, Jian-Sheng

2015-06-01

To establish a preprocessing method for cell morphometry in microscopic images of A549 cells in epithelial-mesenchymal transition (EMT). Adobe Photoshop CS2 (Adobe Systems, Inc.) was used for preprocessing the images. First, all images were processed for size uniformity and high distinguishability between the cell and background area. Then, a blank image with the same size and grids was established and cross points of the grids were added into a distinct color. The blank image was merged into a processed image. In the merged images, the cells with 1 or more cross points were chosen, and then the cell areas were enclosed and were replaced in a distinct color. Except for chosen cellular areas, all areas were changed into a unique hue. Three observers quantified roundness of cells in images with the image preprocess (IPP) or without the method (Controls), respectively. Furthermore, 1 observer measured the roundness 3 times with the 2 methods, respectively. The results between IPPs and Controls were compared for repeatability and reproducibility. As compared with the Control method, among 3 observers, use of the IPP method resulted in a higher number and a higher percentage of same-chosen cells in an image. The relative average deviation values of roundness, either for 3 observers or 1 observer, were significantly higher in Controls than in IPPs (p < 0.01 or 0.001). The values of intraclass correlation coefficient, both in Single Type or Average, were higher in IPPs than in Controls both for 3 observers and 1 observer. Processed with Adobe Photoshop, a chosen cell from an image was more objective, regular, and accurate, creating an increase of reproducibility and repeatability on morphometry of A549 cells in epithelial to mesenchymal transition.

Up-Regulation of Pro-Inflammatory Cytokines and Chemokine Production in Avian Influenza H9N2 Virus-Infected Human Lung Epithelial Cell Line (A549).

PubMed

Farzin, Hamidreza; Toroghi, Reza; Haghparast, Alireza

2016-01-01

Influenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1β, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1β (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1β proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1β (p < 0.05 in 18 hours and p < 0.001 in 24-48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.

Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

NASA Astrophysics Data System (ADS)

Brown, David M.; Varet, Julia; Johnston, Helinor; Chrystie, Alison; Stone, Vicki

2015-10-01

Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks' balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle's activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed.

Anacardic acid, a histone acetyltransferase inhibitor, modulates LPS-induced IL-8 expression in a human alveolar epithelial cell line A549

PubMed Central

Takizawa, Hajime

2013-01-01

Objective and design: The histone acetylation processes, which are believed to play a critical role in the regulation of many inflammatory genes, are reversible and regulated by histone acetyltransferases (HATs), which promote acetylation, and histone deacetylases (HDACs), which promote deacetylation. We studied the effects of lipopolysaccharide (LPS) on histone acetylation and its role in the regulation of interleukin (IL)-8 expression.  Material: A human alveolar epithelial cell line A549 was used in vitro. Methods: Histone H4 acetylation at the IL-8 promoter region was assessed by a chromatin immunoprecipitation (ChIP) assay. The expression and production of IL-8 were evaluated by quantitative polymerase chain reaction and specific immunoassay. Effects of a HDAC inhibitor, trichostatin A (TSA), and a HAT inhibitor, anacardic acid, were assessed.  Results: Escherichia coli-derived LPS showed a dose- and time-dependent stimulatory effect on IL-8 protein production and mRNA expression in A549 cells in vitro. LPS showed a significant stimulatory effect on histone H4 acetylation at the IL-8 promoter region by ChIP assay. Pretreatment with TSA showed a dose-dependent stimulatory effect on IL-8 release from A549 cells as compared to LPS alone. Conversely, pretreatment with anacardic acid inhibited IL-8 production and expression in A549 cells.  Conclusion: These data suggest that LPS-mediated proinflammatory responses in the lungs might be modulated via changing chromatin remodeling by HAT inhibition. PMID:24627774

Role of {alpha}{sub v}{beta}{sub 5} integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pande, Priyadarshini; Mosleh, Tariq A.; Aust, Ann E.

Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by {alpha}{sub v}{beta}{sub 5} integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin {alpha}{sub v}{beta}{sub 5} blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin {alpha}{sub v}{beta}{sub 5} receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changesmore » in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin {alpha}{sub v}{beta}{sub 5} receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.« less

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

PubMed

Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

2013-09-24

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial

Inhibition of the expression of aquaporin‑1 by RNA interference in pulmonary epithelial cells and its effects on water transport.

PubMed

Zhang, Qiuyue; Fu, Jianhua; Xue, Xindong

2016-01-01

In the present study, the effect of aquaporin‑1 (AQP1) on fluid transportation in pulmonary epithelial cells, and the role of AQP1 in alveolar fluid clearance were investigated to provide an experimental foundation to elucidate the pathogenesis of hyperoxic lung edema. An siRNA transfection technique was used to silence AQP1 in the A549 cell line. The transfected cells were randomized into a hyperoxia exposure and an air control group, with a negative control group set for each group. Cell volume was determined using flow cytometry, and Pf values were used to determine osmotic water permeability. Cell volume was found to be reduced in the AQP1‑silenced A549 cells, compared with the negative control group 72 h following air exposure. In addition, cell volume was reduced in the AQP1‑silenced A549 cells, compared with the negative control group 48 and 72 h following hyperoxia exposure. The osmotic water permeability of the AQP1‑silenced cells was reduced in the air control and hyperoxia exposure groups, compared with the negative control group 48 and 72 h following exposure. The volume and cell membrane osmotic water permeability of the A549 cells were reduced, compared with those in the control group following AQP1‑silencing, which indicated that the downregulation of AQP1 impedes extracellular to intracellular fluid transportation. Therefore, the disturbance in alveolar fluid clearance resulting from the downregulation of AQP1 following hyperoxia exposure may be one of the key mechanisms responsible for hyperoxic lung edema.

Inflammatory effects induced by selected limonene oxidation products: 4-OPA, IPOH, 4-AMCH in human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines.

PubMed

Lipsa, Dorelia; Leva, Paolo; Barrero-Moreno, Josefa; Coelhan, Mehmet

2016-11-16

Limonene, a monoterpene abundantly present in most of the consumer products (due to its pleasant citrus smell), easily undergoes ozonolysis leading to several limonene oxidation products (LOPs) such as 4-acetyl-1-methylcyclohexene (4-AMCH), 4-oxopentanal (4-OPA) and 3-isopropenyl-6-oxoheptanal (IPOH). Toxicological studies have indicated that human exposure to limonene and ozone can cause adverse airway effects. However, little attention has been paid to the potential health impact of specific LOPs, in particular of IPOH, 4-OPA and 4-AMCH. This study evaluates the cytotoxic effects of the selected LOPs on human bronchial epithelial (16HBE14o-) and alveolar epithelial (A549) cell lines by generating concentration-response curves using the neutral red uptake assay and analyzing the inflammatory response with a series of cytokines/chemokines. The cellular viability was mostly reduced by 4-OPA [IC 50 =1.6mM (A549) and 1.45mM (16HBE14o-)] when compared to IPOH [IC 50 =3.5mM (A549) and 3.4mM (16HBE14o-)] and 4-AMCH [IC 50 could not be calculated]. As a result from the inflammatory response, IPOH [50μM] induced an increase of both IL-6 and IL-8 secretion in A549 (1.5-fold change) and in 16HBE14o- (2.8- and 7-fold change respectively). 4-OPA [50μM] treatment of A549 increased IL-6 (1.4-times) and IL-8 (1.3-times) levels, while in 16HBE14o- had an opposite effect. A549 treated with 4-AMCH [50μM] elevate both IL-6 and IL-8 levels by 1.2-times, while in 16HBE14o- had an opposite effect. Based on our results, lung cellular injury characterized by inflammatory cytokine release was observed for both cell lines treated with the selected chemicals at concentrations that did not affect their cellular viability. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Epithelial to mesenchymal transition-related proteins ZEB1, β-catenin, and β-tubulin-III in idiopathic pulmonary fibrosis.

PubMed

Chilosi, Marco; Caliò, Anna; Rossi, Andrea; Gilioli, Eliana; Pedica, Federica; Montagna, Licia; Pedron, Serena; Confalonieri, Marco; Doglioni, Claudio; Ziesche, Rolf; Grubinger, Markus; Mikulits, Wolfgang; Poletti, Venerino

2017-01-01

Epithelial to mesenchymal transition has been suggested as a relevant contributor to pulmonary fibrosis, but how and where this complex process is triggered in idiopathic pulmonary fibrosis is not fully understood. Beta-tubulin-III (Tubβ3), ZEB1, and β-catenin are partially under the negative control of miR-200, a family of micro-RNAs playing a major role in epithelial to mesenchymal transition, that are reduced in experimental lung fibrosis and idiopathic pulmonary fibrosis. We wonder whether in situ expression of these proteins is increased in idiopathic pulmonary fibrosis, to better understand the significance of miR-200 feedback loop and epithelial to mesenchymal transition. We investigated the immunohistochemical and immunofluorescent expression and precise location of ZEB1, Tubβ3, and β-catenin in tissue samples from 34 idiopathic pulmonary fibrosis cases and 21 controls (5 normal lungs and 16 other interstitial lung diseases). In 100% idiopathic pulmonary fibrosis samples, the three proteins were concurrently expressed in fibroblastic foci, as well in damaged epithelial cells overlying these lesions and in pericytes within neo-angiogenesis areas. These results were also confirmed by immunofluorescence assay. In controls the abnormal expression of the three proteins was absent or limited. This is the first study that relates concurrent expression of Tubβ3, ZEB1, and β-catenin to abnormal epithelial and myofibroblast differentiation in idiopathic pulmonary fibrosis, providing indirect but robust evidence of miR-200 deregulation and epithelial to mesenchymal transition activation in idiopathic pulmonary fibrosis. The abnormal expression and localization of these proteins in bronchiolar fibro-proliferative lesions are unique for idiopathic pulmonary fibrosis, and might represent a disease-specific marker in challenging lung biopsies.

Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

PubMed Central

Sakai, Norihiko; Tager, Andrew M.

2013-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA.

PubMed

Sadik, Christian D; Bachmann, Malte; Pfeilschifter, Josef; Mühl, Heiko

2009-08-01

U1-snRNA is an integral part of the U1 ribonucleoprotein pivotal for pre-mRNA splicing. Toll-like receptor (TLR) signaling has recently been associated with immunoregulatory capacities of U1-snRNA. Using lung A549 epithelial/carcinoma cells, we report for the first time on interferon regulatory factor (IRF)-3 activation initiated by endosomally delivered U1-snRNA. This was associated with expression of the IRF3-inducible genes interferon-beta (IFN-beta), CXCL10/IP-10 and indoleamine 2,3-dioxygenase. Mutational analysis of the U1-snRNA-activated IFN-beta promoter confirmed the crucial role of the PRDIII element, previously proven pivotal for promoter activation by IRF3. Notably, expression of these parameters was suppressed by bafilomycin A(1), an inhibitor of endosomal acidification, implicating endosomal TLR activation. Since resiquimod, an agonist of TLR7/8, failed to stimulate A549 cells, data suggest TLR3 to be of prime relevance for cellular activation. To assess the overall regulatory potential of U1-snRNA-activated epithelial cells on cytokine production, co-cultivation with peripheral blood mononuclear cells (PBMC) was performed. Interestingly, A549 cells activated by U1-snRNA reinforced phytohemagglutinin-induced interleukin-10 release by PBMC but suppressed that of tumor necrosis factor-alpha, indicating an anti-inflammatory potential of U1-snRNA. Since U1-snRNA is enriched in apoptotic bodies and epithelial cells are capable of performing efferocytosis, the present data in particular connect to immunobiological aspects of apoptosis at host/environment interfaces.

Exosome cargo reflects TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

PubMed

Kim, Jiyeon; Kim, Tae Yeon; Lee, Myung Shin; Mun, Ji Young; Ihm, Chunhwa; Kim, Soon Ae

2016-09-16

It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-β1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-β1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of β-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Metabolic pathway catalyzed by Vanin-1 pantetheinase plays a suppressive role in influenza virus replication in human alveolar epithelial A549 cells.

PubMed

Yamashita, Nobuko; Yashiro, Masato; Ogawa, Hirohito; Namba, Hikaru; Nosaka, Nobuyuki; Fujii, Yousuke; Morishima, Tsuneo; Tsukahara, Hirokazu; Yamada, Masao

2017-08-05

Our previous analysis of gene expression profiles in the peripheral blood from patients with influenza A (H1N1) pdm09 pneumonia revealed elevated transcription levels of the vanin-1 (vascular non-inflammatory molecule 1, VNN1) gene, which encodes an epithelial ectoenzyme with pantetheinase activity involved in recycling coenzyme A. Here, to elucidate the role of VNN1 in influenza A virus (IAV) H1N1 infection, we investigated the change of VNN1 expression in the context of IAV infection and the effects of its related substances, i.e., its direct substrate pantetheine and its two metabolites pantothenic acid and cysteamine on the replication of IAV in the human alveolar epithelial carcinoma cell line A549. The messenger RNA expression of VNN1 in A549 cells was significantly increased (by 4.9-fold) after IAV infection under an elevated concentration of pantetheine. Moreover, VNN1 mRNA levels were elevated by > 100-fold in response to pro-inflammatory cytokines, especially TNF-α and IL-1β. Pantetheine significantly reduced the IAV replication and IAV Matrix 1 (M1) mRNA levels when it was administered prior to and during infection. In addition, cysteamine treatment during IAV infection significantly reduced the viral replication and IAV M1 mRNA levels, whereas pantothenic acid did not. These findings suggest that the metabolic pathway catalyzed by VNN1 pantetheinase plays a suppressive role in IAV infection in the respiratory tract, especially in severe conditions under hypercytokinemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Diacetyl Induces Amphiregulin Shedding in Pulmonary Epithelial Cells and in Experimental Bronchiolitis Obliterans

PubMed Central

Sun, Jesse; Fischer, Bernard M.; Voynow, Judith A.; Kummarapurugu, Apparao B.; Zhang, Helen L.; Nugent, Julia L.; Beasley, Robert F.; Martinu, Tereza; Gwinn, William M.; Morgan, Daniel L.; Palmer, Scott M.

2014-01-01

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air–liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α–converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO. PMID:24816162

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Caveolin-1 siRNA increases the pulmonary microvascular and alveolar epithelial permeability in rats.

PubMed

Gao, Chengjin; Li, Rongrong; Huan, Jingning; Li, Wei

2011-01-01

Increased pulmonary microvascular and epithelial permeability are important contributors to pulmonary edema in acute lung injury. In this study, we used small interfering RNA (siRNA) to knock down caveolin-1 expression in rat lungs and to confirm the important role of caveolin-1 in regulating pulmonary edema. After pulmonary injection of siRNA against caveolin-1 messenger RNA incorporated in liposomes with three concentrations of 0.4, 0.8, and 1.2 mg/kg, the gene silencing rate and the effects of caveolin-1 siRNA on aquaporin (AQP)-1, AQP-5, and epithelial sodium channel (ENaC) were detected. For pulmonary permeability analysis, Evans blue fluorimetry, ratios of albumin concentrations between blood and bronchoalveolar lavage, and wet/dry weight ratios were measured. The impacts of caveolin-1 suppression on interendothelial junctions were evaluated by the performance of electron microscopy and the analysis of vascular endothelial (VE)-cadherin Western blot. Alveolar wall thickness analysis and chest fluoroscopy were performed to determine the pulmonary edema degree. After 72 hours of injection, the gene silencing rate of caveolin-1 siRNA is about 87%. AQP-1, AQP-5, ENaC-α, ENaC-β, ENaC-γ, and VE-cadherin protein levels were decreased by 63%, 66%, 80%, 90%, 89%, and 50%, respectively. Caveolin-1 siRNA also resulted in increasing microvascular and epithelial permeability and pulmonary edema. These data suggest that caveolin-1 plays an important part in regulating the pulmonary permeability by modifying AQP-1, AQP-5, ENaC, and VE-cadherin.

Cellular uptake and toxic effects of fine and ultrafine metal-sulfate particles in human A549 lung epithelial cells.

PubMed

Könczöl, Mathias; Goldenberg, Ella; Ebeling, Sandra; Schäfer, Bianca; Garcia-Käufer, Manuel; Gminski, Richard; Grobéty, Bernard; Rothen-Rutishauser, Barbara; Merfort, Irmgard; Gieré, Reto; Mersch-Sundermann, Volker

2012-12-17

Ambient airborne particulate matter is known to cause various adverse health effects in humans. In a recent study on the environmental impacts of coal and tire combustion in a thermal power station, fine crystals of PbSO(4) (anglesite), ZnSO(4)·H(2)O (gunningite), and CaSO(4) (anhydrite) were identified in the stack emissions. Here, we have studied the toxic potential of these sulfate phases as particulates and their uptake in human alveolar epithelial cells (A549). Both PbSO(4) and CaSO(4) yielded no loss of cell viability, as determined by the WST-1 and NR assays. In contrast, a concentration-dependent increase in cytotoxicity was observed for Zn sulfate. For all analyzed sulfates, an increase in the production of reactive oxygen species (ROS), assessed by the DCFH-DA assay and EPR, was observed, although to a varying extent. Again, Zn sulfate was the most active compound. Genotoxicity assays revealed concentration-dependent DNA damage and induction of micronuclei for Zn sulfate and, to a lower extent, for CaSO(4), whereas only slight effects could be found for PbSO(4). Moreover, changes of the cell cycle were observed for Zn sulfate and PbSO(4). It could be shown further that Zn sulfate increased the nuclear factor kappa-B (NF-κB) DNA binding activity and activated JNK. During our TEM investigations, no effect on the appearance of the A549 cells exposed to CaSO(4) compared to the nonexposed cells was observed, and in our experiments, only one CaSO(4) particle was detected in the cytoplasm. In the case of exposure to Zn sulfate, no particles were found in the cytoplasm of A549 cells, but we observed a concentration-dependent increase in the number and size of dark vesicles (presumably zincosomes). After exposure to PbSO(4), the A549 cells contained isolated particles as well as agglomerates both in vesicles and in the cytoplasm. Since these metal-sulfate particles are emitted into the atmosphere via the flue gas of coal-fired power stations, they may be

Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

PubMed Central

Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

2014-01-01

As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

Leptin promotes pulmonary fibrosis development by inhibiting autophagy via PI3K/Akt/mTOR pathway.

PubMed

Gui, Xianhua; Chen, Hongwei; Cai, Hourong; Sun, Lingyun; Gu, Luo

2018-04-06

Leptin, a protein-related product of the obesity gene, plays an important role in the pathogenesis of fibrotic diseases including pulmonary fibrosis. As a highly conservative process, autophagy regulates various biological functions. Otherwise, insufficient autophagy has been described in alveolar epithelial cells (AEC) to cope with the progression of pulmonary fibrosis. Hence, this study is to investigate the effects of leptin on fibrosis in TGF-β1 induced epithelial mesenchymal transition (EMT) and the potential roles of autophagy in this processes. Our results showed that the elevated leptin level in serum correlated with the severity of lung fibrosis and leptin significantly promoted the EMT in A549 cells as evidenced by promoting collagen I and α-SMA production. Additionally, treatment with leptin decreased autophagosome formation, inhibited the lipidation of LC3I to LC3II, and up-regulated the expression of p62 via activating PI3K/Akt/mTOR pathway, which is indicative of inhibition of autophagy by leptin. Finally, rapmycin pretreatment reversed the pro-fibrogenic effects of leptin. Taken together, our study suggested that leptin accelerated the EMT of A549 cells through inhibiting autophagy via PI3K/Akt/mTOR pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Up-Regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

PubMed Central

Pardo, Annie; Gibson, Kevin; Cisneros, José; Richards, Thomas J; Yang, Yinke; Becerril, Carina; Yousem, Samueal; Herrera, Iliana; Ruiz, Victor; Selman, Moisés; Kaminski, Naftali

2005-01-01

Background Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung. Methods and Findings Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to αvβ3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-αvβ3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7. Conclusions Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease. PMID:16128620

Investigations of Pulmonary Epithelial Cell Damage due to Air-Liquid Interfacial Stresses in a Microgravity Environment

NASA Technical Reports Server (NTRS)

Gaver, Donald P., III; Bilek, A. M.; Kay, S.; Dee, K. C.

2004-01-01

Pulmonary airway closure is a potentially dangerous event that can occur in microgravity environments and may result in limited gas exchange for flight crew during long-term space flight. Repetitive airway collapse and reopening subjects the pulmonary epithelium to large, dynamic, and potentially injurious mechanical stresses. During ventilation at low lung volumes and pressures, airway instability leads to repetitive collapse and reopening. During reopening, air must progress through a collapsed airway, generating stresses on the airway walls, potentially damaging airway tissues. The normal lung can tolerate repetitive collapse and reopening. However, combined with insufficient or dysfunctional pulmonary surfactant, repetitive airway collapse and reopening produces severe lung injury. Particularly at risk is the pulmonary epithelium. As an important regulator of lung function and physiology, the degree of pulmonary epithelial damage influences the course and outcome of lung injury. In this paper we present experimental and computational studies to explore the hypothesis that the mechanical stresses associated with airway reopening inflict injury to the pulmonary epithelium.

Mechanisms of crystalline silica-induced pulmonary toxicity revealed by global gene expression profiling

PubMed Central

Sellamuthu, Rajendran; Umbright, Christina; Li, Shengqiao; Kashon, Michael; Joseph, Pius

2015-01-01

A proper understanding of the mechanisms underlying crystalline silica-induced pulmonary toxicity has implications in the management and potential prevention of the adverse health effects associated with silica exposure including silicosis, cancer and several auto-immune diseases. Human lung type II epithelial cells and rat lungs exposed to crystalline silica were employed as experimental models to determine global gene expression changes in order to understand the molecular mechanisms underlying silica-induced pulmonary toxicity. The differential gene expression profile induced by silica correlated with its toxicity in the A549 cells. The biological processes perturbed by silica exposure in the A549 cells and rat lungs, as identified by the bioinformatics analysis of the differentially expressed genes, demonstrated significant similarity. Functional categorization of the differentially expressed genes identified cancer, cellular movement, cellular growth and proliferation, cell death, inflammatory response, cell cycle, cellular development, and genetic disorder as top ranking biological functions perturbed by silica exposure in A549 cells and rat lungs. Results of our study, in addition to confirming several previously identified molecular targets and mechanisms involved in silica toxicity, identified novel molecular targets and mechanisms potentially involved in silica-induced pulmonary toxicity. Further investigations, including those focused on the novel molecular targets and mechanisms identified in the current study may result in better management and, possibly, reduction and/or prevention of the potential adverse health effects associated with crystalline silica exposure. PMID:22087542

Airway epithelial cell response to human metapneumovirus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, X.; Liu, T.; Spetch, L.

2007-11-10

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and typemore » I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.« less

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

PubMed

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy.

Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells

PubMed Central

Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

2016-01-01

We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

Airway epithelial stem cells and the pathophysiology of chronic obstructive pulmonary disease.

PubMed

Randell, Scott H

2006-11-01

Characteristic pathologic changes in chronic obstructive pulmonary disease (COPD) include an increased fractional volume of bronchiolar epithelial cells, fibrous thickening of the airway wall, and luminal inflammatory mucus exudates, which are positively correlated with airflow limitation and disease severity. The mechanisms driving general epithelial expansion, mucous secretory cell hyperplasia, and mucus accumulation must relate to the effects of initial toxic exposures on patterns of epithelial stem and progenitor cell proliferation and differentiation, eventually resulting in a self-perpetuating, and difficult to reverse, cycle of injury and repair. In this review, current concepts in stem cell biology and progenitor-progeny relationships related to COPD are discussed, focusing on the factors, pathways, and mechanisms leading to mucous secretory cell hyperplasia and mucus accumulation in the airways. A better understanding of alterations in airway epithelial phenotype in COPD will provide a logical basis for novel therapeutic approaches.

Tannic acid attenuates TGF-β1-induced epithelial-to-mesenchymal transition by effectively intervening TGF-β signaling in lung epithelial cells.

PubMed

Pattarayan, Dhamotharan; Sivanantham, Ayyanar; Krishnaswami, Venkateshwaran; Loganathan, Lakshmanan; Palanichamy, Rajaguru; Natesan, Subramanian; Muthusamy, Karthikeyan; Rajasekaran, Subbiah

2018-03-01

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and an irreversible lung disorder characterized by the accumulation of fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) is thought to be one of the possible sources for a substantial increase in the number of fibroblasts/myofibroblasts in IPF lungs. Tannic acid (TA), a natural dietary polyphenolic compound has been shown to possess diverse pharmacological effects. However, whether TA can inhibit TGF-β1-mediated EMT in lung epithelial cells remains enigmatic. Both the human adenocarcinomic alveolar epithelial (A549) and normal bronchial epithelial (BEAS-2B) cells were treated with TGF-β1 with or without TA. Results showed that TA addition, markedly inhibited TGF-β1-induced EMT as assessed by reduced expression of N-cadherin, type-1-collagen, fibronectin, and vimentin. Furthermore, TA inhibited TGF-β1-induced cell proliferation through inducing cell cycle arrest at G0/G1 phase. TGF-β1-induced increase in the phosphorylation of Smad (Smad2 and 3), Akt as well as that of mitogen activated protein kinase (ERK1/2, JNK1/2, and p38) mediators was effectively inhibited by TA. On the other hand, TA reduced the TGF-β1-induced increase in TGF-β receptors expression. Using molecular docking approach, FTIR, HPLC and Western blot analyses, we further identified the direct binding of TA to TGF-β1. Finally, we conclude that TA might directly interact with TGF-β1, thereby repressing TGF-β signaling and subsequent EMT process in lung epithelial cells. Further animal studies are needed to clarify its potential therapeutic benefit in pulmonary fibrosis. © 2017 Wiley Periodicals, Inc.

Silencing the Snail-dependent RNA splice regulator ESRP1 drives malignant transformation of human pulmonary epithelial cells. | Office of Cancer Genomics

Cancer.gov

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated including in non-small cell lung cancer (NSCLC). Here we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo.

Withaferin A inhibits tumor necrosis factor-alpha-induced expression of cell adhesion molecules by inactivation of Akt and NF-kappaB in human pulmonary epithelial cells.

PubMed

Oh, Jung Hwa; Kwon, Taeg Kyu

2009-05-01

We here investigated the functional effect of withaferin A on airway inflammation and its action mechanism. Withaferin A inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human lung epithelial A549 cells stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to lung epithelial A549 cells. In addition, withaferin A inhibited TNF-alpha-induced expression of adhesion molecules (ICAM-1 and VCAM-1) protein and mRNA in a dose-dependent manner. Withaferin A prevented DNA binding activity of nuclear factor-kappaB (NF-kappaB) and nuclear translocation of NF-kappaB. It also inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK), which are upstream in the regulation of adhesion molecules by TNF-alpha. Furthermore, withaferin A inhibited U937 monocyte adhesion to A549 cells stimulated by TNF-alpha, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-alpha. Taken together, these results suggest that withaferin A inhibits cell adhesion through inhibition of ICAM-1 and VCAM-1 expression, at least in part, by blocking Akt and down-regulating NF-kappaB activity.

Decreased levels of irisin, a skeletal muscle cell-derived myokine, are related to emphysema associated with chronic obstructive pulmonary disease.

PubMed

Sugiyama, Yukari; Asai, Kazuhisa; Yamada, Kazuhiro; Kureya, Yuko; Ijiri, Naoki; Watanabe, Tetsuya; Kanazawa, Hiroshi; Hirata, Kazuto

2017-01-01

Cigarette smoking-induced oxidant-antioxidant imbalance is a factor that contributes to the pathogenesis of COPD through epithelial cell apoptosis. Irisin is a skeletal muscle cell-derived myokine associated with physical activity. Irisin is also known to decrease oxidant-induced apoptosis in patients with diabetes mellitus. However, the correlation between irisin and emphysema in COPD and its role in epithelial cell apoptosis remains unknown. Forty patients with COPD were enrolled in this study. Pulmonary function tests and measurements of the percentage of low-attenuation area on high-resolution computed tomography images were performed, and the results were evaluated for correlation with serum irisin levels. The effect of irisin on cigarette-smoke extract-induced A549 cell apoptosis and the expression of Nrf2, a transcription factor for antioxidants, was also examined in vitro. Serum irisin levels were significantly correlated with lung diffusing capacity for carbon monoxide divided by alveolar volume ( r =0.56, P <0.01) and percentage of low-attenuation area ( r =-0.79, P <0.01). Moreover, irisin significantly enhanced Nrf2 expression ( P <0.05) and reduced cigarette-smoke extract-induced A549 cell apoptosis ( P <0.05). Decreased serum irisin levels are related to emphysema in patients with COPD and involved in epithelial apoptosis, resulting in emphysema. Irisin could be a novel treatment for emphysema in patients with COPD.

STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation

PubMed Central

Pedroza, Mesias; Le, Thuy T.; Lewis, Katherine; Karmouty-Quintana, Harry; To, Sarah; George, Anuh T.; Blackburn, Michael R.; Tweardy, David J.; Agarwal, Sandeep K.

2016-01-01

Lung fibrosis is the hallmark of the interstitial lung diseases. Alveolar epithelial cell (AEC) injury is a key step that contributes to a profibrotic microenvironment. Fibroblasts and myofibroblasts subsequently accumulate and deposit excessive extracellular matrix. In addition to TGF-β, the IL-6 family of cytokines, which signal through STAT-3, may also contribute to lung fibrosis. In the current manuscript, the extent to which STAT-3 inhibition decreases lung fibrosis is investigated. Phosphorylated STAT-3 was elevated in lung biopsies from patients with idiopathic pulmonary fibrosis and bleomycin (BLM)-induced fibrotic murine lungs. C-188-9, a small molecule STAT-3 inhibitor, decreased pulmonary fibrosis in the intraperitoneal BLM model as assessed by arterial oxygen saturation (control, 84.4 ± 1.3%; C-188-9, 94.4 ± 0.8%), histology (Ashcroft score: untreated, 5.4 ± 0.25; C-188-9, 3.3 ± 0.14), and attenuated fibrotic markers such as diminished α–smooth muscle actin, reduced collagen deposition. In addition, C-188-9 decreased the expression of epithelial injury markers, including hypoxia-inducible factor-1α (HIF-1α) and plasminogen activator inhibitor-1 (PAI-1). In vitro studies show that inhibition of STAT-3 decreased IL-6– and TGF-β–induced expression of multiple genes, including HIF-1α and PAI-1, in AECs. Furthermore, C-188-9 decreased fibroblast-to-myofibroblast differentiation. Finally, TGF-β stimulation of lung fibroblasts resulted in SMAD2/SMAD3-dependent phosphorylation of STAT-3. These findings demonstrate that STAT-3 contributes to the development of lung fibrosis and suggest that STAT-3 may be a therapeutic target in pulmonary fibrosis.—Pedroza, M., Le, T. T., Lewis, K., Karmouty-Quintana, H., To, S., George, A. T., Blackburn, M. R., Tweardy, D. J., Agarwal, S. K. STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation. PMID:26324850

Airway Epithelial Barrier Dysfunction in Chronic Obstructive Pulmonary Disease: Role of Cigarette Smoke Exposure.

PubMed

Aghapour, Mahyar; Raee, Pourya; Moghaddam, Seyed Javad; Hiemstra, Pieter S; Heijink, Irene H

2018-02-01

The epithelial lining of the airway forms the first barrier against environmental insults, such as inhaled cigarette smoke, which is the primary risk factor for the development of chronic obstructive pulmonary disease (COPD). The barrier is formed by airway epithelial junctions, which are interconnected structures that restrict permeability to inhaled pathogens and environmental stressors. Destruction of the epithelial barrier not only exposes subepithelial layers to hazardous agents in the inspired air, but also alters the normal function of epithelial cells, which may eventually contribute to the development of COPD. Of note, disruption of epithelial junctions may lead to modulation of signaling pathways involved in differentiation, repair, and proinflammatory responses. Epithelial barrier dysfunction may be particularly relevant in COPD, where repeated injury by cigarette smoke exposure, pathogens, inflammatory mediators, and impaired epithelial regeneration may compromise the barrier function. In the current review, we discuss recent advances in understanding the mechanisms of barrier dysfunction in COPD, as well as the molecular mechanisms that underlie the impaired repair response of the injured epithelium in COPD and its inability to redifferentiate into a functionally intact epithelium.

The Pseudomonas aeruginosa exopolysaccharide Psl facilitates surface adherence and NF-kappaB activation in A549 cells.

PubMed

Byrd, Matthew S; Pang, Bing; Mishra, Meenu; Swords, W Edward; Wozniak, Daniel J

2010-06-29

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213-8221, 2006). Using an NF-kappaB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-kappaB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-kappaB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-kappaB activation, likely as a result of increasing contact between bacterial cells and epithelial cells.

The Pseudomonas aeruginosa Exopolysaccharide Psl Facilitates Surface Adherence and NF-κB Activation in A549 Cells

PubMed Central

Byrd, Matthew S.; Pang, Bing; Mishra, Meenu; Swords, W. Edward; Wozniak, Daniel J.

2010-01-01

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213–8221, 2006). Using an NF-κB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-κB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-κB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-κB activation, likely as a result of increasing contact between bacterial cells and epithelial cells. PMID:20802825

Hybrid Lipid/Polymer Nanoparticles for Pulmonary Delivery of siRNA: Development and Fate Upon In Vitro Deposition on the Human Epithelial Airway Barrier.

PubMed

d'Angelo, Ivana; Costabile, Gabriella; Durantie, Estelle; Brocca, Paola; Rondelli, Valeria; Russo, Annapina; Russo, Giulia; Miro, Agnese; Quaglia, Fabiana; Petri-Fink, Alke; Rothen-Rutishauser, Barbara; Ungaro, Francesca

2017-10-16

Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system. A panel of DPPC/PLGA hNPs was prepared by emulsion/solvent diffusion and fully characterized. A combination of model siRNAs against the sodium transepithelial channel (ENaC) was entrapped in optimized hNPs comprising or not poly(ethylenimine) (PEI) as third component. siRNA-loaded hNPs were characterized for encapsulation efficiency, release kinetics, aerodynamic properties, and stability in artificial mucus (AM). The fate and cytotoxicity of hNPs upon aerosolization on a triple cell co-culture model (TCCC) mimicking human epithelial airway barrier were assessed. Finally, the effect of siRNA-loaded hNPs on ENaC protein expression at 72 hours was evaluated in A549 cells. Optimized muco-inert hNPs encapsulating model siRNA with high efficiency were produced. The developed hNPs displayed a hydrodynamic diameter of ∼150 nm, a low polydispersity index, a negative ζ potential close to -25 mV, and a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI. siRNA formulations showed optimal in vitro aerosol performance after delivery with a vibrating mesh nebulizer. Furthermore, small-angle X-ray scattering analyses highlighted an excellent stability upon incubation with AM, confirming the potential of hNPs for direct aerosolization on mucus-lined airways. Studies in TCCC confirmed that fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute proinflammatory effect. Finally, a prolonged inhibition of ENaC protein expression was observed in A549 cells upon treatment with siRNA-loaded hNPs. Results demonstrate the great potential

COMPARATIVE TOXICITY OF DIFFERENT EMISSION PARTICLES IN MURINE PULMONARY EPITHELIAL CELLS AND MACROPHAGES

EPA Science Inventory

Comparative Toxicity of Different Emission Particles in Murine Pulmonary Epithelial Cells and Macrophages. T Stevens1, M Daniels2, P Singh2, M I Gilmour2. 1 UNC, Chapel Hill 27599 2Experimental Toxicology Division, NHEERL, RTP, NC 27711

Epidemiological studies have shown ...

Airborne particulate matter in vitro exposure induces cytoskeleton remodeling through activation of the ROCK-MYPT1-MLC pathway in A549 epithelial lung cells.

PubMed

Chirino, Yolanda I; García-Cuellar, Claudia María; García-García, Carlos; Soto-Reyes, Ernesto; Osornio-Vargas, Álvaro Román; Herrera, Luis A; López-Saavedra, Alejandro; Miranda, Javier; Quintana-Belmares, Raúl; Pérez, Irma Rosas; Sánchez-Pérez, Yesennia

2017-04-15

Airborne particulate matter with an aerodynamic diameter ≤10μm (PM 10 ) is considered a risk factor for the development of lung cancer. Little is known about the cellular mechanisms by which PM 10 is associated with cancer, but there is evidence that its exposure can lead to an acquired invasive phenotype, apoptosis evasion, inflammasome activation, and cytoskeleton remodeling in lung epithelial cells. Cytoskeleton remodeling occurs through actin stress fiber formation, which is partially regulated through ROCK kinase activation, we aimed to investigate if this protein was activated in response to PM 10 exposure in A549 lung epithelial cells. Results showed that 10μg/cm 2 of PM 10 had no influence on cell viability but increased actin stress fibers, cytoplasmic ROCK expression, and phosphorylation of myosin phosphatase-targeting 1 (MYPT1) and myosin light chain (MLC) proteins, which are targeted by ROCK. The inhibition of ROCK prevented actin stress fiber formation and the phosphorylation of MYPT1 and MLC, suggesting that PM 10 activated the ROCK-MYPT1-MLC pathway in lung epithelial cells. The activation of ROCK1 has been involved in the acquisition of malignant phenotypes, and its induction by PM 10 exposure could contribute to the understanding of PM 10 as a risk factor for cancer development through the mechanisms associated with invasive phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar-Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Sakamoto, Atsushi; Matsumaru, Takehisa; Yamamura, Norio; Suzuki, Shinobu; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2015-09-01

Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells.

PubMed

Aueviriyavit, Sasitorn; Phummiratch, Duangkamol; Kulthong, Kornphimol; Maniratanachote, Rawiwan

2012-10-01

Titanium dioxide nanoparticles (TiO(2)NPs) are increasingly being used in various industrial applications including the production of paper, plastics, cosmetics and paints. With the increasing number of nano-related products, the concern of governments and the general public about the health and environmental risks, especially with regard to occupational and other environmental exposure, are gradually increasing. However, there is insufficient knowledge about the actual affects upon human health and the environment, as well as a lack of suitable biomarkers for assessing TiO(2)NP-induced cytotoxicity. Since the respiratory tract is likely to be the main exposure route of industrial workers to TiO(2)NPs, we investigated the cytotoxicity of the anatase and rutile crystalline forms of TiO(2)NPs in A549 cells, a human alveolar type II-like epithelial cell line. In addition, we evaluated the transcript and protein expression levels of two heat shock protein (HSP) members, Grp78 and Hsp70, to ascertain their suitability as biomarkers of TiO(2)NP-induced toxicity in the respiratory system. Ultrastructural observations confirmed the presence of TiO(2)NPs inside cells. In vitro exposure of A549 cells to the anatase or rutile forms of TiO(2)NPs led to cell death and induced intracellular ROS generation in a dose-dependent manner, as determined by the MTS and dichlorofluorescein (DCF) assays, respectively. In contrast, the transcript and protein expression levels of Hsp70 and Grp78 did not change within the same TiO(2)NPs dose range (25-500 μg/ml). Thus, whilst TiO(2)NPs can cause cytotoxicity in A549 cells, and thus potentially in respiratory cells, Hsp70 and Grp78 are not suitable biomarkers for evaluating the acute toxicological effects of TiO(2)NPs in the respiratory system.

Normal reference values for regional pulmonary peripheral airspace epithelial permeability. Influence of pneumonectomy and the smoking habit.

PubMed

Todisco, T; Dottorini, M; Rossi, F; Baldoncini, A; Palumbo, R

1989-01-01

Peripheral airspace epithelial permeability (PAEP) to diethylentriaminopentacetate (DTPA), an index of pulmonary integrity, was measured in 3 groups of subjects for different purposes: (1) to establish vertical regional reference values; (2) to determine the physiological role of acute doubling of total pulmonary blood flow; (3) to quantify the pulmonary epithelial damage in smokers and the possibility of lung protection by an agent stimulating surfactant production. This study broadens previous knowledge of PAEP. First of all, regional reference values are given for young normal nonsmoking subjects and the existence of a vertical gradient of PAEP is confirmed. Furthermore, this study shows that this gradient is independent of the vertical blood flow gradient, since an acute increase of total blood flow in pneumonectomized patients does not modify the regional distribution of PAEP. Finally, it is confirmed that the cigarette smoker's lung is more permeable than the controls and that probably a drug-stimulating surfactant production gives some protection against damage due to chronic smoking.

Endogenous acetylcholine increases alveolar epithelial fluid transport via activation of alveolar epithelial Na,K-ATPase in mice.

PubMed

Li, Xia; Yan, Xi Xin; Li, Hong Lin; Li, Rong Qin

2015-10-01

The contribution of endogenous acetylcholine to alveolar fluid clearance (AFC) and related molecular mechanisms were explored. AFC was measured in Balb/c mice after vagotomy and vagus nerve stimulation. Effects of acetylcholine chloride on AFC in Kunming mice and Na,K-ATPase function in A549 alveolar epithelial cells also were determined. AFC significantly decreased in mice with left cervical vagus nerve transection compared with controls (48.69 ± 2.57 vs. 66.88 ± 2.64, P ≤ 0.01), which was reversed by stimulation of the peripheral (60.81 ± 1.96, P ≤ 0.01). Compared with control, acetylcholine chloride dose-dependently increased AFC and elevated Na,K-ATPase activity, and these increases were blocked or reversed by atropine. These effects were accompanied by recruitment of Na,K-ATPase α1 to the cell membrane. Thus, vagus nerves participate in alveolar epithelial fluid transport by releasing endogenous acetylcholine in the infusion-induced pulmonary edema mouse model. Effects of endogenous acetylcholine on AFC are likely mediated by Na,K-ATPase function through activation of muscarinic acetylcholine receptors on alveolar epithelia. Copyright © 2015 Elsevier B.V. All rights reserved.

Injurious effects of wool and grain dusts on alveolar epithelial cells and macrophages in vitro.

PubMed Central

Brown, D M; Donaldson, K

1991-01-01

Epidemiological studies of workers in wool textile mills have shown a direct relation between the concentration of wool dust in the air and respiratory symptoms. Injurious effects of wool dust on the bronchial epithelium could be important in causing inflammation and irritation. A pulmonary epithelial cell line in vitro was therefore used to study the toxic effects of wool dust. Cells of the A549 epithelial cell line were labelled with 51Cr and treated with whole wool dusts and extracts of wool, after which injury was assessed. Also, the effects of grain dust, which also causes a form of airway obstruction, were studied. The epithelial injury was assessed by measuring 51Cr release from cells as an indication of lysis, and by monitoring cells which had detached from the substratum. No significant injury to A549 cells was caused by culture with any of the dusts collected from the air but surface "ledge" dust caused significant lysis at some doses. Quartz, used as a toxic control dust, caused significant lysis at the highest concentration of 100 micrograms/well. To determine whether any injurious material was soluble the dusts were incubated in saline and extracts collected. No extracts caused significant injury to epithelial cells. A similar lack of toxicity was found when 51Cr labelled control alveolar macrophages were targets for injury. Significant release of radiolabel was evident when macrophages were exposed to quartz at concentrations of 10 and 20 micrograms/well, there being no significant injury with either wool or grain dusts. These data suggest that neither wool nor grain dust produce direct injury to epithelial cells, and further studies are necessary to explain inflammation leading to respiratory symptoms in wool and grain workers. PMID:2015211

Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers.

PubMed

Selman, Moisés; Pardo, Annie

2006-06-01

Idiopathic pulmonary fibrosis (IPF), a progressive and relentless lung scarring of unknown etiology, has been recognized as the most lethal interstitial lung disease. Despite the growing interest in IPF, the precise molecular mechanisms underlying the development of fibrosis and leading to the irreversible destruction of the lung are still unknown. Recently, it has been proposed that IPF, instead of being a chronic inflammatory disorder, results from multiple cycles of epithelial cell injury and activation. In turn, active alveolar epithelial cells provoke the migration, proliferation, and activation of mesenchymal cells with the formation of fibroblastic/myofibroblastic foci and the exaggerated accumulation of extracellular matrix, mirroring abnormal wound repair. In this article, some characteristics of the alveolar epithelium are briefly outlined, and the fibrogenic mechanisms specifically operated by active abnormal epithelial cells are examined.

Mechanisms for cellular NO oxidation and nitrite formation in lung epithelial cells.

PubMed

Zhao, Xue-Jun; Wang, Ling; Shiva, Sruti; Tejero, Jesus; Myerburg, Mike M; Wang, Jun; Frizzell, Sam; Gladwin, Mark T

2013-08-01

Airway lining fluid contains relatively high concentrations of nitrite, and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 h under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low-oxygen conditions. The addition of oxyhemoglobin to the A549 cell medium decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and nitrate, suggesting an enzymatic activity is required. This NO oxidation activity was highest in membrane-bound proteins with molecular size <100kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation. We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into the medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via p-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in either nitrite or cellular S

Mechanisms for Cellular NO Oxidation and Nitrite Formation in Lung Epithelial Cells

PubMed Central

Zhao, Xue-Jun; Wang, Ling; Shiva, Sruti; Tejero, Jesus; Wang, Jun; Frizzell, Sam; Gladwin, Mark T.

2013-01-01

Airway lining fluid contains relatively high concentrations of nitrite and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 hrs under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low oxygen conditions. The addition of oxy-hemoglobin (oxy-Hb) to the A549 cell media decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and bitrate, suggesting an enzymatic activity is required. This NO oxidation activity was found to be highest in membrane bound proteins with molecular sizes < 100 kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation (NO+). We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via ρ-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in

Pulmonary edema in meningococcal septicemia associated with reduced epithelial chloride transport.

PubMed

Eisenhut, Michael; Wallace, Helen; Barton, Paul; Gaillard, Erol; Newland, Paul; Diver, Michael; Southern, Kevin W

2006-03-01

To test the hypothesis that meningococcal septicemia-related pulmonary edema is associated with a systemic abnormality of epithelial sodium and chloride transport and to investigate an association with hormones regulating Na transport. Prospective observational study. The 24-bed pediatric intensive care unit and pediatric wards of Royal Liverpool Children's Hospital. Consecutive children admitted to the pediatric intensive care unit and pediatric wards with a diagnosis of meningococcal septicemia and children (controls) with noninfectious critical illness receiving ventilatory support in the pediatric intensive care unit. We measured sweat and saliva electrolytes, renal electrolyte excretion, nasal potential difference, and aldosterone, thyroxine, and cortisol levels. Pulmonary edema was diagnosed by chest radiography and its severity quantified by calculation of ventilation index at admission and duration of mechanical ventilation. We recruited 17 patients with severe meningococcal septicemia (nine patients with pulmonary edema), 14 patients with mild meningococcal septicemia, and 20 controls. Sweat and saliva Na and Cl concentrations and renal Na excretion were significantly (p < .05) higher in patients with pulmonary edema compared with controls. Nasal potential difference and amiloride response in patients with pulmonary edema were not significantly different to controls, but response to a low Cl solution was reduced in the nasal airway of patients with pulmonary edema (p < .05). Sweat and saliva chloride concentrations correlated significantly and better with ventilation index and duration of ventilation than sodium concentrations. Aldosterone, thyroxine, and cortisol levels were not significantly different between groups. We have confirmed that meningococcal septicemia-related pulmonary edema is associated with reduced systemic sodium and chloride transport. Features of reduced Cl transport were most closely associated with markers of respiratory compromise

Mycobacterium tuberculosis strains exhibit differential and strain-specific molecular signatures in pulmonary epithelial cells.

PubMed

Mvubu, Nontobeko Eunice; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

2016-12-01

Although pulmonary epithelial cells are integral to innate and adaptive immune responses during Mycobacterium tuberculosis infection, global transcriptomic changes in these cells remain largely unknown. Changes in gene expression induced in pulmonary epithelial cells infected with M. tuberculosis F15/LAM4/KZN, F11, F28, Beijing and Unique genotypes were investigated by RNA sequencing (RNA-Seq). The Illumina HiSeq 2000 platform generated 50 bp reads that were mapped to the human genome (Hg19) using Tophat (2.0.10). Differential gene expression induced by the different strains in infected relative to the uninfected cells was quantified and compared using Cufflinks (2.1.0) and MeV (4.0.9), respectively. Gene expression varied among the strains with the total number of genes as follows: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A subset of 292 genes was commonly induced by all strains, where 52 genes were down-regulated while 240 genes were up-regulated. Differentially expressed genes were compared among the strains and the number of induced strain-specific gene signatures were as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (186) and H37Rv (125). Strain-specific molecular gene signatures associated with functional pathways were observed only for the Unique and H37Rv strains while certain biological functions may be associated with other strain signatures. This study demonstrated that strains of M. tuberculosis induce differential gene expression and strain-specific molecular signatures in pulmonary epithelial cells. Specific signatures induced by clinical strains of M. tuberculosis can be further explored for novel host-associated biomarkers and adjunctive immunotherapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pulmonary vasculature directed adenovirus increases epithelial lining fluid alpha-1 antitrypsin levels.

PubMed

Buggio, Maurizio; Towe, Christopher; Annan, Anand; Kaliberov, Sergey; Lu, Zhi Hong; Stephens, Calvin; Arbeit, Jeffrey M; Curiel, David T

2016-01-01

Gene therapy for inherited serum deficiency disorders has previously been limited by the balance between obtaining adequate expression and causing hepatic toxicity. Our group has previously described modifications of a replication deficient human adenovirus serotype 5 that increase pulmonary vasculature transgene expression. In the present study, we use a modified pulmonary targeted adenovirus to express human alpha-1 antitrypsin (A1AT) in C57BL/6 J mice. Using the targeted adenovirus, we were able to achieve similar increases in serum A1AT levels with less liver viral uptake. We also increased pulmonary epithelial lining fluid A1AT levels by more than an order of magnitude compared to that of untargeted adenovirus expressing A1AT in a mouse model. These gains are achieved along with evidence of decreased systemic inflammation and no evidence for increased inflammation within the vector-targeted end organ. In addition to comprising a step towards clinically viable gene therapy for A1AT, maximization of protein production at the site of action represents a significant technical advancement in the field of systemically delivered pulmonary targeted gene therapy. It also provides an alternative to the previous limitations of hepatic viral transduction and associated toxicities. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of whole cigarette smoke induced oxidative stress in A549 and BEAS-2B cells.

PubMed

Zhang, Shimin; Li, Xiang; Xie, Fuwei; Liu, Kejian; Liu, Huimin; Xie, Jianping

2017-09-01

Cigarette smoke is a complex and oxidative aerosol. Previous researches on the hazards of cigarette smoke mainly focused on the adverse bioeffects induced by its condensates or gas vapor phase, which ignored the dynamic processes of smoking and the cigarette smoke aging. To overcome these disadvantages, we performed air-liquid interface exposure of whole smoke, which used native and unmodified smoke and ensured the exposure similar to physiological inhalation. Our results indicated that whole cigarette smoke induced lung epithelial cells (A549) and bronchial epithelial cells (BEAS-2B) damages in cytotoxicity assays (methyl thiazoly tetrazolium and neutral red uptake assays). In addition, A549 and BEAS-2B cells showed oxidative damages in whole smoke exposure, with concentration change of several biomarkers (reduced and oxidized glutathione, malondialdehyde, 4-hydroxyhydroxy-2-nonenal, extracellular superoxide dismutase, and 8-hydroxyl deoxyguanosine). These results indicate that whole smoke-induced oxidative stress occurs in two different kinds of cells at air-liquid interface. Copyright © 2017 Elsevier B.V. All rights reserved.

Computational modeling predicts simultaneous targeting of fibroblasts and epithelial cells is necessary for treatment of pulmonary fibrosis

DOE PAGES

Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; ...

2016-06-23

Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited withmore » enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGFβ1 receptor ligand signaling in fibroblasts. PGE 2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE 2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. In conclusion, a two-hit therapeutic

Computational modeling predicts simultaneous targeting of fibroblasts and epithelial cells is necessary for treatment of pulmonary fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.

Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited withmore » enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGFβ1 receptor ligand signaling in fibroblasts. PGE 2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE 2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. In conclusion, a two-hit therapeutic

Global secretome characterization of A549 human alveolar epithelial carcinoma cells during Mycoplasma pneumoniae infection

PubMed Central

2014-01-01

Background Mycoplasma pneumoniae (M. pneumoniae) is one of the major etiological agents for community-acquired pneumonia (CAP) in all age groups. The early host response to M. pneumoniae infection relies on the concerted release of proteins with various biological activities. However, no comprehensive analysis of the secretory proteins has been conducted to date regarding the host response upon M. pneumoniae infection. Results We employed the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based label-free quantitative proteomic technology to identify and characterize the members of the human alveolar epithelial carcinoma A549 cell secretome during M. pneumoniae infection. A total of 256 proteins were identified, with 113 being differentially expressed (>1.5-fold change), among which 9 were only expressed in control cells, 10 only in M. pneumoniae-treated cells, while 55 were up-regulated and 39 down-regulated by M. pneumoniae. The changed expression of some of the identified proteins was validated by RT-PCR and immunoblot analysis. Cellular localization analysis of the secretome data revealed 59.38% of the proteins were considered as “putative secretory proteins”. Functional analysis revealed that the proteins affected upon M. pneumoniae infection were mainly related to metabolic process, stress response, and immune response. We further examined the level of one up-regulated protein, IL-33, in clinical samples. The result showed that IL-33 levels were significantly higher in the plasma and bronchoalveolar lavage fluid (BALF) of M. pneumoniae pneumonia (MPP) patients. Conclusions The present study provided systematic information about the changes in the expression of secretory proteins during M. pneumoniae infection, which is useful for the discovery of specific biomarkers and targets for pharmacological intervention. PMID:24507763

G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

NASA Astrophysics Data System (ADS)

Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

2016-10-01

The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

Down-regulated peroxisome proliferator-activated receptor γ (PPARγ) in lung epithelial cells promotes a PPARγ agonist-reversible proinflammatory phenotype in chronic obstructive pulmonary disease (COPD).

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Zhang, Yingze; Sciurba, Frank C; Mallampalli, Rama K; Duncan, Steven R; Reddy, Raju C

2014-03-07

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition and a leading cause of death, with no available cure. We assessed the actions in pulmonary epithelial cells of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor with anti-inflammatory effects, whose role in COPD is largely unknown. We found that PPARγ was down-regulated in lung tissue and epithelial cells of COPD patients, via both reduced expression and phosphorylation-mediated inhibition, whereas pro-inflammatory nuclear factor-κB (NF-κB) activity was increased. Cigarette smoking is the main risk factor for COPD, and exposing airway epithelial cells to cigarette smoke extract (CSE) likewise down-regulated PPARγ and activated NF-κB. CSE also down-regulated and post-translationally inhibited the glucocorticoid receptor (GR-α) and histone deacetylase 2 (HDAC2), a corepressor important for glucocorticoid action and whose down-regulation is thought to cause glucocorticoid insensitivity in COPD. Treating epithelial cells with synthetic (rosiglitazone) or endogenous (10-nitro-oleic acid) PPARγ agonists strongly up-regulated PPARγ expression and activity, suppressed CSE-induced production and secretion of inflammatory cytokines, and reversed its activation of NF-κB by inhibiting the IκB kinase pathway and by promoting direct inhibitory binding of PPARγ to NF-κB. In contrast, PPARγ knockdown via siRNA augmented CSE-induced chemokine release and decreases in HDAC activity, suggesting a potential anti-inflammatory role of endogenous PPARγ. The results imply that down-regulation of pulmonary epithelial PPARγ by cigarette smoke promotes inflammatory pathways and diminishes glucocorticoid responsiveness, thereby contributing to COPD pathogenesis, and further suggest that PPARγ agonists may be useful for COPD treatment.

MicroRNA regulatory networks reflective of polyhexamethylene guanidine phosphate-induced fibrosis in A549 human alveolar adenocarcinoma cells.

PubMed

Shin, Da Young; Jeong, Mi Ho; Bang, In Jae; Kim, Ha Ryong; Chung, Kyu Hyuck

2018-05-01

Polyhexamethylene guanidine phosphate (PHMG-phosphate), an active component of humidifier disinfectant, is suspected to be a major cause of pulmonary fibrosis. Fibrosis, induced by recurrent epithelial damage, is significantly affected by epigenetic regulation, including microRNAs (miRNAs). The aim of this study was to investigate the fibrogenic mechanisms of PHMG-phosphate through the profiling of miRNAs and their target genes. A549 cells were treated with 0.75 μg/mL PHMG-phosphate for 24 and 48 h and miRNA microarray expression analysis was conducted. The putative mRNA targets of the miRNAs were identified and subjected to Gene Ontology analysis. After exposure to PHMG-phosphate for 24 and 48 h, 46 and 33 miRNAs, respectively, showed a significant change in expression over 1.5-fold compared with the control. The integrated analysis of miRNA and mRNA microarray results revealed the putative targets that were prominently enriched were associated with the epithelial-mesenchymal transition (EMT), cell cycle changes, and apoptosis. The dose-dependent induction of EMT by PHMG-phosphate exposure was confirmed by western blot. We identified 13 putative EMT-related targets that may play a role in PHMG-phosphate-induced fibrosis according to the Comparative Toxicogenomic Database. Our findings contribute to the comprehension of the fibrogenic mechanism of PHMG-phosphate and will aid further study on PHMG-phosphate-induced toxicity. Copyright © 2018 Elsevier B.V. All rights reserved.

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation

PubMed Central

Zhao, Yingxin; Sun, Hong; Zhang, Yueqing; Yang, Jun; Brasier, Allan R.

2016-01-01

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases. PMID:27793799

The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

PubMed Central

Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

2015-01-01

Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

Genotoxicity of fine and coarse fraction ambient particulate matter in immortalised normal (TT1) and cancer‐derived (A549) alveolar epithelial cells

PubMed Central

Enlo‐Scott, Zachary; Nagy, Eszter; Mudway, Ian S.; Tetley, Teresa D.; Arlt, Volker M.; Phillips, David H.; Gollapudi, B.

2018-01-01

Human exposure to airborne particulate matter (PM) is associated with adverse cardiopulmonary health effects, including lung cancer. Ambient PM represents a heterogeneous mixture of chemical classes including transition metals, polycyclic aromatic hydrocarbons (PAHs) and their derivatives such as nitro‐PAHs, many of which are classified as putative carcinogens. As the primary site of human exposure to PM is the lungs, we investigated the response of two alveolar epithelial cell lines, the tumour‐derived A549 and newly described TT1 cells, to fine and coarse PM collected from background and roadside locations. We show that coarse PM elicits a genotoxic response in the TT1 cells, with the strongest signal associated with the background sample. This response could be recapitulated using the organic extract derived from this sample. No responses were observed in PM‐challenged A549 cells. Fine PM failed to elicit a genotoxic response in either cell line despite the higher PAH concentrations within this fraction. Consistent with the lack of a simplistic association between PM PAH content and the observed genotoxic response, TT1 cells treated with benzo[a]pyrene (BaP) demonstrated no increase in the selected markers. In contrast, a pattern of response was observed in TT1 cells challenged with 3‐nitrobenzanthrone (3‐NBA) similar to that with coarse PM. Together, these data illustrated the suitability of the TT1 cell line for assessing PM‐induced genotoxicity and challenge the contention that fine roadside PM poses the higher cancer risk. Furthermore, the response to 3‐NBA and not BaP suggests a major contribution of nitro‐PAHs to the overall toxicity of PM. Environ. Mol. Mutagen. 59:290–301, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society PMID:29368350

Protocatechuic aldehyde ameliorates experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Liang, E-mail: countryspring@sina.com; Ji, Yunxia, E-mail: 413499057@qq.com; Kang, Zechun, E-mail: davidjiangwl@163.com

An abnormal high mobility group box 1 (HMGB1) activation and a decrease in receptor for advanced glycation end-product (RAGE) play a key role in the pathogenesis of pulmonary fibrosis. Protocatechuic aldehyde (PA) is a naturally occurring compound, which is extracted from the degradation of phenolic acids. However, whether PA has anti-fibrotic functions is unknown. In this study, the effects of PA on the transforming growth factor-β1 (TGF-β1)-mediated epithelial–mesenchymal transition (EMT) in A549 cells, on the apoptosis of human type I alveolar epithelial cells (AT I), on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on bleomycin (BLM)-induced pulmonarymore » fibrosis in vivo were investigated. PA treatment resulted in a reduction of EMT in A549 cells with a decrease in vimentin and HMGB, an increase of E-cadherin and RAGE, a reduction of HLF-1 proliferation with a decrease of fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor (PDGF). Apoptosis of AT I was attenuated with an increase of RAGE. PA ameliorated BLM-induced pulmonary fibrosis in rats with a reduction of histopathological scores and collagen deposition, and a lower FGF-2, PDGF, α-smooth muscle actin (α-SMA) and HMGB1 expression, whereas higher RAGE was found in BLM-instilled lungs. Through the decrease of HGMB1 and the regulation of RAGE, PA reversed the EMT, inhibited HLF-1 proliferation as well as reduced apoptosis in AT I, and prevented pulmonary fibrosis in vivo. Collectively, our results demonstrate that PA prevents experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway. - Highlights: • PA prevents EMT, reduces the apoptosis of AT1 in vitro. • PA decreases proliferation of HLF-1, reduces PDGF and FGF expression in vitro. • PA prevents experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.« less

Effects of calcitriol (1, 25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice.

PubMed

Gui, Boxiang; Chen, Qin; Hu, Chuanxia; Zhu, Caihui; He, Guimei

2017-01-23

H9N2 influenza viruses circulate globally and are considered to have pandemic potential. The hyper-inflammatory response elicited by these viruses is thought to contribute to disease severity. Calcitriol plays an important role in modulating the immune response to viral infections. However, its unknown whether calcitriol can attenuate the inflammatory response elicited by H9N2 influenza virus infection. Human lung A549 epithelial cells were treated with calcitriol (100 nM) and then infected with an H9N2 influenza virus, or infected and then treated with calcitriol (30 nM). Culture supernatants were collected every 24 h post infection and the viral growth kinetics and inflammatory response were evaluated. Calcitriol (5 mg/kg) was administered daily by intraperitoneal injection to BABL/c mice for 15 days following H9N2 influenza virus infection. Mice were monitored for clinical signs of disease, lung pathology and inflammatory responses. Calcitriol treatment prior to and post infection with H9N2 influenza significantly decreased expression of the influenza M gene, IL-6, and IFN-β in A549 cells, but did not affect virus replication. In vivo, we found that calcitriol treatment significantly downregulated pulmonary inflammation in mice 2 days post-infection, but increased the inflammatory response 4 to 6 days post-infection. In contrast, the antiviral cytokine IFN-β was significantly higher in calcitriol-treated mice than in the untreated infected mice at 2 days post-infection, but lower than in untreated infected mice on days 4 and 8 post-infection. The elevated levels of pro-inflammatory cytokines and the decreased levels of antiviral cytokine are consistent with the period of maximum body weight loss and the lung damage in calcitriol-treated mice. These results suggest that calcitriol treatment might have a negative impact on the innate immune response elicited by H9N2 infection in mice, especially at the later stage of influenza virus infection. This study

Integrin-Mediated Transforming Growth Factor-β Activation Regulates Homeostasis of the Pulmonary Epithelial-Mesenchymal Trophic Unit

PubMed Central

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L.

2006-01-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-β is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, αvβ6 and αvβ8, are responsible for almost all of the TGF-β activation in the EMTU. Both αvβ8 and αvβ6 contribute to fetal tracheal epithelial activation of TGF-β, whereas only αvβ8 contributes to fetal tracheal fibroblast activation of TGF-β. Interestingly, fetal tracheal epithelial αvβ8-mediated TGF-β activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in αvβ8-mediated activation of TGF-β. Autocrine αvβ8-mediated TGF-β activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-β within the EMTU. PMID:16877343

Integrin-mediated transforming growth factor-beta activation regulates homeostasis of the pulmonary epithelial-mesenchymal trophic unit.

PubMed

Araya, Jun; Cambier, Stephanie; Morris, Alanna; Finkbeiner, Walter; Nishimura, Stephen L

2006-08-01

Trophic interactions between pulmonary epithelial and mesenchymal cell types, known as the epithelial-mesenchymal trophic unit (EMTU), are crucial in lung development and lung disease. Transforming growth factor (TGF)-beta is a key factor in mediating these interactions, but it is expressed in a latent form that requires activation to be functional. Using intact fetal tracheal tissue and primary cultures of fetal tracheal epithelial cells and fibroblasts, we demonstrate that a subset of integrins, alpha(v)beta(6) and alpha(v)beta(8), are responsible for almost all of the TGF-beta activation in the EMTU. Both alpha(v)beta(8) and alpha(v)beta(6) contribute to fetal tracheal epithelial activation of TGF-beta, whereas only alpha(v)beta(8) contributes to fetal tracheal fibroblast activation of TGF-beta. Interestingly, fetal tracheal epithelial alpha(v)beta(8)-mediated TGF-beta activation can be enhanced by phorbol esters, likely because of the increased activity of MT1-MMP, an essential co-factor in alpha(v)beta(8)-mediated activation of TGF-beta. Autocrine alpha(v)beta(8)-mediated TGF-beta activation by fetal tracheal fibroblasts results in suppression of both transcription and secretion of hepatocyte growth factor, which is sufficient to affect phosphorylation of the airway epithelial hepatocyte growth factor receptor, c-Met, as well as airway epithelial proliferation in a co-culture model of the EMTU. These findings elucidate the function and complex regulation of integrin-mediated activation of TGF-beta within the EMTU.

Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

PubMed

Wang, Yiqiang; Hedblom, Andreas; Koerner, Steffi K; Li, Mailin; Jernigan, Finith E; Wegiel, Barbara; Sun, Lijun

2016-12-01

A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC 50 : 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC 50 : 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC 50 : 0.55μM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10μM, indicating specificity towards cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

β-Liddle mutation of the epithelial sodium channel increases alveolar fluid clearance and reduces the severity of hydrostatic pulmonary oedema in mice

PubMed Central

Randrianarison, Nadia; Escoubet, Brigitte; Ferreira, Chrystophe; Fontayne, Alexandre; Fowler-Jaeger, Nicole; Clerici, Christine; Hummler, Edith; Rossier, Bernard C; Planès, Carole

2007-01-01

Transepithelial sodium transport via alveolar epithelial Na+ channels and Na+,K+-ATPase constitutes the driving force for removal of alveolar oedema fluid. Decreased activity of the amiloride-sensitive epithelial Na+ channel (ENaC) in the apical membrane of alveolar epithelial cells impairs sodium-driven alveolar fluid clearance (AFC) and predisposes to pulmonary oedema. We hypothesized that hyperactivity of ENaC in the distal lung could improve AFC and facilitate the resolution of pulmonary oedema. AFC and lung fluid balance were studied at baseline and under conditions of hydrostatic pulmonary oedema in the β-Liddle (L) mouse strain harbouring a gain-of-function mutation (R566stop) within the Scnn1b gene. As compared with wild-type (+/+), baseline AFC was increased by 2- and 3-fold in heterozygous (+/L) and homozygous mutated (L/L) mice, respectively, mainly due to increased amiloride-sensitive AFC. The β2-agonist terbutaline stimulated AFC in +/+ and +/L mice, but not in L/L mice. Acute volume overload induced by saline infusion (40% of body weight over 2 h) significantly increased extravascular (i.e. interstitial and alveolar) lung water as assessed by the bloodless wet-to-dry lung weight ratio in +/+ and L/L mice, as compared with baseline. However, the increase was significantly larger in +/+ than in L/L groups (P= 0.01). Volume overload also increased the volume of the alveolar epithelial lining fluid in +/+ mice, indicating the presence of alveolar oedema, but not in L/L mice. Cardiac function as evaluated by echocardiography was comparable in both groups. These data show that constitutive ENaC activation improved sodium-driven AFC in the mouse lung, and attenuated the severity of hydrostatic pulmonary oedema. PMID:17430990

Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells.

PubMed

Crespo, Rosana; Villaverde, M Luciana; Girotti, Juan R; Güerci, Alba; Juárez, M Patricia; de Bravo, Margarita G

2011-06-14

Ulomoides dermestoides (Fairmaire, 1893) is a cosmopolitan tenebrionid beetle reared by Argentine people who consume them alive as an alternative medicine in the treatment of different illnesses such as asthma, Parkinson's, diabetes, arthritis, HIV and specially cancer. To evaluate the cytotoxicity and DNA damage of the major volatile components released by Ulomoides dermestoides on human lung carcinoma epithelial cell line A549. The defence compounds of Ulomoides dermestoides were extracted with dichloromethane and analyzed and quantified by capillary gas chromatography. The toxicity effects of the beetle's extract against A549 cell line were evaluated. Cytotoxicity was evaluated by MTT test and Trypan blue assay and genotoxicity was evaluated by the comet assay. The synthetic compounds, individually or combined, were also tested in A549 cells and normal mononuclear human cells. The defence compounds of Ulomoides dermestoides extracted with dichloromethane (methyl-1,4-benzoquinones, ethyl-1,4-benzoquinones and 1-pentadecene as major components) showed cytotoxic activity on A549 cells demonstrated by MTT test and Trypan blue assay, with IC(50) values of 0.26equivalent/ml and 0.34equivalent/ml, respectively (1equivalent=amount of components extracted per beetle). The inhibition of A549 cell proliferation with the synthetic blend (1,4-benzoquinone and 1-pentadecene) or 1,4-benzoquinone alone was similar to that obtained with the insect extract. 1-Pentadecene showed no inhibitory effect. Low doses of insect extract or synthetic blend (0.15equivalent/ml) inhibited mononuclear cell proliferation by 72.2±2.7% and induced significant DNA damage both in tumor and mononuclear cells. Results of this study demonstrated that defence compounds of Ulomoides dermestoides reduced cell viability and induced DNA damage. We also concluded that the insect benzoquinones are primarily responsible for inducing cytotoxicity and genotoxicity in culture cells. Copyright © 2011 Elsevier

Epithelial and endothelial cell plasticity in chronic obstructive pulmonary disease (COPD).

PubMed

Sohal, Sukhwinder Singh

2017-03-01

Chronic Obstructive Pulmonary Disease (COPD) is mainly caused by smoking and presents with shortness of breath that is progressive and irreversible. It is a worldwide health problem and the fourth most common cause of chronic disability and mortality (even in developed countries). It is a complex disease involving both the airway and lung parenchyma. Small-airway fibrosis is the main contributor to physiological airway dysfunction in COPD. One potential mechanism contributing to small-airway fibrosis is epithelial mesenchymal transition (EMT). When associated with angiogenesis (EMT-type-3), EMT may well also be linked to the development of airway epithelial cancer, which is closely associated with COPD and predominantly observed in large airways. Vascular remodeling has also been widely reported in smokers and patients with COPD but the mechanisms behind it are poorly understood. It is quite possible that the process of endothelial to mesenchymal transition (EndMT) is also active in COPD lungs, in addition to EMT. Understanding these pathological mechanisms will greatly enhance our knowledge of the immunopathology of smoking-related lung disease. Only by understanding these processes can new therapies be developed. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

PubMed

Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

2017-04-01

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

PubMed Central

2012-01-01

Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents. PMID:22917272

Protective role of gambogic acid in experimental pulmonary fibrosis in vitro and in vivo.

PubMed

Qu, Yubei; Zhang, Guanghua; Ji, Yunxia; Zhua, Haibo; Lv, Changjun; Jiang, Wanglin

2016-04-15

Idiopathic pulmonary fibrosis (IPF) is a progressive disorder with poor prognosis. The treatment options for IPF are very limited. Gambogic acid (GA) has anticancer effect and anti-proliferative activity which is extracted from a dried yellow resin of the Garcinia hanburyi Hook.f. [Clusiaceae (Guttiferae)] in Southeast Asia. However, the anti-fibrotic activities of GA have not been previously investigated. In this study, the effects of GA on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells and endothelial-mesenchymal transition (EndoMT) in human pulmonary microvascular endothelial cells (HPMECs), on the proliferation of human lung fibroblasts (HLF-1) were investigated in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis was investigated in vivo. In TGF-β1 stimulated A549 cells, treatment with GA resulted in a reduction of EMT with a decrease in vimentin and p-Smad3 and an increase in E-cadherin instead. In TGF-β1 stimulated HPMECs, treatment with GA resulted in a reduction of EndoMT with a decrease in vimentin, and an increase in VE-cadherin instead. In the hypoxic HPMECs, treatment with GA reduced Vasohibin-2 (VASH-2), whereas increased VASH-1. In TGF-β1 stimulated HLF-1, treatment with GA reduced HLF-1 proliferation with a decrease in platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF-2) expressions. In vivo, treatment with GA for 2 weeks resulted in an amelioration of the BLM-induced pulmonary fibrosis in rats with a lower VASH-2. Instead, it was observed a higher VASH-1 expression at early stage of fibrosis at 1 mg/kg, with reductions of the pathological score, collagen deposition, α-SMA, PDGF and FGF-2 expressions at fibrotic stage at 0.5 mg/kg and 1 mg/kg. In summary, GA reversed EMT and EndoMT, as well as HLF-1 proliferation in vitro and prevented pulmonary fibrosis in vivo by modulating VASH-2/VASH-1 and suppressing the TGF-β1/Smad3 pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

Comparison of Active Drug Concentrations in the Pulmonary Epithelial Lining Fluid and Interstitial Fluid of Calves Injected with Enrofloxacin, Florfenicol, Ceftiofur, or Tulathromycin

PubMed Central

Foster, Derek M.; Martin, Luke G.; Papich, Mark G.

2016-01-01

Bacterial pneumonia is the most common reason for parenteral antimicrobial administration to beef cattle in the United States. Yet there is little information describing the antimicrobial concentrations at the site of action. The objective of this study was to compare the active drug concentrations in the pulmonary epithelial lining fluid and interstitial fluid of four antimicrobials commonly used in cattle. After injection, plasma, interstitial fluid, and pulmonary epithelial lining fluid concentrations and protein binding were measured to determine the plasma pharmacokinetics of each drug. A cross-over design with six calves per drug was used. Following sample collection and drug analysis, pharmacokinetic calculations were performed. For enrofloxacin and metabolite ciprofloxacin, the interstitial fluid concentration was 52% and 78% of the plasma concentration, while pulmonary fluid concentrations was 24% and 40% of the plasma concentration, respectively. The pulmonary concentrations (enrofloxacin + ciprofloxacin combined) exceeded the MIC90 of 0.06 μg/mL at 48 hours after administration. For florfenicol, the interstitial fluid concentration was almost 98% of the plasma concentration, and the pulmonary concentrations were over 200% of the plasma concentrations, exceeding the breakpoint (≤ 2 μg/mL), and the MIC90 for Mannheimia haemolytica (1.0 μg/mL) for the duration of the study. For ceftiofur, penetration to the interstitial fluid was only 5% of the plasma concentration. Pulmonary epithelial lining fluid concentration represented 40% of the plasma concentration. Airway concentrations exceeded the MIC breakpoint for susceptible respiratory pathogens (≤ 2 μg/mL) for a short time at 48 hours after administration. The plasma and interstitial fluid concentrations of tulathromcyin were lower than the concentrations in pulmonary fluid throughout the study. The bronchial concentrations were higher than the plasma or interstitial concentrations, with over 900

Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation

PubMed Central

Rajavelu, Priya; Chen, Gang; Xu, Yan; Kitzmiller, Joseph A.; Korfhagen, Thomas R.; Whitsett, Jeffrey A.

2015-01-01

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef–/– mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen. PMID:25866971

Epithelial-myoepithelial tumour of the lung: a case report referring to its molecular histogenesis

PubMed Central

2011-01-01

Tracheobronchial submucous glands can be considered the pulmonary equivalent of minor salivary glands and therefore they can develop most of the tumours originated in these. Nevertheless, in spite of the wide distribution of this kind of glands along the tracheobronchial tree, pulmonary salivary gland-like neoplasms are not very frequent. Among them, the most frequent are mucoepidermoid and adenoid cystic carcinomas. On the contrary, pulmonary neoplasms showing a mixture of epithelial and myoepithelial elements are extraordinary infrequent, with only 11 cases collected from literature. We present the case of a 76 year-old woman with no interesting pathological history, to whom a pulmonary nodule is detected during a study of unknown origin neutropenia. An upper right lobectomy is performed. After macro and microscopic study, the diagnosis of pulmonary epithelial-myoepithelial tumour is made. It is a low malignant potential tumour with capacity to locally recur and less frequently to metastasize. Our case has the peculiarity of not being connected neither to visceral pleura nor to bronchial tree; we have not found this characteristic in any literature reviewed case. These tumours have been named in a lot of different ways, including adenomyoepithelioma, epithelial-myoepithelial tumour, epithelial-myoepithelial carcinoma or epithelial-myoepithelial tumour of uncertain malignant potential. The p27/kip-1 protein plays a fundamental role in the development of these neoplasms. As we have verified in our case, its aberrant cytoplasmic location, besides its proved oncogenic function, would favour the proliferation of stem cells, which would explain both dual phenotype with presence of myoepithelial cells without connection with the bronchial tree, and TTF-1 immunostaining in epithelial cells. PMID:21798017

Epithelial-myoepithelial tumour of the lung: a case report referring to its molecular histogenesis.

PubMed

Muñoz, Guillermo; Felipo, Francesc; Marquina, Isabel; Del Agua, Celia

2011-07-28

Tracheobronchial submucous glands can be considered the pulmonary equivalent of minor salivary glands and therefore they can develop most of the tumours originated in these. Nevertheless, in spite of the wide distribution of this kind of glands along the tracheobronchial tree, pulmonary salivary gland-like neoplasms are not very frequent. Among them, the most frequent are mucoepidermoid and adenoid cystic carcinomas. On the contrary, pulmonary neoplasms showing a mixture of epithelial and myoepithelial elements are extraordinary infrequent, with only 11 cases collected from literature.We present the case of a 76 year-old woman with no interesting pathological history, to whom a pulmonary nodule is detected during a study of unknown origin neutropenia. An upper right lobectomy is performed.After macro and microscopic study, the diagnosis of pulmonary epithelial-myoepithelial tumour is made. It is a low malignant potential tumour with capacity to locally recur and less frequently to metastasize. Our case has the peculiarity of not being connected neither to visceral pleura nor to bronchial tree; we have not found this characteristic in any literature reviewed case.These tumours have been named in a lot of different ways, including adenomyoepithelioma, epithelial-myoepithelial tumour, epithelial-myoepithelial carcinoma or epithelial-myoepithelial tumour of uncertain malignant potential.The p27/kip-1 protein plays a fundamental role in the development of these neoplasms. As we have verified in our case, its aberrant cytoplasmic location, besides its proved oncogenic function, would favour the proliferation of stem cells, which would explain both dual phenotype with presence of myoepithelial cells without connection with the bronchial tree, and TTF-1 immunostaining in epithelial cells.

Lung Epithelial Cells Coordinate Innate Lymphocytes and Immunity against Pulmonary Fungal Infection.

PubMed

Hernández-Santos, Nydiaris; Wiesner, Darin L; Fites, J Scott; McDermott, Andrew J; Warner, Thomas; Wüthrich, Marcel; Klein, Bruce S

2018-04-11

Lung epithelial cells (LECs) are strategically positioned in the airway mucosa to provide barrier defense. LECs also express pattern recognition receptors and a myriad of immune genes, but their role in immunity is often concealed by the activities of "professional" immune cells, particularly in the context of fungal infection. Here, we demonstrate that NF-κB signaling in LECs is essential for immunity against the pulmonary fungal pathogen Blastomyces dermatitidis. LECs orchestrate innate antifungal immunity by augmenting the numbers of interleukin-17A (IL-17A)- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing innate lymphocytes, specifically "natural" Th17 (nTh17) cells. Innate lymphocyte-derived IL-17A and GM-CSF in turn enable phagocyte-driven fungal killing. LECs regulate the numbers of nTh17 cells via the production of chemokines such as CCL20, a process dependent on IL-1α-IL-1 receptor (IL-1R) signaling on LECs. Therefore, LECs orchestrate IL-17A- and GM-CSF-mediated immunity in an IL-1R-dependent manner and represent an essential component of innate immunity to pulmonary fungal pathogens. Copyright © 2018 Elsevier Inc. All rights reserved.

Reactive oxygen species mediated DNA damage in human lung alveolar epithelial (A549) cells from exposure to non-cytotoxic MFI-type zeolite nanoparticles.

PubMed

Bhattacharya, Kunal; Naha, Pratap C; Naydenova, Izabela; Mintova, Svetlana; Byrne, Hugh J

2012-12-17

Increasing utilization of engineered nanoparticles in the field of electronics and biomedical applications demands an assessment of risk associated with deliberate or accidental exposure. Metal based nanoparticles are potentially most important of all the nanoparticles in terms of health risks. Microporous alumino-silicates and pure silicates named as zeolites and zeo-type materials with variety of structures, chemical compositions, particle sizes and morphologies have a significant number of industrial uses such as in catalysis, sorption and ion-exchange processes. In particular, the nanosized particles due to their unique properties are used in hybrid organic-inorganic materials for photography, photonics, electronics, labeling, imaging, and sensing. The aim of the current study is to investigate pure silica MFI-type zeolites nanoparticles with sizes of 50nm and 100nm (samples MFI-50 and MFI-100) under suspended conditions and their toxicological effects on human lung alveolar (A549) cells under in vitro conditions. Live cell imaging showed that the nanoparticles precipitated from the colloidal suspension of cell culture media as large agglomerates, coming in contact with the cell surface through sedimentation. A cellular proliferative capacity test showed the zeolite nanoparticles to exhibit no significant cytotoxicity below a concentration of 100μg/ml. However, both the MFI-50 and MFI-100 nanoparticles induced high intracellular reactive oxygen species (ROS) generation and elevated mitochondrial membrane potential in the A549 cells over the measured time period of 12h and at concentrations up to ≤50μg/ml. DNA fragmentation analysis using the comet assay showed that the MFI-50 and MFI-100 nanoparticles cause genotoxicity in a concentration dependent manner. Furthermore, the rate at which maximum genomic damage was caused by MFI-100 nanoparticles in the A549 cells was found to be high as compared to the MFI-50 nanoparticles. However, the damage caused by the

Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika

2005-12-30

Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzymemore » inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.« less

Triptolide suppresses paraquat induced idiopathic pulmonary fibrosis by inhibiting TGFB1-dependent epithelial mesenchymal transition.

PubMed

Chen, Hong; Chen, Qun; Jiang, Chun-Ming; Shi, Guang-Yue; Sui, Bo-Wen; Zhang, Wei; Yang, Li-Zhen; Li, Zhu-Ying; Liu, Li; Su, Yu-Ming; Zhao, Wen-Cheng; Sun, Hong-Qiang; Li, Zhen-Zi; Fu, Zhou

2018-03-01

Idiopathic pulmonary fibrosis (IPF) and tumor are highly similar to abnormal cell proliferation that damages the body. This malignant cell evolution in a stressful environment closely resembles that of epithelial-mesenchymal transition (EMT). As a popular EMT-inducing factor, TGFβ plays an important role in the progression of multiple diseases. However, the drugs that target TGFB1 are limited. In this study, we found that triptolide (TPL), a Chinese medicine extract, exerts an anti-lung fibrosis effect by inhibiting the EMT of lung epithelial cells. In addition, triptolide directly binds to TGFβ and subsequently increase E-cadherin expression and decrease vimentin expression. In in vivo studies, TPL improves the survival state and inhibits lung fibrosis in mice. In summary, this study revealed the potential therapeutic effect of paraquat induced TPL in lung fibrosis by regulating TGFβ-dependent EMT progression. Copyright © 2017 Elsevier B.V. All rights reserved.

The Expression of AQP1 IS Modified in Lung of Patients With Idiopathic Pulmonary Fibrosis: Addressing a Possible New Target.

PubMed

Galán-Cobo, Ana; Arellano-Orden, Elena; Sánchez Silva, Rocío; López-Campos, José Luis; Gutiérrez Rivera, César; Gómez Izquierdo, Lourdes; Suárez-Luna, Nela; Molina-Molina, María; Rodríguez Portal, José A; Echevarría, Miriam

2018-01-01

Activation of the epithelial-mesenchymal transition process (EMT) by which alveolar cells in human lung tissue undergo differentiation giving rise to a mesenchymal phenotype (fibroblast/miofibroblasts) has been well recognized as a key element in the origin of idiopathic pulmonary fibrosis (IPF). Here we analyzed expression of AQP1 in lung biopsies of patients diagnosed with IPF, and compared it to biopsies derived from patients with diverse lung pneumonies, such as hypersensitivity pneumonitis, sarcoidosis or normal lungs. Immunostaining for AQP1 showed a clear increment of AQP1 localized in the alveolar epithelium in biopsies from IPF patients alone. Moreover, to examine the possible participation of AQP1 in the pathophysiology of IPF, we evaluated its role in the pro-fibrotic transformation induced by transforming growth factor (TGF-β) in vitro . Human alveolar epithelial cells (A549), and fibroblasts derived from an IPF patient (LL29), or fibroblasts from healthy normal lung tissue (MRC-5), were treated with TGF-β, and levels of expression of AQP1, as well as those of E-cadherin, vimentin, α-SMA and collagen were analyzed by RT-qPCR, western blot and immunohistochemistry. An increase of AQP1 mRNA and protein after TGF-β treatment (4-72h) was observed either in A549 or IPF fibroblast-LL29 but not in MRC-5 fibroblasts. A gradual reduction of E-cadherin, and increased expression of vimentin, with no changes in α-SMA levels were observed in A549. Whereas in LL29 and MRC-5, TGF-β1 elicited a large production of collagen and α-SMA that was significantly greater in IPF fibroblast-LL29. Changes observed are consistent with activation of EMT by TGF-β, but whether modifications in AQP1 expression are responsible or independent events occurring at the same time is still unknown. Our results suggest that AQP1 plays a role in the pro-fibrotic TGF-β action and contributes to the etiology and pathophysiology of IPF. Understanding AQP1's role will help us comprehend

Targeting Interleukin-13 with Tralokinumab Attenuates Lung Fibrosis and Epithelial Damage in a Humanized SCID Idiopathic Pulmonary Fibrosis Model

PubMed Central

Zhang, Huilan; Oak, Sameer R.; Coelho, Ana Lucia; Herath, Athula; Flaherty, Kevin R.; Lee, Joyce; Bell, Matt; Knight, Darryl A.; Martinez, Fernando J.; Sleeman, Matthew A.; Herzog, Erica L.; Hogaboam, Cory M.

2014-01-01

The aberrant fibrotic and repair responses in the lung are major hallmarks of idiopathic pulmonary fibrosis (IPF). Numerous antifibrotic strategies have been used in the clinic with limited success, raising the possibility that an effective therapeutic strategy in this disease must inhibit fibrosis and promote appropriate lung repair mechanisms. IL-13 represents an attractive target in IPF, but its disease association and mechanism of action remains unknown. In the present study, an overexpression of IL-13 and IL-13 pathway markers was associated with IPF, particularly a rapidly progressive form of this disease. Targeting IL-13 in a humanized experimental model of pulmonary fibrosis using tralokinumab (CAT354) was found to therapeutically block aberrant lung remodeling in this model. However, targeting IL-13 was also found to promote lung repair and to restore epithelial integrity. Thus, targeting IL-13 inhibits fibrotic processes and enhances repair processes in the lung. PMID:24325475

Oxidative stress mediated apoptosis induced by nickel ferrite nanoparticles in cultured A549 cells.

PubMed

Ahamed, Maqusood; Akhtar, Mohd Javed; Siddiqui, Maqsood A; Ahmad, Javed; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; AlSalhi, Mohamad S; Alrokayan, Salman A

2011-05-10

Due to the interesting magnetic and electrical properties with good chemical and thermal stabilities, nickel ferrite nanoparticles are being utilized in many applications including magnetic resonance imaging, drug delivery and hyperthermia. Recent studies have shown that nickel ferrite nanoparticles produce cytotoxicity in mammalian cells. However, there is very limited information concerning the toxicity of nickel ferrite nanoparticles at the cellular and molecular level. The aim of this study was to investigate the cytotoxicity, oxidative stress and apoptosis induction by well-characterized nickel ferrite nanoparticles (size 26 nm) in human lung epithelial (A549) cells. Nickel ferrite nanoparticles induced dose-dependent cytotoxicity in A549 cells demonstrated by MTT, NRU and LDH assays. Nickel ferrite nanoparticles were also found to induce oxidative stress evidenced by generation of reactive oxygen species (ROS) and depletion of antioxidant glutathione (GSH). Further, co-treatment with the antioxidant L-ascorbic acid mitigated the ROS generation and GSH depletion due to nickel ferrite nanoparticles suggesting the potential mechanism of oxidative stress. Quantitative real-time PCR analysis demonstrated that following the exposure of A549 cells to nickel ferrite nanoparticles, the level of mRNA expressions of cell cycle checkpoint protein p53 and apoptotic proteins (bax, caspase-3 and caspase-9) were significantly up-regulated, whereas the expression of anti-apoptotic proteins (survivin and bcl-2) were down-regulated. Moreover, activities of caspase-3 and caspase-9 enzymes were also significantly higher in nickel ferrite nanoparticles exposed cells. To the best of our knowledge this is the first report showing that nickel ferrite nanoparticles induced apoptosis in A549 cells through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

PM2.5 induces Nrf2-mediated defense mechanisms against oxidative stress by activating PIK3/AKT signaling pathway in human lung alveolar epithelial A549 cells.

PubMed

Deng, Xiaobei; Rui, Wei; Zhang, Fang; Ding, Wenjun

2013-06-01

It has been well documented in in vitro studies that ambient airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM(2.5)) is capable of inducing oxidative stress, which plays a key role in PM(2.5)-mediated cytotoxicity. Although nuclear factor erythroid-2-related factor 2 (Nrf2) has been shown to regulate the intracellular defense mechanisms against oxidative stress, a potential of the Nrf2-mediated cellular defense against oxidative stress induced by PM(2.5) remains to be determined. This study was aimed to explore the potential signaling pathway of Nrf2-mediated defense mechanisms against PM(2.5)-induced oxidative stress in human type II alveolar epithelial A549 cells. We exposed A549 cells to PM(2.5) particles collected from Beijing at a concentration of 16 μg/cm(2). We observed that PM(2.5) triggered an increase of intracellular reactive oxygen species (ROS) in a time-dependent manner during a period of 2 h exposure. We also found that Nrf2 overexpression suppressed and Nrf2 knockdown increased PM(2.5)-induced ROS generation. Using Western blot and confocal microscopy, we found that PM(2.5) exposure triggered significant translocation of Nrf2 into nucleus, resulting in AKT phosphorylation and significant transcription of ARE-driven phases II enzyme genes, such as NAD(P)H:quinone oxidoreductase (NQO-1), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase catalytic subunit (GCLC) in A549 cells. Evaluation of signaling pathways showed that a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), but not an ERK 1/2 inhibitor (PD98059) or a p38 MAPK (SB203580), significantly down-regulated PM(2.5)-induced Nrf2 nuclear translocation and HO-1 mRNA expression, indicating PI3K/AKT is involved in the signaling pathway leads to the PM(2.5)-induced nuclear translocation of Nrf2 and subsequent Nrf2-mediated HO-1 transcription. Taken together, our results suggest that PM(2.5)-induced ROS may function as signaling molecules to activate Nrf

CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot

Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonicalmore » Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.« less

MTOR Suppresses Cigarette Smoke-Induced Epithelial Cell Death and Airway Inflammation in Chronic Obstructive Pulmonary Disease.

PubMed

Wang, Yong; Liu, Juan; Zhou, Jie-Sen; Huang, Hua-Qiong; Li, Zhou-Yang; Xu, Xu-Chen; Lai, Tian-Wen; Hu, Yue; Zhou, Hong-Bin; Chen, Hai-Pin; Ying, Song-Min; Li, Wen; Shen, Hua-Hao; Chen, Zhi-Hua

2018-04-15

Airway epithelial cell death and inflammation are pathological features of chronic obstructive pulmonary disease (COPD). Mechanistic target of rapamycin (MTOR) is involved in inflammation and multiple cellular processes, e.g., autophagy and apoptosis, but little is known about its function in COPD pathogenesis. In this article, we illustrate how MTOR regulates cigarette smoke (CS)-induced cell death, airway inflammation, and emphysema. Expression of MTOR was significantly decreased and its suppressive signaling protein, tuberous sclerosis 2 (TSC2), was increased in the airway epithelium of human COPD and in mouse lungs with chronic CS exposure. In human bronchial epithelial cells, CS extract (CSE) activated TSC2, inhibited MTOR, and induced autophagy. The TSC2-MTOR axis orchestrated CSE-induced autophagy, apoptosis, and necroptosis in human bronchial epithelial cells; all of which cooperatively regulated CSE-induced inflammatory cytokines IL-6 and IL-8 through the NF-κB pathway. Mice with a specific knockdown of Mtor in bronchial or alveolar epithelial cells exhibited significantly augmented airway inflammation and airspace enlargement in response to CS exposure, accompanied with enhanced levels of autophagy, apoptosis, and necroptosis in the lungs. Taken together, these data demonstrate that MTOR suppresses CS-induced inflammation and emphysema-likely through modulation of autophagy, apoptosis, and necroptosis-and thus suggest that activation of MTOR may represent a novel therapeutic strategy for COPD. Copyright © 2018 by The American Association of Immunologists, Inc.

Gpr177 regulates pulmonary vasculature development.

PubMed

Jiang, Ming; Ku, Wei-yao; Fu, Jiang; Offermanns, Stefan; Hsu, Wei; Que, Jianwen

2013-09-01

Establishment of the functional pulmonary vasculature requires intimate interaction between the epithelium and mesenchyme. Previous genetic studies have led to inconsistent conclusions about the contribution of epithelial Wnts to pulmonary vasculature development. This discrepancy is possibly due to the functional redundancy among different Wnts. Here, we use Shh-Cre to conditionally delete Gpr177 (the mouse ortholog of Drosophila Wntless, Wls), a chaperon protein important for the sorting and secretion of Wnt proteins. Deletion of epithelial Gpr177 reduces Wnt signaling activity in both the epithelium and mesenchyme, resulting in severe hemorrhage and abnormal vasculature, accompanied by branching defects and abnormal epithelial differentiation. We then used multiple mouse models to demonstrate that Wnt/β-catenin signaling is not only required for the proliferation and differentiation of mesenchyme, but also is important for the maintenance of smooth muscle cells through the regulation of the transcription factor Kruppel-like factor 2 (Klf2). Together, our studies define a novel mechanism by which epithelial Wnts regulate the normal development and maintenance of pulmonary vasculature. These findings provide insight into the pathobiology of congenital lung diseases, such as alveolar capillary dysplasia (ACD), that have abnormal alveolar development and dysmorphic pulmonary vasculature.

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling.

PubMed

Tamminen, Jenni A; Myllärniemi, Marjukka; Hyytiäinen, Marko; Keski-Oja, Jorma; Koli, Katri

2012-07-01

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity. Copyright © 2012 Wiley Periodicals, Inc.

Microarray identifies ADAM family members as key responders to TGF-beta1 in alveolar epithelial cells.

PubMed

Keating, Dominic T; Sadlier, Denise M; Patricelli, Andrea; Smith, Sinead M; Walls, Dermot; Egan, Jim J; Doran, Peter P

2006-09-01

The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF) remain elusive. Transforming Growth Factor beta 1(TGF-beta1) is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549) in response to TGF-beta1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-beta1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM) proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO) databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing) family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity) of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-beta1, suggesting a potential role for these molecules in ECM accumulation in IPF.

Peroxisome proliferator-activated receptor-{gamma} agonists inhibit the replication of respiratory syncytial virus (RSV) in human lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arnold, Ralf; Koenig, Wolfgang

2006-07-05

We have previously shown that peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) agonists inhibited the inflammatory response of RSV-infected human lung epithelial cells. In this study, we supply evidence that specific PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone, troglitazone, Fmoc-Leu) efficiently blocked the RSV-induced cytotoxicity and development of syncytia in tissue culture (A549, HEp-2). All PPAR{gamma} agonists under study markedly inhibited the cell surface expression of the viral G and F protein on RSV-infected A549 cells. This was paralleled by a reduced cellular amount of N protein-encoding mRNA determined by real-time RT-PCR. Concomitantly, a reduced release of infectious progeny virus into the cell supernatants ofmore » human lung epithelial cells (A549, normal human bronchial epithelial cells (NHBE)) was observed. Similar results were obtained regardless whether PPAR{gamma} agonists were added prior to RSV infection or thereafter, suggesting that the agonists inhibited viral gene expression and not the primary adhesion or fusion process.« less

Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2015-07-01

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. Copyright © 2015 Elsevier B.V. All rights reserved.

Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway.

PubMed

Gao, Rundi; Chen, Ruilin; Cao, Yu; Wang, Yuan; Song, Kang; Zhang, Ya; Yang, Junchao

2017-03-01

Pulmonary fibrosis is characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type II epithelial cells RLE-6TN in a concentration-dependent manner; reduces the expression of Collagen I, α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Protective effects of pulmonary epithelial lining fluid on oxidative stress and DNA single-strand breaks caused by ultrafine carbon black, ferrous sulphate and organic extract of diesel exhaust particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, Hsiao-Chi; Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan; Cheng, Yi-Ling

Pulmonary epithelial lining fluid (ELF) is the first substance to make contact with inhaled particulate matter (PM) and interacts chemically with PM components. The objective of this study was to determine the role of ELF in oxidative stress, DNA damage and the production of proinflammatory cytokines following physicochemical exposure to PM. Ultrafine carbon black (ufCB, 15 nm; a model carbonaceous core), ferrous sulphate (FeSO{sub 4}; a model transition metal) and a diesel exhaust particle (DEP) extract (a model organic compound) were used to examine the acellular oxidative potential of synthetic ELF and non-ELF systems. We compared the effects of exposuremore » to ufCB, FeSO{sub 4} and DEP extract on human alveolar epithelial Type II (A549) cells to determine the levels of oxidative stress, DNA single-strand breaks and interleukin-8 (IL-8) production in ELF and non-ELF systems. The effects of ufCB and FeSO{sub 4} on the acellular oxidative potential, cellular oxidative stress and DNA single-strand breakage were mitigated significantly by the addition of ELF, whereas there was no decrease following treatment with the DEP extract. There was no significant effect on IL-8 production following exposure to samples that were suspended in ELF/non-ELF systems. The results of the present study indicate that ELF plays an important role in the initial defence against PM in the pulmonary environment. Experimental components, such as ufCB and FeSO{sub 4}, induced the production of oxidative stress and led to DNA single-strand breaks, which were moderately prevented by the addition of ELF. These findings suggest that ELF plays a protective role against PM-driven oxidative stress and DNA damage. -- Highlights: ► To determine the role of ELF in ROS, DNA damage and IL-8 after exposure to PM. ► ufCB, FeSO{sub 4} and DEP extract were used to examine the protective effects of ELF. ► PM-driven oxidative stress and DNA single-strand breakage were mitigated by ELF. ► The

Different signal pathways regulate IL-1β-induced mature and primary miRNA-146a expression in human alveolar epithelial cells.

PubMed

Jiang, Xiaoying

2014-09-01

It was known that IL-1β-induced rapid expression of miR-146a, which regulated the secretion of inflammatory chemokines in human A549 alveolar epithelial cells. However, little is known about the level of primary miR-146a and the downstream biogenesis of miR-146a in A549 cells. We examined the levels of primary miR-146a and mature miR-146a in A549 cells following treatment with pharmacological inhibitors of IKK-2 (TPCA-1), MEK-1/2 (PD098059), JNK-1/2 (SP600125), p38 MAPK (SB 203580) and PI-3k (LY294002). Our studies showed that exposure to PD98059, TPCA-1 and LY294002 resulted in a dose-dependent reduction in the expression of mature miR-146a while the primary miR-146a expression was not changed by any inhibitor. Western blot showed that IL-1β induced an increase of TRBP at 30 min, following by an extended expression at 24 h compared to the non-IL-1β controls in A549 cells. In conclusion, our studies indicated that miR-146a expression in alveolar epithelial cells was regulated at the post-transcriptional level via a MEK-1/2 and IKK2 pathway, and also for the first time via PI-3k pathway. The longer expression of TRBP following stimulation with IL-1β suggests that TRBP might play a role in the process of regulating the processing of primary miR-146a to mature miR-146a in human alveolar epithelial cells.

Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

PubMed

Amin, Shayista; Thywissen, Andreas; Heinekamp, Thorsten; Saluz, Hans Peter; Brakhage, Axel A

2014-07-01

Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

PubMed Central

WAN, QIAOFENG; WANG, HAO; LIN, YUAN; GU, LIGANG; HAN, MEI; YANG, ZHIWEI; ZHANG, YANLI; MA, RUI; WANG, LI; WANG, ZHISHENG

2013-01-01

This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10−4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30–60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4–48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection. PMID:24649026

31 CFR 549.303 - Entity.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Entity. 549.303 Section 549.303 Money... CONTROL, DEPARTMENT OF THE TREASURY LEBANON SANCTIONS REGULATIONS General Definitions § 549.303 Entity. The term entity means a partnership, association, trust, joint venture, corporation, group, subgroup...

Ion Transport by Pulmonary Epithelia

PubMed Central

Hollenhorst, Monika I.; Richter, Katrin; Fronius, Martin

2011-01-01

The lung surface of air-breathing vertebrates is formed by a continuous epithelium that is covered by a fluid layer. In the airways, this epithelium is largely pseudostratified consisting of diverse cell types such as ciliated cells, goblet cells, and undifferentiated basal cells, whereas the alveolar epithelium consists of alveolar type I and alveolar type II cells. Regulation and maintenance of the volume and viscosity of the fluid layer covering the epithelium is one of the most important functions of the epithelial barrier that forms the outer surface area of the lungs. Therefore, the epithelial cells are equipped with a wide variety of ion transport proteins, among which Na+, Cl−, and K+ channels have been identified to play a role in the regulation of the fluid layer. Malfunctions of pulmonary epithelial ion transport processes and, thus, impairment of the liquid balance in our lungs is associated with severe diseases, such as cystic fibrosis and pulmonary oedema. Due to the important role of pulmonary epithelial ion transport processes for proper lung function, the present paper summarizes the recent findings about composition, function, and ion transport properties of the airway epithelium as well as of the alveolar epithelium. PMID:22131798

Protein kinase D is increased and activated in lung epithelial cells and macrophages in idiopathic pulmonary fibrosis.

PubMed

Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

2014-01-01

Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.

Anti-pulmonary fibrotic activity of salvianolic acid B was screened by a novel method based on the cyto-biophysical properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Miao; Zheng, Mingjing; Xu, Hanying

Various methods have been used to evaluate anti-fibrotic activity of drugs. However, most of them are complicated, labor-intensive and lack of efficiency. This study was intended to develop a rapid method for anti-fibrotic drugs screening based on biophysical properties. A549 cells in vitro were stimulated with transforming growth factor-β1 (TGF-β1), and fibrogenesis was confirmed by conventional immunological assays. Meanwhile, the alterations of cyto-biophysical properties including morphology, roughness and stiffness were measured utilizing atomic force microscopy (AFM). It was found that fibrogenesis was accompanied with changes of cellular biophysical properties. TGF-β1-stimulated A549 cells became remarkably longer, rougher and stiffer than the control.more » Then, the effect of N-acetyl-L-cysteine (NAC) as a positive drug on ameliorating fibrogenesis in TGF-β1-stimulated A549 cells was verified respectively by immunological and biophysical markers. The result of Principal Component Analysis showed that stiffness was a leading index among all biophysical markers during fibrogenesis. Salvianolic acid B (SalB), a natural anti-oxidant, was detected by AFM to protect TGF-β1-stimulated A549 cells against stiffening. Then, SalB treatment was provided in preventive mode on a rat model of bleomycin (BLM) -induced pulmonary fibrosis. The results showed that SalB treatment significantly ameliorated BLM-induced histological alterations, blocked collagen accumulations and reduced α-SMA expression in lung tissues. All these results revealed the anti-pulmonary fibrotic activity of SalB. Detection of cyto-biophysical properties were therefore recommended as a rapid method for anti-pulmonary fibrotic drugs screening. - Highlights: • Fibrogenesis was accompanied with the changes of cyto-biophysical properties. • Cyto-biophysical properties could be markers for anti-fibrotic drugs screening. • Stiffness is a leading index among all biophysical markers. • Sal

Toxicity of wood smoke particles in human A549 lung epithelial cells: the role of PAHs, soot and zinc.

PubMed

Dilger, Marco; Orasche, Jürgen; Zimmermann, Ralf; Paur, Hanns-Rudolf; Diabaté, Silvia; Weiss, Carsten

2016-12-01

Indoor air pollution is associated with increased morbidity and mortality. Specifically, the health impact of emissions from domestic burning of biomass and coal is most relevant and is estimated to contribute to over 4 million premature deaths per year worldwide. Wood is the main fuel source for biomass combustion and the shift towards renewable energy sources will further increase emissions from wood combustion even in developed countries. However, little is known about the constituents of wood smoke and biological mechanisms that are responsible for adverse health effects. We exposed A549 lung epithelial cells to collected wood smoke particles and found an increase in cellular reactive oxygen species as well as a response to bioavailable polycyclic aromatic hydrocarbons. In contrast, cell vitality and regulation of the pro-inflammatory cytokine interleukin-8 were not affected. Using a candidate approach, we could recapitulate WSP toxicity by the combined actions of its constituents soot, metals and PAHs. The soot fraction and metals were found to be the most important factors for ROS formation, whereas the PAH response can be mimicked by the model PAH benzo[a]pyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than B[a]P individually applied in suspension. As PAHs initiate multiple adverse outcome pathways and are prominent carcinogens, their role as key pollutants in wood smoke and its health effects warrants further investigation. The presented results suggest that each of the investigated constituents soot, metals and PAHs are major contributors to WSP toxicity. Mitigation strategies to prevent adverse health effects of wood combustion should therefore not only aim at reducing the emitted soot and PAHs but also the metal content, through the use of more efficient combustion appliances, and particle precipitation techniques, respectively.

Sustainability of CD24 expression, cell proliferation and migration, cisplatin-resistance, and caspase-3 expression during mesenchymal-epithelial transition induced by the removal of TGF-β1 in A549 lung cancer cells.

PubMed

Kim, Seong-Kwan; Park, Jin-A; Zhang, Dan; Cho, Sang-Hyun; Yi, Hee; Cho, Soo-Min; Chang, Byung-Joon; Kim, Jin-Suk; Shim, Jae-Han; Abd El-Aty, A M; Shin, Ho-Chul

2017-08-01

Epithelial-mesenchymal transition (EMT) is a notable mechanism underlying cancer cell metastasis. Transforming growth factor β1 (TGF-β1) has been used to induce EMT; however, there is a lack of information regarding the role of TGF-β1 in mesenchymal-epithelial transition (MET). In the present study, EMT was induced in A549 lung cancer cells using TGF-β1 (TGF-β1-treated group) and MET was induced sequentially from the TGF-β1-treated group by removing the TGF-β1 (MET/return group). Untreated A549 lung cancer cells were used as a control. Characteristic features, including cancer stem cell markers [cluster of differentiation (CD)24, CD44 and CD133], cell proliferation and migration and diverse intracellular mechanisms, were observed in all groups. Using western blot analysis, the TGF-β1-treated group demonstrated increased vimentin and reduced E-cadherin expression, whereas the MET/return group demonstrated the opposite trend. Among cancer stem cell markers, the population of CD24 low cells was reduced in the TGF-β1-treated group. Furthermore, the G2/M phase cell cycle population, cisplatin-sensitivity, and cell proliferation and migration ability were increased in the TGF-β1-treated group. These features were unaltered in the MET/return group when compared to the TGF-β1-treated group. Immunoblotting revealed an increase in the levels of SMAD3, phosphorylated SMAD3, phosphorylated extracellular signal-regulated kinase and caspase-3, and a decrease in active caspase-3 levels in the TGF-β1-treated group. Increased caspase-3 and reduced active caspase-3 levels were observed in the MET/return group, similar to those in the TGF-β1-treated group; however, levels of other signalling proteins were unchanged compared with the control group. EMT induced by TGF-β1 was not preserved; however, stemness-associated properties (CD24 expression, caspase-3 expression, cell proliferation and cisplatin-resistance) were sustained following removal of TGF-β1.

Nicotine transport in lung and non-lung epithelial cells.

PubMed

Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

2017-11-01

Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [ 3 H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus.

PubMed

Chang, Hong-Bin; Chen, Bing-Huei

2015-01-01

The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.

Emodin suppresses TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells through Notch signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Rundi; Chen, Ruilin; Cao, Yu

Pulmonary fibrosis is characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicament on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been established yet. In the present study, we investigated the hypothesis that Emodin plays an inhibitory role in TGF-β1 induced epithelial-mesenchymal transition (EMT) of alveolar epithelial cell, and Emodin exerts its effect through the Notch signaling pathway. Emodin inhibits the proliferation of Rat alveolar type II epithelial cells RLE-6TN inmore » a concentration-dependent manner; reduces the expression of Collagen I, α-SMA and Vimentin, promotes the expression of E-cadherin. Moreover, Emodin could regulate the expression patterns of the Notch signaling pathway-related factors and reduce the Notch-1 nucleus translocation. Knockdown of Notch-1 enhances the inhibitory effect of Emodin on TGF-β1-induced EMT in RLE-6TN cells. In conclusion, the data of the present study suggests that Emodin suppresses TGF-β1-induced EMT in alveolar epithelial cells through Notch signaling pathway and shows the potential to be effective in the treatment of pulmonary fibrosis. - Highlights: • Emodin inhibits TGF-β1-induced EMT in alveolar epithelial cells. • Emodin regulates the expression patterns of the Notch signaling pathway-related factors. • Emodin inhibits TGF-β1-induced Notch-1 nucleus translocation and activation.« less

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

PubMed

Thomas, Richard J; Brooks, Tim J

2004-02-01

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

Enhancement of recombinant myricetin on the radiosensitivity of lung cancer A549 and H1299 cells

PubMed Central

2014-01-01

Objective Myricetin, a common dietary flavonoid is widely distributed in fruits and vegetables, and is used as a health food supplement based on its immune function, anti-oxidation, anti-tumor, and anti-inflammatory properties. The aim of this study was to investigate the effects of myricetin on combination with radiotherapy enhance radiosensitivity of lung cancer A549 and H1299 cells. Methods A549 cells and H1299 cells were exposed to X-ray with or without myricetin treatment. Colony formation assays, CCK-8 assay, flow cytometry and Caspase-3 level detection were used to evaluate the radiosensitization activity of myricetin on cell proliferation and apoptosis in vitro. Nude mouse tumor xenograft model was built to assessed radiosensitization effect of myricetin in vivo. Results Compared with the exposed group without myricetin treatment, the groups treated with myricetin showed significantly suppressed cell surviving fraction and proliferation, increased the cell apoptosis and increased Caspase-3 protein expression after X-ray exposure in vitro. And in vivo assay, growth speed of tumor xenografts was significantly decreased in irradiated mice treated with myricetin. Conclusions The study demonstrated both in vitro and in vivo evidence that combination of myricetin with radiotherapy can enhance tumor radiosensitivity of pulmonary carcinoma A549 and H1299 cells, and myricetin could be a potential radiosensitizer for lung cancer therapy. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5791518001210633 PMID:24650056

Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus

PubMed Central

Chang, Hong-Bin; Chen, Bing-Huei

2015-01-01

The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells. PMID:26345201

28 CFR 549.71 - Inmates affected.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Inmates affected. 549.71 Section 549.71 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.71 Inmates affected. This subpart applies to: (a) Any individual...

28 CFR 549.71 - Inmates affected.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Inmates affected. 549.71 Section 549.71 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.71 Inmates affected. This subpart applies to: (a) Any individual...

28 CFR 549.71 - Inmates affected.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Inmates affected. 549.71 Section 549.71 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.71 Inmates affected. This subpart applies to: (a) Any individual...

28 CFR 549.71 - Inmates affected.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Inmates affected. 549.71 Section 549.71 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.71 Inmates affected. This subpart applies to: (a) Any individual...

28 CFR 549.71 - Inmates affected.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Inmates affected. 549.71 Section 549.71 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.71 Inmates affected. This subpart applies to: (a) Any individual...

25 CFR 700.549 - Employee organizations.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 25 Indians 2 2010-04-01 2010-04-01 false Employee organizations. 700.549 Section 700.549 Indians... Employee Responsibility and Conduct § 700.549 Employee organizations. An employee may not knowingly be a member of an organization of Government employees that advocates the overthrow of the United States...

Sequential assessment of pulmonary epithelial diethylene triamine penta-acetate clearance and intrapulmonary transferrin accumulation during Escherichia coli peritonitis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishizaka, A.; Stephens, K.E.; Segall, G.M.

1990-03-01

The individual roles of pulmonary capillary endothelial and alveolar epithelial permeability in the pathogenesis of the adult respiratory distress syndrome (ARDS) are unclear. We developed a method for the sequential assessment of pulmonary macromolecule accumulation and small solute clearance in vivo using a gamma camera. We measured the exponential clearance coefficient of 111In-labeled diethylene triamine penta-acetate (111In-DTPA) to assess airway clearance of small solutes. We also calculated the exponential equilibration coefficient of 111In-labeled transferrin (111In-TF) to assess intrapulmonary accumulation of transferrin. We determined these parameters in guinea pigs with Escherichia coli peritonitis and compared them with a saline-treated control group,more » oleic-acid-treated groups, and a group treated with low molecular weight dextran Ringer solution. The pulmonary DTPA clearance and the intrapulmonary transferrin accumulation were significantly increased in the peritonitis group (29.4 +/- 8.2 x 10(-3) min-1, p less than 0.02, and 15.1 +/- 3.1 x 10(-3) min-1, p less than 0.02) when compared with the control group (3.1 +/- 0.8 x 10(-3) min-1 and 4.5 +/- 0.5 x 10(-3) min-1). These changes developed within 5.5 h of the initial insult. Neither increased extravascular lung water nor elevated pulmonary artery and left atrial pressures were detected in the peritonitis group. The low molecular weight dextran Ringer group did not show a significant increase in the pulmonary DTPA clearance and the intrapulmonary transferrin accumulation.« less

Malnutrition causes a reduction in alveolar epithelial sodium and chloride transport which predisposes to death from lung injury.

PubMed

Eisenhut, Michael

2007-01-01

All forms of malnutrition have been associated with increased severity of pneumonia, an increased pneumonia associated mortality and an increased risk of pulmonary fluid overload. Malnutrition was found to be associated with increased sweat sodium and chloride concentrations. A reduction of systemic sodium and chloride transport reflected in sweat sodium and chloride levels has been linked to increased severity of pulmonary edema in children with septicemia. Malnutrition causes a reduction in alveolar epithelial sodium and chloride transport which predisposes to death from lung injury. SUPPORTING EVIDENCE FOR THE HYPOTHESIS: Malnutrition caused reduced pulmonary fluid clearance in the rat model. Amiloride insensitive pulmonary fluid clearance in malnourished rats was reduced. The reduction in fluid clearance was reversible by beta agonists which increases epithelial sodium and chloride transport. Reduction of alveolar ion and fluid transport capacity explains the predisposition to death from pulmonary edema associated with intravenous fluids and blood transfusions in inpatients with malnutrition. Reduced alveolar epithelial ion transport impairs absorption of intra-alveolar inflammatory exudate in pneumonia leading to a increased severity of respiratory compromise and increased mortality. MEANS TO TEST THE HYPOTHESIS: Nasal potential difference measurements could compare airway epithelial sodium and chloride transport in patients with and without malnutrition and malnutrition associated lung disease. Sweat sodium and chloride concentrations could be compared in patients with and without respiratory disease associated with malnutrition and correlated with the severity of respiratory compromise.

Budesonide suppresses pulmonary antibacterial host defense by down-regulating cathelicidin-related antimicrobial peptide in allergic inflammation mice and in lung epithelial cells

PubMed Central

2013-01-01

Background Glucocorticoids are widely regarded as the most effective treatment for asthma. However, the direct impact of glucocorticoids on the innate immune system and antibacterial host defense during asthma remain unclear. Understanding the mechanisms underlying this process is critical to the clinical application of glucocorticoids for asthma therapy. After sensitization and challenge with ovalbumin (OVA), BALB/c mice were treated with inhaled budesonide and infected with Pseudomonas aeruginosa (P. aeruginosa). The number of viable bacteria in enflamed lungs was evaluated, and levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum were measured. A lung epithelial cell line was pretreated with budesonide. Levels of cathelicidin-related antimicrobial peptide (CRAMP) were measured by immunohistochemistry and western blot analysis. Intracellular bacteria were observed in lung epithelial cells. Results Inhaled budesonide enhanced lung infection in allergic mice exposed to P. aeruginosa and increased the number of viable bacteria in lung tissue. Higher levels of IL-4 and lower levels of IFN-γ were observed in the serum. Budesonide decreased the expression of CRAMP, increased the number of internalized P. aeruginosa in OVA-challenged mice and in lung epithelial cell lines. These data indicate that inhaled budesonide can suppress pulmonary antibacterial host defense by down-regulating CRAMP in allergic inflammation mice and in cells in vitro. Conclusions Inhaled budesonide suppressed pulmonary antibacterial host defense in an asthmatic mouse model and in lung epithelium cells in vitro. This effect was dependent on the down-regulation of CRAMP. PMID:23387852

Type I interferon promotes alveolar epithelial type II cell survival during pulmonary Streptococcus pneumoniae infection and sterile lung injury in mice.

PubMed

Maier, Barbara B; Hladik, Anastasiya; Lakovits, Karin; Korosec, Ana; Martins, Rui; Kral, Julia B; Mesteri, Ildiko; Strobl, Birgit; Müller, Mathias; Kalinke, Ulrich; Merad, Miriam; Knapp, Sylvia

2016-09-01

Protecting the integrity of the lung epithelial barrier is essential to ensure respiration and proper oxygenation in patients suffering from various types of lung inflammation. Type I interferon (IFN-I) has been associated with pulmonary epithelial barrier function, however, the mechanisms and involved cell types remain unknown. We aimed to investigate the importance of IFN-I with respect to its epithelial barrier strengthening function to better understand immune-modulating effects in the lung with potential medical implications. Using a mouse model of pneumococcal pneumonia, we revealed that IFN-I selectively protects alveolar epithelial type II cells (AECII) from inflammation-induced cell death. Mechanistically, signaling via the IFN-I receptor on AECII is sufficient to promote AECII survival. The net effects of IFN-I are barrier protection, together with diminished tissue damage, inflammation, and bacterial loads. Importantly, we found that the protective role of IFN-I can also apply to sterile acute lung injury, in which loss of IFN-I signaling leads to a significant reduction in barrier function caused by AECII cell death. Our data suggest that IFN-I is an important mediator in lung inflammation that plays a protective role by antagonizing inflammation-associated cell obstruction, thereby strengthening the integrity of the epithelial barrier. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis

PubMed Central

Franz-Bacon, Karin; DiTirro, Danielle N.; Ly, Tai Wei; Bacon, Kevin B.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease. PMID:27144281

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis.

PubMed

Boehme, Stefen A; Franz-Bacon, Karin; DiTirro, Danielle N; Ly, Tai Wei; Bacon, Kevin B

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

Dexamethasone-(C21-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549).

PubMed

Coyne, Cody P; Narayanan, Lakshmi

2016-01-01

Corticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma), autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively "target" delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems. The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR)-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide)-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549) known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1. The dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide)-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin did not significantly modify the ex vivo antineoplastic cytotoxicity of dexamethasone against pulmonary adenocarcinoma at and between the standardized dexamethasone equivalent concentrations of 10(-9) M and 10(-5) M. Rapid increases in

Characterization of the 2009 Pandemic A/Beijing/501/2009 H1N1 Influenza Strain in Human Airway Epithelial Cells and Ferrets

PubMed Central

Xing, Li; Li, Zhiwei; Wang, Wei; Zhao, Yan; Yan, Yiwu; Gu, Hongjing; Liu, Xin; Zhao, Zhongpeng; Zhang, Shaogeng; Wang, Xiliang; Jiang, Chengyu

2012-01-01

Background A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood. Methodology/Principal Finding In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus in vitro and in vivo may have been mediated by different mechanisms. Conclusion/Significance Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed in vitro and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe. PMID:23049974

Nanomaterial induction of oxidative stress in lung epithelial cells and macrophages

NASA Astrophysics Data System (ADS)

Wang, Lin; Pal, Anoop K.; Isaacs, Jacqueline A.; Bello, Dhimiter; Carrier, Rebecca L.

2014-09-01

Oxidative stress in the lung epithelial A549 cells and macrophages J774A.1 due to contact with commercially important nanomaterials [i.e., nano-silver (nAg), nano-alumina (nAl2O3), single-wall carbon nanotubes (CNT), and nano-titanium oxide anatase (nTiO2)] was evaluated. Nanomaterial-induced intracellular oxidative stress was analyzed by both H2DCFDA fluorescein probe and GSH depletion, extracellular oxidative stress was assessed by H2HFF fluorescein probes, and the secretion of chemokine IL-8 by A549 cells due to elevation of cellular oxidative stress was also monitored, in order to provide a comprehensive in vitro study on nanomaterial-induced oxidative stress in lung. In addition, results from this study were also compared with an acellular "ferric reducing ability of serum" (FRAS) assay and a prokaryotic cell-based assay in evaluating oxidative damage caused by the same set of nanomaterials, for comparison purposes. In general, it was found that nanomaterial-induced oxidative stress is highly cell-type dependent. In A549 lung epithelial cells, nAg appeared to induce highest level of oxidative stress and cell death followed by CNT, nTiO2, and nAl2O3. Different biological oxidative damage (BOD) assays' (i.e., H2DCFA, GSH, and IL-8 release) results generally agreed with each other, and the same trends of nanomaterial-induced BOD were also observed in acellular FRAS and prokaryotic E. coli K12-based assay. In macrophage J774A.1 cells, nAl2O3 and nTiO2 appeared to induce highest levels of oxidative stress. These results suggest that epithelial and macrophage cell models may provide complimentary information when conducting cell-based assays to evaluate nanomaterial-induced oxidative damage in lung.

[Study on thaspine in inducing apoptosis of A549 cell].

PubMed

Zhang, Yan-min; He, Lang-chong

2007-04-01

To investigate the effect of thaspine on the cellular proliferation, apoptosis and cell cycle in A549 cell line. A549 cell was cultured with different concentrations of thaspine. Cellular proliferation was detected with MTT, apoptosis and cell cycle were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. Thaspine could inhibit the proliferation and induce apoptosis of A549 cell in a time-dose dependent manner. Cell cycle was significantly stopped at the S phase by thaspine with FCM technology. Under electronic microscope, the morphology of A549 cell showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body when the cell was treated with thaspine. Thaspine has the effects of anti-tumor and inducing apoptosis.

28 CFR 549.52 - Informed consent.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Informed consent. 549.52 Section 549.52 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Plastic Surgery § 549.52 Informed consent. Approved plastic surgery procedures may not be performed...

28 CFR 549.52 - Informed consent.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Informed consent. 549.52 Section 549.52 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Plastic Surgery § 549.52 Informed consent. Approved plastic surgery procedures may not be performed...

28 CFR 549.52 - Informed consent.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Informed consent. 549.52 Section 549.52 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Plastic Surgery § 549.52 Informed consent. Approved plastic surgery procedures may not be performed...

28 CFR 549.52 - Informed consent.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Informed consent. 549.52 Section 549.52 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Plastic Surgery § 549.52 Informed consent. Approved plastic surgery procedures may not be performed...

28 CFR 549.52 - Informed consent.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Informed consent. 549.52 Section 549.52 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Plastic Surgery § 549.52 Informed consent. Approved plastic surgery procedures may not be performed...

Lung function declines in patients with pulmonary sarcoidosis and increased respiratory epithelial permeability to sup 99m Tc-DTPA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chinet, T.; Dusser, D.; Labrune, S.

1990-02-01

Respiratory epithelial clearance of {sup 99m}Tc-DTPA (RC-Tc-DTPA) and pulmonary function tests (PFT) were determined at intervals of 6 or 12 months in 37 untreated, nonsmoking patients with sarcoidosis over a period of 6 to 36 months. PFT included the measurements of total lung capacity (TLC), vital capacity (VC), FEV1, and diffusing capacity for carbon monoxide. No difference was found between the respiratory clearance of {sup 113m}In-DTPA (2.25 +/- 1.00%/min) and RC-Tc-DTPA (2.29 +/- 1.11%/min) in eight patients with pulmonary sarcoidosis. Pulmonary function decreased 15% or more in at least 2 function tests during 11 follow-up periods, but it remained stablemore » during 47 follow-up periods. In patients whose lung function deteriorated, RC-Tc-DTPA increased to 3.51 +/- 1.55%/min; in contrast, in patients whose lung function remained stable, regardless of the initial values, RC-Tc-DTPA was normal (1.00 +/- 0.50%/min; p less than 0.001). In eight patients who were treated with corticosteroids, RC-Tc-DTPA decreased from 3.48 +/- 1.31%/min to 1.56 +/- 0.64%/min (p less than 0.001), and PFT improved. We conclude that in nonsmokers with pulmonary sarcoidosis, increased RC-Tc-DTPA is not related to dissociation of 99mTc from DTPA, RC-Tc-DTPA is increased when pulmonary function decreases, and, when increased, RC-Tc-DTPA decreases with corticosteroid therapy.« less

[Houttuynia Cordata induces expression of human beta-defensin-2 mRNA in pulmonary epithelial cells in vitro].

PubMed

Luo, Li; Dong, Bi-rong; Teng, Li-hua

2008-07-01

To explore the effects of Houttuynia Cordata on expression of human beta-defensin-2 (HBD-2) in pulmonary epithelial cells (SPC-A-1) in vitro; and to observe the correlationship between the level of HBD-2 mRNA and the concentrations or treatment times of Houttuynia Cordata. The SPC-A-1 cells were cultured with different concentrations of Houttuynia Cordata in vitro, including 0, 12.5, 25, 50, 100 and 200 microg/ml. And then, the SPC-A-1 cells were cultured with the optimal concentration of Houttuynia Cordata in different lengths of time, including 1, 2, 4, 8, 16 and 24 hours. After the treatment, the mRNA level of HBD-2 in pulmonary epithelial cells was detected by means of semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). After being cultured with Houttuynia Cordata, the expression of HBD-2 mRNA had positive correlation with the stimulus concentrations (rs=0.829, P=0.042) and stimulus time (rs=0.914, P=0.003). The highest expression of HBD-2 mRNA was induced by 100 microg/ml Houttuynia Cordata after 8-hour treatment. In comparison with the normal control group and the interleukin-1beta group, 100 microg/ml Houttuynia Cordata could significantly up-regulate the expression of HBD-2 mRNA in SPC-A-1 cells after 8-hour treatment (P<0.01). Houttuynia Cordata can up-regulate expression of HBD-2 mRNA in SPC-A-1 cells, and the highest expression level of HBD-2 mRNA can be obtained by culture with 100 microg/ml Houttuynia Cordata for 8 hours.

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

PubMed Central

Cooper, James Ross; Abdullatif, Muhammad Bilal; Burnett, Edward C.; Kempsell, Karen E.; Conforti, Franco; Tolley, Howard; Collins, Jane E.; Davies, Donna E.

2016-01-01

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. PMID:27792742

Effect of pulmonary surfactant on the dissolution, stability and uptake of zinc oxide nanowires by human respiratory epithelial cells

PubMed Central

Theodorou, Ioannis G.; Ruenraroengsak, Pakatip; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng (Jim); Chung, Kian Fan; Tetley, Teresa D.; Ryan, Mary P.; Porter, Alexandra E.

2017-01-01

Inhaled nanoparticles have high deposition rates in the alveolar region of the lung but the effects of pulmonary surfactant (PS) on nanoparticle bioreactivity are unclear. Here, the impact of PS on the stability and dissolution of ZnO nanowires (ZnONWs) was investigated, and linked with their bioreactivity in vitro with human alveolar epithelial type 1-like cells (TT1). Pre-incubation of ZnONWs with Curosurf® (a natural porcine PS) decreased their dissolution at acidic pH, through the formation of a phospholipid corona. Confocal live cell microscopy confirmed that Curosurf® lowered intracellular dissolution, thus delaying the onset of cell death compared to bare ZnONWs. Despite reducing dissolution, Curosurf® significantly increased the uptake of ZnONWs within TT1 cells, ultimately increasing their toxicity after 24h. Although serum, improved ZnONW dispersion in suspension similar to Curosurf®, it had no effect on ZnONW internalization and toxicity, indicating a unique role of PS in promoting particle uptake. In the absence of PS, ZnONW length had no effect on dissolution kinetics or degree of cellular toxicity, indicating a less important role of length in determining ZnONW bioreactivity. This work provides unique findings on the effects of PS on the stability and toxicity of ZnONWs, which could be important in the study of pulmonary toxicity and epithelial-endothelial translocation of nanoparticles in general. PMID:27441789

Regulation of Proinflammatory Cytokines in Human Lung Epithelial Cells Infected with Mycoplasma pneumoniae

PubMed Central

Yang, Jun; Hooper, W. Craig; Phillips, Donald J.; Talkington, Deborah F.

2002-01-01

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1β mRNA also increased after infection and IL-1β protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level. PMID:12065506

Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells.

PubMed

Molina-Molina, M; Machahua-Huamani, C; Vicens-Zygmunt, V; Llatjós, R; Escobar, I; Sala-Llinas, E; Luburich-Hernaiz, P; Dorca, J; Montes-Worboys, A

2018-04-27

Pirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. Rapamycin, an inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone. Primary lung fibroblasts from IPF patients and human alveolar epithelial cells (A549) were treated in vitro with pirfenidone and rapamycin in the presence or absence of transforming growth factor β1 (TGF-β). Extracellular matrix protein and gene expression of markers involved in lung fibrosis (tenascin-c, fibronectin, collagen I [COL1A1], collagen III [COL3A1] and α-smooth muscle actin [α-SMA]) were analyzed. A cell migration assay in pirfenidone, rapamycin and TGF-β-containing media was performed. Gene and protein expression of tenascin-c and fibronectin of fibrotic fibroblasts were reduced by pirfenidone or rapamycin treatment. Pirfenidone-rapamycin treatment did not revert the epithelial to mesenchymal transition pathway activated by TGF-β. However, the drug combination significantly abrogated fibroblast to myofibroblast transition. The inhibitory effect of pirfenidone on fibroblast migration in the scratch-wound assay was potentiated by rapamycin combination. These findings indicate that the combination of pirfenidone and rapamycin widen the inhibition range of fibrogenic markers and prevents fibroblast migration. These results would open a new line of research for an anti-fibrotic combination therapeutic approach.

Effect of age on pulmonary epithelial permeability in unanesthetized lambs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchison, A.A.; McNicol, K.J.; Loughlin, G.M.

1985-01-01

Pulmonary epithelial permeability was measured 1) in unanesthetized sheep, and 2) longitudinally in growing lambs. Awake sheep were intubated and a solution of /sup 51/Cr-ethylenediaminetetraacetic acid and /sup 125/I-antipyrine was instilled in the intrathoracic trachea via the nasotracheal tube. Arterial blood was drawn 1-25 minutes after the instillation. The ratios of the counts of /sup 51/Cr to /sup 125/I at 7, 10, and 13 min were calculated and averaged for each animal. Data from six adult sheep showed that the mean +/- SE of the permeability ratio was 0.012 +/- 0.003 and was reproducible over three months. When measured twicemore » within two hours, the second ratio was significantly higher than the first. One hour of general anesthesia with methoxyflurane did not alter the permeability ratio significantly. Ten lambs were studied longitudinally 10 hours and 5, 10, 20, and 30 days after delivery. Within the first 24 hours of life the permeability ratio was, significantly greater than the adult value. At five days there was no significant difference between lambs and adult sheep. Throughout the first month of life, the permeability ratio in lambs remained at at the adult level.« less

Protein S is protective in pulmonary fibrosis.

PubMed

Urawa, M; Kobayashi, T; D'Alessandro-Gabazza, C N; Fujimoto, H; Toda, M; Roeen, Z; Hinneh, J A; Yasuma, T; Takei, Y; Taguchi, O; Gabazza, E C

2016-08-01

Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis. Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar

WNTLESS IS REQUIRED FOR PERIPHERAL LUNG DIFFERENTIATION AND PULMONARY VASCULAR DEVELOPMENT

PubMed Central

Cornett, Bridget; Snowball, John; Varisco, Brian M.; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-01-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. PMID:23523683

Cytotoxicity and gene expression profiling of polyhexamethylene guanidine hydrochloride in human alveolar A549 cells.

PubMed

Jung, Ha-Na; Zerin, Tamanna; Podder, Biswajit; Song, Ho-Yeon; Kim, Yong-Sik

2014-06-01

In Korea, lung disease of children and pregnant women associated with humidifier disinfectant use has become a major concern. A common sterilizer is polyhexamethylene guanidine (PHMG), a member of the guanidine family of antiseptics. This study was done to elucidate the putative cytotoxic effect of PHMG and the PHMG-mediated altered gene expression in human alveolar epithelial A549 cells in vitro. Cell viability analyses revealed the potent cytotoxicity of PHMG, with cell death evident at as low as 5 μg/mL. Death was dose- and time-dependent, and was associated with formation of intracellular reactive oxygen species, and apoptosis significantly, at even 2 μg/mL concentration. The gene expression profile in A549 cells following 24 h exposure to 5 μg/mL of PHMG was investigated using DNA microarray analysis. Changes in gene expression relevant to the progression of cell death included induction of genes related to apoptosis, autophagy, fibrosis, and cell cycle. However, the expressions of genes encoding antioxidant and detoxifying enzymes were down-regulated or not affected. The altered expression of selected genes was confirmed by quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The collective data suggest that PHMG confers cellular toxicity through the generation of intracellular reactive oxygen species and alteration of gene expression. Copyright © 2014 Elsevier Ltd. All rights reserved.

β2 adrenergic agonist suppresses eosinophil-induced epithelial-to-mesenchymal transition of bronchial epithelial cells.

PubMed

Kainuma, Keigo; Kobayashi, Tetsu; D'Alessandro-Gabazza, Corina N; Toda, Masaaki; Yasuma, Taro; Nishihama, Kota; Fujimoto, Hajime; Kuwabara, Yu; Hosoki, Koa; Nagao, Mizuho; Fujisawa, Takao; Gabazza, Esteban C

2017-05-02

Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils. Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line. Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol. Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

Copper doping enhanced the oxidative stress-mediated cytotoxicity of TiO2 nanoparticles in A549 cells.

PubMed

Ahmad, J; Siddiqui, M A; Akhtar, M J; Alhadlaq, H A; Alshamsan, A; Khan, S T; Wahab, R; Al-Khedhairy, A A; Al-Salim, A; Musarrat, J; Saquib, Q; Fareed, M; Ahamed, M

2018-05-01

Physicochemical properties of titanium dioxide nanoparticles (TiO 2 NPs) can be tuned by doping with metals or nonmetals. Copper (Cu) doping improved the photocatalytic behavior of TiO 2 NPs that can be applied in various fields such as environmental remediation and nanomedicine. However, interaction of Cu-doped TiO 2 NPs with human cells is scarce. This study was designed to explore the role of Cu doping in cytotoxic response of TiO 2 NPs in human lung epithelial (A549) cells. Characterization data demonstrated the presence of both TiO 2 and Cu in Cu-doped TiO 2 NPs with high-quality lattice fringes without any distortion. The size of Cu-doped TiO 2 NPs (24 nm) was lower than pure TiO 2 NPs (30 nm). Biological results showed that both pure and Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in a dose-dependent manner. Low mitochondrial membrane potential and higher caspase-3 enzyme (apoptotic markers) activity were also observed in A549 cells exposed to pure and Cu-doped TiO 2 NPs. We further observed that cytotoxicity caused by Cu-doped TiO 2 NPs was higher than pure TiO 2 NPs. Moreover, antioxidant N-acetyl cysteine effectively prevented the reactive oxygen species generation, glutathione depletion, and cell viability reduction caused by Cu-doped TiO 2 NPs. This is the first report showing that Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in A549 cells. This study warranted further research to explore the role of Cu doping in toxicity mechanisms of TiO 2 NPs.

Wntless is required for peripheral lung differentiation and pulmonary vascular development.

PubMed

Cornett, Bridget; Snowball, John; Varisco, Brian M; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-07-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Dexamethasone-(C21-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549)

PubMed Central

Coyne, Cody P; Narayanan, Lakshmi

2016-01-01

Purpose Corticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma), autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively “target” delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems. Materials and methods The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR)-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide)-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549) known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1. Results The dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide)-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin did not significantly modify the ex vivo antineoplastic cytotoxicity of dexamethasone against pulmonary adenocarcinoma at and between the standardized dexamethasone equivalent concentrations of 10�

Myb permits multilineage airway epithelial cell differentiation

PubMed Central

Pan, Jie-hong; Adair-Kirk, Tracy L.; Patel, Anand C.; Huang, Tao; Yozamp, Nicholas S.; Xu, Jian; Reddy, E. Premkumar; Byers, Derek E.; Pierce, Richard A.; Holtzman, Michael J.; Brody, Steven L.

2014-01-01

The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63+ basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps is poorly defined. Here we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb+ cells were identified as p63− and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63− population with failed maturation of Foxj1+ ciliated cells, as well as Scbg1a1+ and Muc5ac+ secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb+ cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63− Myb+ population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease. PMID:25103188

Murine epithelial cells: isolation and culture.

PubMed

Davidson, Donald J; Gray, Michael A; Kilanowski, Fiona M; Tarran, Robert; Randell, Scott H; Sheppard, David N; Argent, Barry E; Dorin, Julia R

2004-08-01

We describe an air-liquid interface primary culture method for murine tracheal epithelial cells on semi-permeable membranes, forming polarized epithelia with a high transepithelial resistance, differentiation to ciliated and secretory cells, and physiologically appropriate expression of key genes and ion channels. We also describe the isolation of primary murine nasal epithelial cells for patch-clamp analysis, generating polarised cells with physiologically appropriate distribution and ion channel expression. These methods enable more physiologically relevant analysis of murine airway epithelial cells in vitro and ex vivo, better utilisation of transgenic mouse models of human pulmonary diseases, and have been approved by the European Working Group on CFTR expression.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

PubMed

Flood, P; Alvarez, L; Reynaud, E G

2016-10-11

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

40 CFR 272.502-272.549 - [Reserved

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 27 2014-07-01 2014-07-01 false [Reserved] 272.502-272.549 Section 272.502-272.549 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED STATE HAZARDOUS WASTE MANAGEMENT PROGRAMS Florida §§ 272.502-272.549 [Reserved] ...

40 CFR 272.502-272.549 - [Reserved

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 27 2011-07-01 2011-07-01 false [Reserved] 272.502-272.549 Section 272.502-272.549 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED STATE HAZARDOUS WASTE MANAGEMENT PROGRAMS Florida §§ 272.502-272.549 [Reserved] ...

40 CFR 272.502-272.549 - [Reserved

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 28 2013-07-01 2013-07-01 false [Reserved] 272.502-272.549 Section 272.502-272.549 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED STATE HAZARDOUS WASTE MANAGEMENT PROGRAMS Florida §§ 272.502-272.549 [Reserved] ...

40 CFR 272.502-272.549 - [Reserved

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 28 2012-07-01 2012-07-01 false [Reserved] 272.502-272.549 Section 272.502-272.549 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED STATE HAZARDOUS WASTE MANAGEMENT PROGRAMS Florida §§ 272.502-272.549 [Reserved] ...

40 CFR 272.502-272.549 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 26 2010-07-01 2010-07-01 false [Reserved] 272.502-272.549 Section 272.502-272.549 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES (CONTINUED) APPROVED STATE HAZARDOUS WASTE MANAGEMENT PROGRAMS Florida §§ 272.502-272.549 [Reserved] ...

Perfluorocarbon reduces cell damage from blast injury by inhibiting signal paths of NF-κB, MAPK and Bcl-2/Bax signaling pathway in A549 cells

PubMed Central

Li, Huaidong; Li, Chunsun; Yang, Zhen; Li, Yanqin; She, Danyang; Cao, Lu; Wang, Wenjie; Liu, Changlin; Chen, Liangan

2017-01-01

Background and objective Blast lung injury is a common type of blast injury and has very high mortality. Therefore, research to identify medical therapies for blast injury is important. Perfluorocarbon (PFC) is used to improve gas exchange in diseased lungs and has anti-inflammatory functions in vitro and in vivo. The aim of this study was to determine whether PFC reduces damage to A549 cells caused by blast injury and to elucidate its possible mechanisms of action. Study design and methods A549 alveolar epithelial cells exposed to blast waves were treated with and without PFC. Morphological changes and apoptosis of A549 cells were recorded. PCR and enzyme-linked immunosorbent assay (ELISA) were used to measure the mRNA or protein levels of IL-1β, IL-6 and TNF-α. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity levels were detected. Western blot was used to quantify the expression of NF-κB, Bax, Bcl-2, cleaved caspase-3 and MAPK cell signaling proteins. Results A549 cells exposed to blast wave shrank, with less cell-cell contact. The morphological change of A549 cells exposed to blast waves were alleviated by PFC. PFC significantly inhibited the apoptosis of A549 cells exposed to blast waves. IL-1β, IL-6 and TNF-α cytokine and mRNA expression levels were significantly inhibited by PFC. PFC significantly increased MDA levels and decreased SOD activity levels. Further studies indicated that NF-κB, Bax, caspase-3, phospho-p38, phosphor-ERK and phosphor-JNK proteins were also suppressed by PFC. The quantity of Bcl-2 protein was increased by PFC. Conclusion Our research showed that PFC reduced A549 cell damage caused by blast injury. The potential mechanism may be associated with the following signaling pathways: 1) the signaling pathways of NF-κB and MAPK, which inhibit inflammation and reactive oxygen species (ROS); and 2) the signaling pathways of Bcl-2/Bax and caspase-3, which inhibit apoptosis. PMID:28323898

COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

EPA Science Inventory

Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

28 CFR 549.10 - Purpose and scope.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Purpose and scope. 549.10 Section 549.10 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Infectious Disease Management § 549.10 Purpose and scope. The Bureau will manage infectious diseases in the...

28 CFR 549.10 - Purpose and scope.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Purpose and scope. 549.10 Section 549.10 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Infectious Disease Management § 549.10 Purpose and scope. The Bureau will manage infectious diseases in the...

28 CFR 549.10 - Purpose and scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Purpose and scope. 549.10 Section 549.10 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Infectious Disease Management § 549.10 Purpose and scope. The Bureau will manage infectious diseases in the...

28 CFR 549.10 - Purpose and scope.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Purpose and scope. 549.10 Section 549.10 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Infectious Disease Management § 549.10 Purpose and scope. The Bureau will manage infectious diseases in the...

Foxm1 transcription factor is required for lung fibrosis and epithelial-to-mesenchymal transition

PubMed Central

Balli, David; Ustiyan, Vladimir; Zhang, Yufang; Wang, I-Ching; Masino, Alex J; Ren, Xiaomeng; Whitsett, Jeffrey A; Kalinichenko, Vladimir V; Kalin, Tanya V

2013-01-01

Alveolar epithelial cells (AECs) participate in the pathogenesis of pulmonary fibrosis, producing pro-inflammatory mediators and undergoing epithelial-to-mesenchymal transition (EMT). Herein, we demonstrated the critical role of Forkhead Box M1 (Foxm1) transcription factor in radiation-induced pulmonary fibrosis. Foxm1 was induced in AECs following lung irradiation. Transgenic expression of an activated Foxm1 transcript in AECs enhanced radiation-induced pneumonitis and pulmonary fibrosis, and increased the expression of IL-1β, Ccl2, Cxcl5, Snail1, Zeb1, Zeb2 and Foxf1. Conditional deletion of Foxm1 from respiratory epithelial cells decreased radiation-induced pulmonary fibrosis and prevented the increase in EMT-associated gene expression. siRNA-mediated inhibition of Foxm1 prevented TGF-β-induced EMT in vitro. Foxm1 bound to and increased promoter activity of the Snail1 gene, a critical transcriptional regulator of EMT. Expression of Snail1 restored TGF-β-induced loss of E-cadherin in Foxm1-deficient cells in vitro. Lineage-tracing studies demonstrated that Foxm1 increased EMT during radiation-induced pulmonary fibrosis in vivo. Foxm1 is required for radiation-induced pulmonary fibrosis by enhancing the expression of genes critical for lung inflammation and EMT. PMID:23288041

28 CFR 549.65 - Refusal to accept treatment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Refusal to accept treatment. 549.65 Section 549.65 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Hunger Strikes, Inmate § 549.65 Refusal to accept treatment. (a) When, as a result of...

Cigarette smoke-induced alveolar epithelial-mesenchymal transition is mediated by Rac1 activation.

PubMed

Shen, Hui-juan; Sun, Yan-hong; Zhang, Shui-juan; Jiang, Jun-xia; Dong, Xin-wei; Jia, Yong-liang; Shen, Jian; Guan, Yan; Zhang, Lin-hui; Li, Fen-fen; Lin, Xi-xi; Wu, Xi-mei; Xie, Qiang-min; Yan, Xiao-feng

2014-06-01

Epithelial-mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT. EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively. Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression. Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

9 CFR 590.549 - Dried egg storage.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Dried egg storage. 590.549 Section 590.549 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE EGG..., and Facility Requirements § 590.549 Dried egg storage. Dried egg storage shall be sufficient to...

9 CFR 590.549 - Dried egg storage.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Dried egg storage. 590.549 Section 590.549 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE EGG..., and Facility Requirements § 590.549 Dried egg storage. Dried egg storage shall be sufficient to...

9 CFR 590.549 - Dried egg storage.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Dried egg storage. 590.549 Section 590.549 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE EGG..., and Facility Requirements § 590.549 Dried egg storage. Dried egg storage shall be sufficient to...

28 CFR 549.64 - Food/liquid intake/output.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Food/liquid intake/output. 549.64 Section 549.64 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Hunger Strikes, Inmate § 549.64 Food/liquid intake/output. (a) Staff shall prepare and...

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells.

PubMed

Phung, Thuy Thi Bich; Sugamata, Ryuichi; Uno, Kazuko; Aratani, Yasuaki; Ozato, Keiko; Kawachi, Shoji; Thanh Nguyen, Liem; Nakayama, Toshinori; Suzuki, Kazuo

2011-12-01

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide.

PubMed

Morris, Christopher J; Smith, Mathew W; Griffiths, Peter C; McKeown, Neil B; Gumbleton, Mark

2011-04-10

With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (p<0.05) to the cumulative transport of less than 0.004% for control phage-clone groups. To characterise phage-independent activity of LTP-1 peptide, the LTP-1 peptide was conjugated to a 53kDa anionic PAMAM dendrimer. Compared to respective peptide-dendrimer control conjugates, the LTP-1-PAMAM conjugate displayed a two-fold (bioavailability up to 31%) greater extent of absorption in the IPRL. The LTP-1 peptide-mediated enhancement of transport, when LTP-1 was either attached to the phage clone or conjugated to dendrimer, was sequence-dependent and could be competitively inhibited by co-instillation of excess synthetic free LTP-1 peptide. The specific nature of the target receptor or mechanism involved in LTP-1 lung transport remains unclear although the enhanced transport is enabled through a mechanism that is non-disruptive with respect to the pulmonary transport of hydrophilic permeability probes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ. Copyright �

31 CFR 549.305 - Interest.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Interest. 549.305 Section 549.305... Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct or indirect. ...

31 CFR 549.305 - Interest.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Interest. 549.305 Section 549.305... Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct or indirect. ...

31 CFR 549.305 - Interest.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Interest. 549.305 Section 549.305... Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct or indirect. ...

31 CFR 549.305 - Interest.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Interest. 549.305 Section 549.305... Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct or indirect. ...

Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

PubMed

Dong, Wen-Wen; Zhang, Yun-Qian; Zhu, Xiao-Yan; Mao, Yan-Fei; Sun, Xue-Jun; Liu, Yu-Jian; Jiang, Lai

2017-05-19

BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

Pulmonary arterial remodeling induced by a Th2 immune response

PubMed Central

Daley, Eleen; Emson, Claire; Guignabert, Christophe; de Waal Malefyt, Rene; Louten, Jennifer; Kurup, Viswanath P.; Hogaboam, Cory; Taraseviciene-Stewart, Laimute; Voelkel, Norbert F.; Rabinovitch, Marlene; Grunig, Ekkehard; Grunig, Gabriele

2008-01-01

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4+ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule α, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH. PMID:18227220

Pirfenidone may revert the epithelial-to-mesenchymal transition in human lung adenocarcinoma.

PubMed

Kurimoto, Ryota; Ebata, Takahiro; Iwasawa, Shunichiro; Ishiwata, Tsukasa; Tada, Yuji; Tatsumi, Koichiro; Takiguchi, Yuichi

2017-07-01

The epithelial-to-mesenchymal transition (EMT) in cancer is associated with invasion, metastasis and chemoresistance. Recent studies have revealed the increased expression of programmed death-ligand 1 (PD-L1) in cells undergoing EMT. The underlying mechanism of EMT involves transforming growth factor-β (TGF-β) and fibroblast growth factor-2 (FGF-2). Pirfenidone and the known EMT-suppressor nintedanib suppress pulmonary fibrosis partially through suppression of TGF-β. The present study aimed to determine whether pirfenidone has the potential to induce EMT-reversion, using nintedanib as a reference. The human lung adenocarcinoma cell lines A-549, HCC-827, and PC-9 were treated with TGF-β and FGF-2 to induce EMT. The EMT-induced cells were further treated with pirfenidone or nintedanib. Phenotypic alterations associated with EMT were assessed by examining the following: i) The expression levels of E-cadherin, vimentin, fibronectin and slug, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescent immunohistochemistry; ii) cell motility via wound-healing assays; and iii) the expression of PD-L1 using RT-qPCR. The combination of TGF-β and FGF-2 successfully induced EMT in all three cell lines, characterized by a significant reduction in E-cadherin expression in the A-549 and HCC-827 cells, increased expression levels of vimentin, fibronectin, slug and PD-L1, and increased cell motility in all three cell lines. Pirfenidone and nintedanib reverted all of these phenotypes, with the exception of unaltered E-cadherin expression in all three cell lines, and inconsistent expression of vimentin in the HCC-827 and PC-9 cells. Thus, pirfenidone and nintedanib have the ability to induce EMT-reversion in human lung adenocarcinoma.

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line.

PubMed

Charão, Mariele F; Baierle, Marília; Gauer, Bruna; Goethel, Gabriela; Fracasso, Rafael; Paese, Karina; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Dias, Bruna B; Matte, Ursula S; Guterres, Silvia S; Pohlmann, Adriana R; Garcia, Solange C

2015-06-01

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ. Copyright © 2015 Elsevier B.V. All rights reserved.

Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells.

PubMed

Gong, Yuanqi; Lan, Haibing; Yu, Zhihong; Wang, Meng; Wang, Shu; Chen, Yu; Rao, Haiwei; Li, Jingying; Sheng, Zhiyong; Shao, Jianghua

2017-09-16

Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits beneficial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS. Copyright © 2017 Elsevier Inc. All rights reserved.

Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

NASA Astrophysics Data System (ADS)

Han, Jae Woong; Gurunathan, Sangiliyandi; Jeong, Jae-Kyo; Choi, Yun-Jung; Kwon, Deug-Nam; Park, Jin-Ki; Kim, Jin-Hoi

2014-09-01

The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate . The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles.

Oxidative stress mediated cytotoxicity of biologically synthesized silver nanoparticles in human lung epithelial adenocarcinoma cell line

PubMed Central

2014-01-01

The goal of the present study was to investigate the toxicity of biologically prepared small size of silver nanoparticles in human lung epithelial adenocarcinoma cells A549. Herein, we describe a facile method for the synthesis of silver nanoparticles by treating the supernatant from a culture of Escherichia coli with silver nitrate. The formation of silver nanoparticles was characterized using various analytical techniques. The results from UV-visible (UV-vis) spectroscopy and X-ray diffraction analysis show a characteristic strong resonance centered at 420 nm and a single crystalline nature, respectively. Fourier transform infrared spectroscopy confirmed the possible bio-molecules responsible for the reduction of silver from silver nitrate into nanoparticles. The particle size analyzer and transmission electron microscopy results suggest that silver nanoparticles are spherical in shape with an average diameter of 15 nm. The results derived from in vitro studies showed a concentration-dependent decrease in cell viability when A549 cells were exposed to silver nanoparticles. This decrease in cell viability corresponded to increased leakage of lactate dehydrogenase (LDH), increased intracellular reactive oxygen species generation (ROS), and decreased mitochondrial transmembrane potential (MTP). Furthermore, uptake and intracellular localization of silver nanoparticles were observed and were accompanied by accumulation of autophagosomes and autolysosomes in A549 cells. The results indicate that silver nanoparticles play a significant role in apoptosis. Interestingly, biologically synthesized silver nanoparticles showed more potent cytotoxicity at the concentrations tested compared to that shown by chemically synthesized silver nanoparticles. Therefore, our results demonstrated that human lung epithelial A549 cells could provide a valuable model to assess the cytotoxicity of silver nanoparticles. PMID:25242904

Edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia injury: heme oxygenase-1 and PI3K/Akt pathway may be involved.

PubMed

Cao, Huifang; Feng, Ying; Ning, Yunye; Zhang, Zinan; Li, Weihao; Li, Qiang

2015-01-01

Hyperoxic acute lung injury (HALI) is a clinical syndrome as a result of prolonged supplement of high concentrations of oxygen. As yet, no specific treatment is available for HALI. The present study aims to investigate the effects of edaravone on hyperoxia-induced oxidative injury and the underlying mechanism. We treated rats and human pulmonary alveolar epithelial cells with hyperoxia and different concentration of edaravone, then examined the effects of edaravone on cell viability, cell injury and two oxidative products. The roles of heme oxygenase-1 (HO-1) and PI3K/Akt pathway were explored using Western blot and corresponding inhibitors. The results showed that edaravone reduced lung biochemical alterations induced by hyperoxia and mortality of rats, dose-dependently alleviated cell mortality, cell injury, and peroxidation of cellular lipid and DNA oxidative damage. It upregulated cellular HO-1 expression and activity, which was reversed by PI3K/Akt pathway inhibition. The administration of zinc protoporphyrin-IX, a HO-1 inhibitor, and LY249002, a PI3K/Akt pathway inhibitor, abolished the protective effects of edaravone in cells. This study indicates that edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1, which is regulated by PI3K/Akt pathway.

31 CFR 549.310 - United States.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false United States. 549.310 Section 549.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... United States. The term United States means the United States, its territories and possessions, and all...

31 CFR 549.310 - United States.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false United States. 549.310 Section 549.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... United States. The term United States means the United States, its territories and possessions, and all...

31 CFR 549.310 - United States.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false United States. 549.310 Section 549.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... United States. The term United States means the United States, its territories and possessions, and all...

31 CFR 549.310 - United States.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false United States. 549.310 Section 549.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... United States. The term United States means the United States, its territories and possessions, and all...

Cellular senescence and autophagy in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

PubMed

Kuwano, Kazuyoshi; Araya, Jun; Hara, Hiromichi; Minagawa, Shunsuke; Takasaka, Naoki; Ito, Saburo; Kobayashi, Kenji; Nakayama, Katsutoshi

2016-11-01

Aging is associated with impairments in homeostasis. Although aging and senescence are not equivalent, the number of senescent cells increases with aging. Cellular senescence plays important roles in tissue repair or remodeling, as well as embryonic development. Autophagy is a process of lysosomal self-degradation that maintains a homeostatic balance between the synthesis, degradation, and recycling of cellular proteins. Autophagy diminishes with aging; additionally, accelerated aging can be attributed to reduced autophagy. Cellular senescence has been widely implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), a disease of accelerated lung aging, presumably by impairing cell repopulation and by aberrant cytokine secretion in the senescence-associated secretory phenotype. The possible participation of autophagy in the pathogenic sequence of COPD has been extensively explored. Although it has been reported that increased autophagy may induce epithelial cell death, an insufficient reserve of autophagy can induce cellular senescence in bronchial epithelial cells of COPD. Furthermore, advanced age is one of the most important risk factors for the development of idiopathic pulmonary fibrosis (IPF). Telomere shortening is found in blood leukocytes and alveolar epithelial cells from patients with IPF. Accelerated senescence of epithelial cells plays a role in IPF pathogenesis by perpetuating abnormal epithelial-mesenchymal interactions. Insufficient autophagy may be an underlying mechanism of accelerated epithelial cell senescence and myofibroblast differentiation in IPF. Herein, we review the molecular mechanisms of cellular senescence and autophagy and summarize the role of cellular senescence and autophagy in both COPD and IPF. Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium via the c-Src Pathway

PubMed Central

Lee, Heung-Man; Kang, Ju-Hyung; Shin, Jae-Min; Lee, Seoung-Ae

2017-01-01

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-β1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-β1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-β1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-β1 on migration of A549 cells and suppressed TGF-β1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-β1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-β1 in upper airway chronic inflammatory disease such as CRS. PMID:28804222

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-β1 in Airway Epithelium via the c-Src Pathway.

PubMed

Lee, Heung-Man; Kang, Ju-Hyung; Shin, Jae-Min; Lee, Seoung-Ae; Park, Il-Ho

2017-01-01

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF- β 1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF- β 1. We found that E-cadherin, vimentin, fibronectin, and α -SMA expression was increased in nasal polyps compared to inferior turbinates. TGF- β 1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α -SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF- β 1 on migration of A549 cells and suppressed TGF- β 1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF- β 1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF- β 1 in upper airway chronic inflammatory disease such as CRS.

Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.

1990-08-01

Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to expressmore » diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.« less

46 CFR 169.549 - Ring lifebuoys and water lights.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Ring lifebuoys and water lights. 169.549 Section 169.549 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Lifesaving and Firefighting Equipment Additional Lifesaving Equipment § 169.549 Ring lifebuoys and water lights. (a)(1) The minimum number of...

46 CFR 169.549 - Ring lifebuoys and water lights.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Ring lifebuoys and water lights. 169.549 Section 169.549 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Lifesaving and Firefighting Equipment Additional Lifesaving Equipment § 169.549 Ring lifebuoys and water lights. (a)(1) The minimum number of...

46 CFR 169.549 - Ring lifebuoys and water lights.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Ring lifebuoys and water lights. 169.549 Section 169.549 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Lifesaving and Firefighting Equipment Additional Lifesaving Equipment § 169.549 Ring lifebuoys and water lights. (a)(1) The minimum number of...

Pulmonary toxicity of a coal liquefaction distillate product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haschek, W.M.; Boling, M.E.; Guerin, M.R.

1979-01-01

The pulmonary toxicity of coal derived liquids are under study. Results on a sample from the ZnCl/sub 2/ hydrocracking process are presented as an example. We obtained data on its distribution and persistence in the lung as well as the tissue reaction elicited. We also examined individual chemical fractions to determine which was responsible for pulmonary toxicity. Fifty ..mu..l of the distillate or fraction was administered intratracheally to adult male SPF Fisher rats which were then killed at varying time-points, ranging from 1 to 60 days after administration. Enzyme analysis of pulmonary lavage fluid, /sup 3/H-thymidine incorporation into pulmonary DNA,more » autoradiography, light and fluorescence microscopy were performed. At necropsy the distillate, a black viscid fluid, could be seen within major airways in some lobes. Extensive consolidation and atelectasis or, less frequently, overinflation was present in affected lobes. Twenty-four hours after administration the distillate remained visible in large and small airways as well as in the adjacent parenchyma producing a suppurative necrotizing bronchiolitis and pneumonitis. Within 4 days a histiocytic-fibroblastic reaction to the distillate resulted in granulomatous lesions in the parenchyma, as well as polypoid hyperplasia and obliterating bronchiolitis in the severely damaged airways. Extensive epithelial changes consisting of goblet and columnar cell hyperplasia, as well as squamous metaplasia with focal dysplasia were present throughout the respiratory tract at this time. Similar lesions were produced by the ether-soluble acid fraction, while the mono- and di-aromatic fraction produced less severe lesions which were localized to the airways. After 60 days the product distillate, primarily localized within granulomas, still persisted within the lung as visualized by fluorescence microscopy. Mucoid bronchiolitis with scattered polypoid hyperplasia, as well as epithelial hyperplasia and metaplasia were

Stimulation of IL-8 release from epithelial cells by gas cooker PM10: a pilot study

PubMed Central

Dick, C; Dennekamp, M; Howarth, S; Cherrie, J; Seaton, A; Donaldson, K; Stone, V

2001-01-01

OBJECTIVE—To measure the effect of matter collected by a method that has a 50% efficiency for particles with an aerodynamic diameter of 10 µm (PM10), generated by gas and electric cooking, on A549 epithelial cells with and without nitrogen dioxide (NO2).
METHOD—Multiple indoor PM10 samples were collected on Teflon filters during the use of gas or electric cookers. Interleukin-8 (IL-8) concentrations were measured with a sandwich enzyme linked immunosorbent assay (ELISA) system.
RESULTS—Treatment of A549 cells with PM10 generated from gas cooking resulted in increased concentrations of IL-8 compared with untreated cells; particles from the electric cooker had no effect. NO2 did not alter the concentration of IL-8.
CONCLUSION—PM10 generated by gas cooking has the potential to cause proinflammatory effects in lung cells. This may have implications for susceptible people.


Keywords: indoor air pollution; PM10; interleukin-8 PMID:11171935

The Mitochondrial Cardiolipin Remodeling Enzyme Lysocardiolipin Acyltransferase Is a Novel Target in Pulmonary Fibrosis

PubMed Central

Huang, Long Shuang; Mathew, Biji; Zhao, Yutong; Noth, Imre; Reddy, Sekhar P.; Harijith, Anantha; Usatyuk, Peter V.; Berdyshev, Evgeny V.; Kaminski, Naftali; Zhou, Tong; Zhang, Wei; Zhang, Yanmin; Rehman, Jalees; Kotha, Sainath R.; Gurney, Travis O.; Parinandi, Narasimham L.; Lussier, Yves A.; Garcia, Joe G. N.

2014-01-01

Rationale: Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis. Objectives: To define a role for LYCAT in human and murine models of pulmonary fibrosis. Methods: We analyzed the correlation of LYCAT expression in peripheral blood mononuclear cells (PBMCs) with the outcomes of pulmonary functions and overall survival, and used the murine models to establish the role of LYCAT in fibrogenesis. We studied the LYCAT action on cardiolipin remodeling, mitochondrial reactive oxygen species generation, and apoptosis of alveolar epithelial cells under bleomycin challenge. Measurements and Main Results: LYCAT expression was significantly altered in PBMCs and lung tissues from patients with idiopathic pulmonary fibrosis (IPF), which was confirmed in two preclinical murine models of IPF, bleomycin- and radiation-induced pulmonary fibrosis. LYCAT mRNA expression in PBMCs directly and significantly correlated with carbon monoxide diffusion capacity, pulmonary function outcomes, and overall survival. In both bleomycin- and radiation-induced pulmonary fibrosis murine models, hLYCAT overexpression reduced several indices of lung fibrosis, whereas down-regulation of native LYCAT expression by siRNA accentuated fibrogenesis. In vitro studies demonstrated that LYCAT modulated bleomycin-induced cardiolipin remodeling, mitochondrial membrane potential, reactive oxygen species generation, and apoptosis of alveolar epithelial cells, potential mechanisms of LYCAT-mediated lung protection. Conclusions: This study is the first to identify modulation of LYCAT expression in fibrotic lungs and offers a novel therapeutic approach for ameliorating lung inflammation and pulmonary fibrosis. PMID

29 CFR 549.3 - Distinction between plan and trust.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 29 Labor 3 2010-07-01 2010-07-01 false Distinction between plan and trust. 549.3 Section 549.3... REQUIREMENTS OF A âBONA FIDE PROFIT-SHARING PLAN OR TRUSTâ § 549.3 Distinction between plan and trust. As used... profits; (b) Profit-sharing trust means any such program or arrangement as qualifies under this part which...

28 CFR 549.70 - Purpose and scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.70 Purpose and scope. (a) The Bureau of Prisons (Bureau) may, under... health care services. (b) Generally, if you are an inmate as described in § 549.71, you must pay a fee...

28 CFR 549.70 - Purpose and scope.

Code of Federal Regulations, 2012 CFR

2012-07-01

... Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.70 Purpose and scope. (a) The Bureau of Prisons (Bureau) may, under... health care services. (b) Generally, if you are an inmate as described in § 549.71, you must pay a fee...

28 CFR 549.70 - Purpose and scope.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.70 Purpose and scope. (a) The Bureau of Prisons (Bureau) may, under... health care services. (b) Generally, if you are an inmate as described in § 549.71, you must pay a fee...

28 CFR 549.70 - Purpose and scope.

Code of Federal Regulations, 2014 CFR

2014-07-01

... Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.70 Purpose and scope. (a) The Bureau of Prisons (Bureau) may, under... health care services. (b) Generally, if you are an inmate as described in § 549.71, you must pay a fee...

28 CFR 549.70 - Purpose and scope.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE INSTITUTIONAL MANAGEMENT MEDICAL SERVICES Fees for Health Care Services § 549.70 Purpose and scope. (a) The Bureau of Prisons (Bureau) may, under... health care services. (b) Generally, if you are an inmate as described in § 549.71, you must pay a fee...

Mesoporous carbon nanomaterials induced pulmonary surfactant inhibition, cytotoxicity, inflammation and lung fibrosis.

PubMed

Chen, Yunan; Yang, Yi; Xu, Bolong; Wang, Shunhao; Li, Bin; Ma, Juan; Gao, Jie; Zuo, Yi Y; Liu, Sijin

2017-12-01

Environmental exposure and health risk upon engineered nanomaterials are increasingly concerned. The family of mesoporous carbon nanomaterials (MCNs) is a rising star in nanotechnology for multidisciplinary research with versatile applications in electronics, energy and gas storage, and biomedicine. Meanwhile, there is mounting concern on their environmental health risks due to the growing production and usage of MCNs. The lung is the primary site for particle invasion under environmental exposure to nanomaterials. Here, we studied the comprehensive toxicological profile of MCNs in the lung under the scenario of moderate environmental exposure. It was found that at a low concentration of 10μg/mL MCNs induced biophysical inhibition of natural pulmonary surfactant. Moreover, MCNs at similar concentrations reduced viability of J774A.1 macrophages and lung epithelial A549 cells. Incubating with nature pulmonary surfactant effectively reduced the cytotoxicity of MCNs. Regarding the pro-inflammatory responses, MCNs activated macrophages in vitro, and stimulated lung inflammation in mice after inhalation exposure, associated with lung fibrosis. Moreover, we found that the size of MCNs played a significant role in regulating cytotoxicity and pro-inflammatory potential of this nanomaterial. In general, larger MCNs induced more pronounced cytotoxic and pro-inflammatory effects than their smaller counterparts. Our results provided valuable information on the toxicological profile and environmental health risks of MCNs, and suggested that fine-tuning the size of MCNs could be a practical precautionary design strategy to increase safety and biocompatibility of this nanomaterial. Copyright © 2017. Published by Elsevier B.V.

31 CFR 549.308 - Property; property interest.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Property; property interest. 549.308 Section 549.308 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... or interests therein, present, future, or contingent. ...

31 CFR 549.308 - Property; property interest.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Property; property interest. 549.308 Section 549.308 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... or interests therein, present, future, or contingent. ...

31 CFR 549.308 - Property; property interest.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Property; property interest. 549.308 Section 549.308 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... or interests therein, present, future, or contingent. ...

31 CFR 549.308 - Property; property interest.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Property; property interest. 549.308 Section 549.308 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... or interests therein, present, future, or contingent. ...

The distribution of immunomodulatory cells in the lungs of patients with idiopathic pulmonary fibrosis

PubMed Central

Nuovo, Gerard J.; Hagood, James S.; Magro, Cynthia M.; Chin, Nena; Kapil, Rubina; Davis, Luke; Marsh, Clay B.; Folcik, Virginia A.

2011-01-01

We have characterized the immune system involvement in the disease processes of idiopathic pulmonary fibrosis in novel ways. To do so, we analyzed lung tissue from 21 cases of idiopathic pulmonary fibrosis and 21 (non-fibrotic, non-cancerous) controls for immune cell and inflammation-related markers. The immunohistochemical analysis of the tissue was grouped by patterns of severity in disease pathology. There were significantly greater numbers of CD68+ and CD80+ cells, and significantly fewer CD3+, CD4+, and CD45RO+ cells in areas of relatively (histologically) normal lung in biopsies from idiopathic pulmonary fibrosis patients compared to controls. In zones of active disease, characterized by epithelial cell regeneration and fibrosis, there were significantly more cells expressing CD4, CD8, CD20, CD68, CD80, CCR6, S100, IL-17, tumor necrosis factor-α, and retinoic acid-related orphan receptors compared to histologically normal lung areas from idiopathic pulmonary fibrosis patients. Inflammation was implicated in these active regions by the cells that expressed retinoid orphan receptor-α, -β, and -γ, CCR6, and IL-17. The regenerating epithelial cells predominantly expressed these pro-inflammatory molecules, as evidenced by co-expression analyses with epithelial cytokeratins. Macrophages in pseudo-alveoli and CD3+ T cells in the fibrotic interstitium also expressed IL-17. Co-expression of IL-17 with retinoid orphan receptors, and epithelial cytoskeletal proteins, CD68, and CD3 in epithelial cells, macrophages, and T-cells, respectively, confirmed the production of IL-17 by these cell types. There was little staining for Foxp3, CD56, or CD34 in any idiopathic pulmonary fibrosis lung regions. The fibrotic regions had fewer immune cells overall. In summary, our study shows participation of innate and adaptive mononuclear cells in active-disease regions of idiopathic pulmonary fibrosis lung, where the regenerating epithelial cells appear to propagate inflammation

Budesonide Inhibits Intracellular Infection with Non-Typeable Haemophilus influenzae Despite Its Anti-Inflammatory Effects in Respiratory Cells and Human Lung Tissue: A Role for p38 MAP Kinase.

PubMed

Wagner, Christopher; Goldmann, Torsten; Rohmann, Kristina; Rupp, Jan; Marwitz, Sebastian; Rotta Detto Loria, Johannes; Limmer, Stefan; Zabel, Peter; Dalhoff, Klaus; Drömann, Daniel

2015-01-01

Inhaled corticosteroids (ICS) are widely used in the treatment of obstructive lung diseases. Recent data suggest a higher pneumonia risk in chronic obstructive pulmonary disease (COPD) patients treated with ICS. Since non-typeable Haemophilus influenzae (NTHi) is the most common pathogen associated with acute exacerbations of COPD, we investigated the effects of budesonide (BUD) on NTHi-induced inflammation and invasive infection. The alveolar epithelial cell line A549 and specimens of human lung tissue (HLT) were used in our experiments. Intracellular infection was determined by a lysis/culture assay of infected cells. Activated p38 mitogen-associated protein kinase (MAPK) was assessed using Western blotting and immunohistochemistry, expression of toll-like receptor 2 (TLR2) was determined by PCR, and CXCL-8 levels were measured using ELISA. Immunohistochemistry was used for detection of CXCL-8, platelet-activating factor receptor (PAF-R) and NTHi. BUD significantly reduced CXCL-8 secretion in A549 cells and lung tissue infected with NTHi. Furthermore, BUD decreased the expression of PAF-R in HLT and A549 cells. In A549 cells and HLT, BUD inhibited intracellular infection and - synergistically with NTHi - increased the expression of TLR2 (in A549 cells). TLR2 stimulation did not influence the intracellular infection of A549 cells, but p38 MAPK inhibition resulted in a significant reduction of infection. The present study adds new insights into the effects of glucocorticoids on pulmonary host defence after NTHi infection. Although the inflammatory response to infection is suppressed by BUD, interestingly, the intracellular infection is also inhibited. This effect seems to depend on the inhibition of p38 MAPK - a key enzyme in many pro-inflammatory pathways - as well as of PAF-R expression. © 2015 S. Karger AG, Basel.

Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Xu-Guang; Department of Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou; Ji, Tian-Xing

Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changesmore » in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar

[Apoptosis inducing effect of Hechanpian on human lung adenocarcinoma A549 cells].

PubMed

Xiong, Shao-Quan; Zhou, Dai-Han; Lin, Li-Zhu

2010-06-01

To study the apoptosis inducing effects of Hechanpian (HCP) on human lung adenocarcinoma A549 cells. HCP containing rat serum was prepared and applied on A549 cells. The cell growth inhibition rate was tested by MTT assay; the effect of HCP on cell apoptosis was observed with Propidium iodide (PI) staining and flow cytometry analysis; the mRNA expression of epidermal growth factor receptor (EGFR) was detected through RT-PCR. The growth of A549 cells was obviously inhibited after being treated by HCP containing serum, and the cells presented an apoptotic change. The cell apoptosis rate after treated by serum containing 10% and 20% HCP was 20.5% and 33.2%, respectively, significantly higher than that in the control (6.1% in cells didn't treated with HCP, P < 0.05). Compared with control, EGFR mRNA expression in HCP treated cells was significantly lower (P < 0.05). HCP has apoptosis inducing effect on A549 cell, and its molecular mechanism is probably correlated with the inhibition of EGFR gene transcription.

40 CFR 80.541-80.549 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

....541-80.549 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) REGULATION OF FUELS AND FUEL ADDITIVES Motor Vehicle Diesel Fuel; Nonroad, Locomotive, and Marine Diesel Fuel; and ECA Marine Fuel Geographic Phase-in Provisions §§ 80.541-80.549 [Reserved] Small Refiner...

SiRNA/DOX lodeded chitosan based nanoparticles: Development, Characterization and in vitro evaluation on A549 lung cancer cell line.

PubMed

Seifi-Najmi, M; Hajivalili, M; Safaralizadeh, R; Sadreddini, S; Esmaeili, S; Razavi, R; Ahmadi, M; Mikaeili, H; Baradaran, B; Shams-Asenjan, K; Yousefi, M

2016-09-30

High-mobility group AT-hook2 (HMGA2), involved in epithelial mesenchymal transition (EMT) process, has a pivotal role in lung cancer metastasis. Lung cancer therapy with HMGA2 suppressing small interfering RNA (siRNA) has been introduced recently while doxorubicin (DOX) has been used as a frequent cancer chemotherapy agent. Both reagents have been faced with obstacles in clinic which make them ineffective. NanoParticles (NPs) provided a platform for efficient co delivery of the anticancer drugs. The aim of this study was production and in vitro characterization of different pharmacological groups (siRNA, DOX or siRNA-DOX) of carboxymethyl dextran thrimethyl chitosan nanoparticles (CMDTMChiNPs) on cytotoxicity, gene expression, apoptosis and migration of metastatic lung cancer cell line (A-549). CMDTMChiNPs were synthesized and encapsulated with siRNA, DOX or siRNA-DOX. Then the effects of HMGA2 siRNA and DOX co delivery was assessed in A549 viability and target genes (HMGA2, Ecadherin, vimentin and MMP9) by MTT and real time PCR, respectively. In addition capability of apoptosis induction and anti-migratory features of formulated NPs were analyzed by flowcytometry and wound healing assays. SiRNA-DOX-CMDTM ChiNPs approximate size were 207±5 with poly dispersity index (PDI) and zeta potential of 0.4 and 16.3±0.3, respectively. NPs loaded with DOX and siRNA were the most efficient drug formulations in A549 cell cytotoxicity, altering of EMT markers, apoptosis induction and migration inhibition. Generally our results showed that co delivery of HMGA2 siRNA and DOX by novel designed CMDTMChiNPs is a new therapeutic approach with great potential efficiency for lung cancer treatment.

Fundamentals of pulmonary drug delivery.

PubMed

Groneberg, D A; Witt, C; Wagner, U; Chung, K F; Fischer, A

2003-04-01

Aerosol administration of peptide-based drugs plays an important role in the treatment of pulmonary and systemic diseases and the unique cellular properties of airway epithelium offers a great potential to deliver new compounds. As the relative contributions from the large airways to the alveolar space are important to the local and systemic availability, the sites and mechanism of uptake and transport of different target compounds have to be characterized. Among the different respiratory cells, the ciliated epithelial cells of the larger and smaller airways and the type I and type II pneumocytes are the key players in pulmonary drug transport. With their diverse cellular characteristics, each of these cell types displays a unique uptake possibility. Next to the knowledge of these cellular aspects, the nature of aerosolized drugs, characteristics of delivery systems and the depositional and pulmonary clearance mechanisms display major targets to optimize pulmonary drug delivery. Based on the growing knowledge on pulmonary cell biology and pathophysiology due to modern methods of molecular biology, the future characterization of pulmonary drug transport pathways can lead to new strategies in aerosol drug therapy.

9 CFR 590.549 - Dried egg storage.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Dried egg storage. 590.549 Section 590.549 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE EGG PRODUCTS INSPECTION INSPECTION OF EGGS AND EGG PRODUCTS (EGG PRODUCTS INSPECTION ACT) Sanitary, Processing...

Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, C.; Nettesheim, P.; Barrett, J.C.

1987-04-01

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injectedmore » into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approx. = 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.« less

Continual exposure to cigarette smoke extracts induces tumor-like transformation of human nontumor bronchial epithelial cells in a microfluidic chip.

PubMed

Li, Encheng; Xu, Zhiyun; Liu, Fen; Wang, Huiling; Wen, Jiabin; Shao, Shujuan; Zhang, Lichuan; Wang, Lei; Liu, Chong; Lu, Jianxin; Wang, Wenxin; Gao, Zhancheng; Wang, Qi

2014-08-01

Heavy cigarette smoking-related chronic obstructive pulmonary disease is an independent risk factor for lung squamous carcinoma. However, the mechanisms underlying the malignant transformation of bronchial epithelial cells are unclear. In our study, human tumor-adjacent bronchial epithelial cells were obtained from 10 cases with smoking-related chronic obstructive pulmonary disease and lung squamous carcinoma and cultured in an established microfluidic chip for continual exposure to cigarette smoke extracts (CSE) to investigate the potential tumor-like transformation and mechanisms. The integrated microfluidic chip included upstream concentration gradient generator and downstream cell culture chambers supplied by flowing medium containing different concentrations of CSE. Our results showed that continual exposure to low doses of CSE promoted cell proliferation whereas to high doses of CSE triggered cell apoptosis. Continual exposure to CSE promoted reactive oxygen species production in human epithelial cells in a dose-dependent manner. More importantly, continual exposure to low dose of CSE promoted the epithelial-to-mesenchymal transition process and anchorage-independent growth, and increased chromosome instability in bronchial epithelial cells, accompanied by activating the GRP78, NF-κB, and PI3K pathways. The established microfluidic chip is suitable for primary culture of human tumor-adjacent bronchial epithelial cells to investigate the malignant transformation. Continual exposure to low doses of CSE promoted tumor-like transformation of human nontumor bronchial epithelial cells by inducing reactive oxygen species production and activating the relevant signaling.

Differential concentration-specific effects of caffeine on cell viability, oxidative stress, and cell cycle in pulmonary oxygen toxicity in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiwari, Kirti Kumar; Chu, Chun; Couroucli, Xanthi

Highlights: • Caffeine at 0.05 mM decreases oxidative stress in hyperoxia. • Caffeine at 1 mM decreases cell viability, increases oxidative stress in hyperoxia. • Caffeine at 1 but not 0.05 mM, abrogates hyperoxia-induced G2/M arrest. - Abstract: Caffeine is used to prevent bronchopulmonary dysplasia (BPD) in premature neonates. Hyperoxia contributes to the development of BPD, inhibits cell proliferation and decreases cell survival. The mechanisms responsible for the protective effect of caffeine in pulmonary oxygen toxicity remain largely unknown. A549 and MLE 12 pulmonary epithelial cells were exposed to hyperoxia or maintained in room air, in the presence of differentmore » concentrations (0, 0.05, 0.1 and 1 mM) of caffeine. Caffeine had a differential concentration-specific effect on cell cycle progression, oxidative stress and viability, with 1 mM concentration being deleterious and 0.05 mM being protective. Reactive oxygen species (ROS) generation during hyperoxia was modulated by caffeine in a similar concentration-specific manner. Caffeine at 1 mM, but not at the 0.05 mM concentration decreased the G2 arrest in these cells. Taken together this study shows the novel funding that caffeine has a concentration-specific effect on cell cycle regulation, ROS generation, and cell survival in hyperoxic conditions.« less

Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rennolds, Jessica; Malireddy, Smitha; Hassan, Fatemat

2012-01-06

Highlights: Black-Right-Pointing-Pointer Cadmium induces secretion of IL-6 and IL-8 by two distinct pathways. Black-Right-Pointing-Pointer Cadmium increases NAPDH oxidase activity leading to Erk activation and IL-8 secretion. Black-Right-Pointing-Pointer Curcumin prevents cadmium-induced secretion of both IL-6 and IL-8 by airway cells. Black-Right-Pointing-Pointer Curcumin could be use to suppress lung inflammation due to cadmium inhalation. -- Abstract: Cadmium is a toxic metal present in the environment and its inhalation can lead to pulmonary disease such as lung cancer and chronic obstructive pulmonary disease. These lung diseases are characterized by chronic inflammation. Here we show that exposure of human airway epithelial cells to cadmiummore » promotes a polarized apical secretion of IL-6 and IL-8, two pivotal pro-inflammatory cytokines known to play an important role in pulmonary inflammation. We also determined that two distinct pathways controlled secretion of these proinflammatory cytokines by human airway epithelial cells as cadmium-induced IL-6 secretion occurs via an NF-{kappa}B dependent pathway, whereas IL-8 secretion involves the Erk1/2 signaling pathway. Interestingly, the natural antioxidant curcumin could prevent both cadmium-induced IL-6 and IL-8 secretion by human airway epithelial cells. In conclusion, curcumin could be used to prevent airway inflammation due to cadmium inhalation.« less

[Construction of BAD Lentivirus Vector and Its Effect on Proliferation in A549 Cell Lines].

PubMed

Huang, Na; He, Yan-qi; Zhu, Jing; Li, Wei-min

2015-05-01

To construct the recombinant lentivirus expressing vector BAD (Bcl-2-associated death protein) gene and to study its effect on A549 cell proliferation. The BAD gene was amplified from plasmid pAV-MCMV-BAD-GFP by PCR. The purified BAD gene fragment was inserted into a lentivirus vector (pLVX-IRES-ZsGreen 1), and the insertion was identified by PCR, restriction endonuclease analysis and DNA sequencing. A549 cells were then transfected with the packaged recombinant lentivirus, and resistant cell clones were selected with flow cytometry. The expression of BAD in A549 cell lines stably transduction with a lentivirus was examined using Western blot. The effect of BAD overexpression on proliferation of A549 cells was evaluated by using CCK-8 kit. Restriction enzyme digestion and DNA sequencing showed that the full-length BAD gene (507 bp) had been successfully subcloned into the lentiviral vector to result in the recombinant vector pLVX-IRES-ZsGreen 1. Monoclonal cell lines BAD-A549 was produced after transfection with the recombinant lentivirus and selected with flow cytometry. Stable expression of BAD protein was verified by Western blot. In vitro, the OD value in BAD group was significantly lower than that of control groups from 120-144 h (P<0. 05). A549 cell lines stably transduced with a lentivirus expressing the BAD gene had been successfully generated. In vitro, BAD overexpression significantly inhibited A549 cells proliferation.

Growth Arrest of Epithelial Cells during Measles Virus Infection Is Caused by Upregulation of Interferon Regulatory Factor 1

PubMed Central

Yokota, Shin-ichi; Okabayashi, Tamaki; Yokosawa, Noriko; Fujii, Nobuhiro

2004-01-01

Natural infection with measles virus (MeV) is initiated when the virus reaches epithelial cells in the respiratory tract, oropharynx, or conjunctivae. Human epithelial cells infected with MeV frequently show growth suppression. In this study, we investigated the possible mechanisms for this suppression. The bronchiolar epithelial cell A549 showed growth arrest in G0/G1 following MeV infection or treatment with gamma interferon (IFN-γ). IFN regulatory factor-1 (IRF-1) was upregulated during MeV infection, although A549 did not produce IFN-γ. Cells of the cervical squamous cell line SiHa persistently infected with various strains of MeV displayed slower growth than uninfected SiHa cells, although the growth rates varied depending on the MeV strain. Transfection of antisense-oriented IRF-1 cDNA released the MeV-infected SiHa cells from growth suppression. Although these infected cells did not produce IFN-γ and suppressed IFN-α/β-induced Jak1 phosphorylation, Jak1 was constitutively phosphorylated. The growth rates negatively correlated with levels of both IRF-1 expression and constitutively phosphorylated Jak1. These results indicate that MeV upregulates IRF-1 in a manner that is independent of IFN but dependent on the JAK/STAT pathway. This induction of IRF-1 appears to suppress cell growth, although the extent seems to vary among MeV strains. PMID:15078941

Anticancer activity of polysaccharide from Glehnia littoralis on human lung cancer cell line A549.

PubMed

Wu, Jun; Gao, Weiping; Song, Zhuoyue; Xiong, Qingping; Xu, Yingtao; Han, Yun; Yuan, Jun; Zhang, Rong; Cheng, Yunbo; Fang, Jiansong; Li, Weirong; Wang, Qi

2018-01-01

The purpose of this study was to investigate the anticancer activity of polysaccharide (PGL) from Glehnia littoralis on human lung cancer cell line A549. Based on MTT assay, the results suggested that PGL could significantly reduce A549 cells proliferation in a time- and dose-dependent manner. In addition, PGL displayed an inhibitory activity for the A549 cells migration in Transwell migration assay. The results from both flow cytometry analysis and Hochst 3342 staining of apoptotic cells indicated that PGL could promote apoptosis, and induce cycle arrest of A549 cells. Moreover, immunofluorescence assay elucidated PGL could also down-regulate expression of proliferating cell nuclear antigen (PCNA). Overall, these results showed that PGL exerts a strong anticancer action through inhibiting the A549 cells migration, proliferation and inducing cell apoptosis. It could be a new source of natural anticancer agent against lung cancer with potential value in supplements and medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

The laryngeal primordium and epithelial lamina. A new interpretation.

PubMed Central

Sañudo, J R; Domenech-Mateu, J M

1990-01-01

The laryngeal primordium is present in both the laryngotracheal sulcus (LTS) and the primitive pulmonary sac (PPS). Its early period of development may be subdivided into two phases. The first phase (Stage 11) is represented by what is traditionally referred to as the LTS, located directly beneath the PP4 on the ventral wall of the foregut (primary segment), and by the PPS which is situated at its caudal end. The LTS will represent the primordium of the upper or membranous infraglottic cavity region; whereas the PPS, will give rise not only to the bronchial tree, but also to the primordium of the trachea and the lower or cartilaginous region of the infraglottic cavity. The second phase (Stages 13 and 14) is distinguished by the cranial growth of the LTS above the PP4 and therefore by its absorption into the floor of the primitive pharynx in the mesobranchial area (secondary segment), which will develop into the primordium of the vestibule of the larynx. Similarly, we observed that in the development of the laryngeal cavity there are two temporally and spatially separate epithelial structures: the epithelial septum and the epithelial lamina. In this respect we differ from other authors who are of the opinion that there is a single structure (the epithelial lamina). The epithelial septum is a primary structure responsible for the final configuration of the LTS, as it contributes to the development of the lower end of the primary segment of the LTS and also to the creation of the secondary segment. The epithelial lamina is a secondary structure which appears inside the LTS as a result of pressure exerted by the mesenchyme on its lateral walls, without having any effect on the morphogenesis of the LTS. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:2081706

Pharmacodynamic and pharmacokinetic assessment of pulmonary rehabilitation mixture for the treatment of pulmonary fibrosis.

PubMed

Zhao, Juanjuan; Ren, Yan; Qu, Yubei; Jiang, Wanglin; Lv, Changjun

2017-06-14

Pulmonary rehabilitation mixture (PRM), a Chinese herbal medicine formula, has been used to treat pulmonary fibrosis for decades. In this study, we systematically evaluated the pharmacodynamic and pharmacokinetic performance of PRM. The pharmacodynamic results showed that PRM could improve the condition of CoCl 2 -stimulated human type II alveolar epithelial cells, human pulmonary microvascular endothelial cells, human lung fibroblasts and pulmonary fibrosis rats induced by bleomycin, PRM treatment reduced the expression of platelet-derived growth factor, fibroblast growth factor, toll-like receptor 4, high-mobility group box protein 1 and hypoxia-inducible factor 1α. In the pharmacokinetic study, an accurate and sensitive ultra-high performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of calycosin, calycosin-7-O-glucoside, formononetin, ononin and mangiferin of PRM in the rat plasma for the first time. The method was then successfully applied to the comparative pharmacokinetic study of PRM in normal and pulmonary fibrosis rats. The five constituents could be absorbed in the blood after the oral administration of PRM and exhibited different pharmacokinetic behaviors in normal and pulmonary fibrosis rats. In summary, PRM exhibited a satisfactory pharmacodynamic and pharmacokinetic performance, which highlights PRM as a potential multi-target oral drug for the treatment of pulmonary fibrosis.

28 CFR 549.64 - Food/liquid intake/output.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Food/liquid intake/output. 549.64 Section... MEDICAL SERVICES Hunger Strikes, Inmate § 549.64 Food/liquid intake/output. (a) Staff shall prepare and...) Staff shall remove any commissary food items and private food supplies of the inmate while the inmate is...

28 CFR 549.64 - Food/liquid intake/output.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Food/liquid intake/output. 549.64 Section... MEDICAL SERVICES Hunger Strikes, Inmate § 549.64 Food/liquid intake/output. (a) Staff shall prepare and...) Staff shall remove any commissary food items and private food supplies of the inmate while the inmate is...

28 CFR 549.64 - Food/liquid intake/output.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 2 2014-07-01 2014-07-01 false Food/liquid intake/output. 549.64 Section... MEDICAL SERVICES Hunger Strikes, Inmate § 549.64 Food/liquid intake/output. (a) Staff shall prepare and...) Staff shall remove any commissary food items and private food supplies of the inmate while the inmate is...

28 CFR 549.64 - Food/liquid intake/output.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 2 2013-07-01 2013-07-01 false Food/liquid intake/output. 549.64 Section... MEDICAL SERVICES Hunger Strikes, Inmate § 549.64 Food/liquid intake/output. (a) Staff shall prepare and...) Staff shall remove any commissary food items and private food supplies of the inmate while the inmate is...

Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

PubMed

Gavara, Núria; Sunyer, Raimon; Roca-Cusachs, Pere; Farré, Ramon; Rotger, Mar; Navajas, Daniel

2006-08-01

Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung.

Anti-invasive effect of Cyclamen pseudibericum extract on A549 non-small cell lung carcinoma cells via inhibition of ZEB1 mediated by miR-200c.

PubMed

Karagur, Ege Riza; Ozay, Cennet; Mammadov, Ramazan; Akca, Hakan

2018-06-01

Scientists are increasingly focusing attention on natural products of plant origin for use as agents in cancer protection and treatment. Cyclamen L. tuber extracts contain saponin glycosides that have been shown to have anti-cancer and other biological activities. The epithelial-to-mesenchymal transition (EMT) is thought to enhance malignant tumour progress. The transcriptional repressor zinc-finger E-box binding homeobox 1 (ZEB1) is an important inducer of EMT in different human tumours and has recently been shown to boost invasion by tumour cells. In this study, we investigated the effects of endemic Cyclamen pseudibericum (CP) saponin-rich tuber extract on the capacity of non-small cell lung cancer line A549 cells to proliferate, invade and migrate and also examined the expression levels of several invasion-migration-related microRNAs (miRNAs) to identify those which directly targeted ZEB1. The cytotoxicity effect of the CP extract on the A549 cancer cells was determined by the luminometric method. The half-minimal (50%) inhibitory concentration dose in the A549 cells was determined to be 41.64 ± 2.35 µg/mL. Using the Matrigel invasion chamber system and the wound healing assay we observed that the CP extract suppressed the invasion and migration capacity of A549 cells, respectively. The expression of miRNAs in A549 cells was evaluated by real-time PCR. Our data showed that overexpression of miRNA miR-200c hindered the EMT by increasing the expression of E-cadherin and decreasing the expression of both N-cadherin and vimentin through the direct targeting of ZEB1. These findings suggest that the saponin-rich tuber extract of CP may have considerable anti-cancer properties in lung cancer. Further studies are required to examine in detail the molecular-based mechanism involved in the EMT process of the extract along with isolation and identification of active saponin components.

Peroxisome-proliferator-activated receptor-{gamma} agonists inhibit the release of proinflammatory cytokines from RSV-infected epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arnold, Ralf; Koenig, Wolfgang

2006-03-15

The epithelial cells of the airways are the target cells for respiratory syncytial virus (RSV) infection and the site of the majority of the inflammation associated with the disease. Recently, peroxisome-proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we investigated the role of PPAR{gamma} agonists (15d-PGJ{sub 2}, ciglitazone and troglitazone) on the synthesis of RSV-induced cytokine release from RSV-infected human lung epithelial cells (A549). We observed that all PPAR{gamma} ligands inhibited dose-dependently the release of TNF-{alpha}, GM-CSF, IL-1{alpha}, IL-6 and the chemokines CXCL8 (IL-8) and CCL5 (RANTES) frommore » RSV-infected A549 cells. Concomitantly, the PPAR{gamma} ligands diminished the cellular amount of mRNA encoding for IL-6, CXCL8 and CCL5 and the RSV-induced binding activity of the transcription factors NF-{kappa}B (p65/p50) and AP-1 (c-fos), respectively. Our data presented herein suggest a potential application of PPAR{gamma} ligands in the anti-inflammatory treatment of RSV infection.« less

31 CFR 549.506 - Investment and reinvestment of certain funds.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Investment and reinvestment of certain funds. 549.506 Section 549.506 Money and Finance: Treasury Regulations Relating to Money and Finance... Licenses, Authorizations, and Statements of Licensing Policy § 549.506 Investment and reinvestment of...

Artesunate induces AIF-dependent apoptosis in A549 cells

NASA Astrophysics Data System (ADS)

Zhou, Chen-juan; Chen, Tong-Sheng

2012-03-01

Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

Xylitol induces cell death in lung cancer A549 cells by autophagy.

PubMed

Park, Eunjoo; Park, Mi Hee; Na, Hee Sam; Chung, Jin

2015-05-01

Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells.

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells.

PubMed

Kim, Eunbi; Na, Sunghun; An, Borim; Yang, Se-Ran; Kim, Woo Jin; Ha, Kwon-Soo; Han, Eun-Taek; Park, Won Sun; Lee, Chang-Min; Lee, Ji Yoon; Lee, Seung-Joon; Hong, Seok-Ho

2017-03-01

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

β2-Microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence.

PubMed

Gao, Na; Wang, Ying; Zheng, Chun-Ming; Gao, Yan-Li; Li, Hui; Li, Yan; Fu, Ting-Ting; Xu, Li-Li; Wang, Wei; Ying, Sun; Huang, Kewu

2017-05-01

β 2 -Microglobulin (β 2 M), the light chain of the major histocompatibility complex class I (MHC I), has been identified as a proaging factor and is involved in the pathogenesis of neurodegenerative disorders by driving cognitive and regenerative impairments. However, little attention has focused on the effect of β 2 M in the development of lung emphysema. Here, we found that concentrations of β 2 M in plasma were significantly elevated in patients with lung emphysema than those in normal control subjects (1.89 ± 0.12 vs. 1.42 ± 0.06 mg/l, P < 0.01). Moreover, the expression of β 2 M was significantly higher in lung tissue of emphysema (39.90 ± 1.97 vs. 23.94 ± 2.11%, P < 0.01). Immunofluorescence showed that β 2 M was mainly expressed in prosurfactant protein C-positive (pro-SPC + ) alveolar epithelial cells and CD14 + macrophages. Exposure to recombinant human β 2 M and cigarette smoke extract (CSE) in vitro enhanced cellular senescence and inhibited proliferation of A549 cells, which was partially reversed by the presence of anti-β 2 M antibody. However, anti-β 2 M antibody did not attenuate the elevated production of IL-1β, IL-6, and TNF-α in A549 cells that were exposed to CSE. Immunofluorescence showed that colocalization of β 2 M, and the hemochromatosis gene (HFE) protein was observed on A549 cells. These data suggest β 2 M might participate in the development of lung emphysema through induction of lung epithelial cell senescence and inhibition. Copyright © 2017 the American Physiological Society.

Lung epithelial stem cells and their niches: Fgf10 takes center stage.

PubMed

Volckaert, Thomas; De Langhe, Stijn

2014-01-01

Throughout life adult animals crucially depend on stem cell populations to maintain and repair their tissues to ensure life-long organ function. Stem cells are characterized by their capacity to extensively self-renew and give rise to one or more differentiated cell types. These powerful stem cell properties are key to meet the changing demand for tissue replacement during normal lung homeostasis and regeneration after lung injury. Great strides have been made over the last few years to identify and characterize lung epithelial stem cells as well as their lineage relationships. Unfortunately, knowledge on what regulates the behavior and fate specification of lung epithelial stem cells is still limited, but involves communication with their microenvironment or niche, a local tissue environment that hosts and influences the behaviors or characteristics of stem cells and that comprises other cell types and extracellular matrix. As such, an intimate and dynamic epithelial-mesenchymal cross-talk, which is also essential during lung development, is required for normal homeostasis and to mount an appropriate regenerative response after lung injury. Fibroblast growth factor 10 (Fgf10) signaling in particular seems to be a well-conserved signaling pathway governing epithelial-mesenchymal interactions during lung development as well as between different adult lung epithelial stem cells and their niches. On the other hand, disruption of these reciprocal interactions leads to a dysfunctional epithelial stem cell-niche unit, which may culminate in chronic lung diseases such as chronic obstructive pulmonary disease (COPD), chronic asthma and idiopathic pulmonary fibrosis (IPF).

Dexamethasone inhibits inflammatory response via down regulation of AP-1 transcription factor in human lung epithelial cells.

PubMed

Patil, Rajeshwari H; Naveen Kumar, M; Kiran Kumar, K M; Nagesh, Rashmi; Kavya, K; Babu, R L; Ramesh, Govindarajan T; Chidananda Sharma, S

2018-03-01

The production of inflammatory mediators by epithelial cells in inflammatory lung diseases may represent an important target for the anti-inflammatory effects of glucocorticoids. Activator protein-1 is a major activator of inflammatory genes and has been proposed as a target for inhibition by glucocorticoids. We have used human pulmonary type-II A549 cells to examine the effect of dexamethasone on the phorbol ester (PMA)/Lipopolysaccharide (LPS) induced pro-inflammatory cytokines and AP-1 factors. A549 cells were treated with and without PMA or LPS or dexamethasone and the cell viability and nitric oxide production was measured by MTT assay and Griess reagent respectively. Expression of pro-inflammatory cytokines and AP-1 factors mRNA were measured using semi quantitative RT-PCR. The PMA/LPS treated cells show significant 2-3 fold increase in the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8 and TNF-α), cyclo‑oxygenase-2 (COX-2) and specific AP-1 factors (c-Jun, c-Fos and Jun-D). Whereas, pretreatment of cells with dexamethasone significantly inhibited the LPS induced nitric oxide production and PMA/LPS induced mRNAs expression of above pro-inflammatory cytokines, COX-2 and AP-1 factors. Cells treated with dexamethasone alone at both the concentrations inhibit the mRNAs expression of IL-1β, IL-6 and TNF-α compared to control. Our study reveals that dexamethasone decreased the mRNAs expression of c-Jun and c-Fos available for AP-1 formation suggested that AP-1 is the probable key transcription factor involved in the anti-inflammatory activity of dexamethasone. This may be an important molecular mechanism of steroid action in asthma and other chronic inflammatory lung diseases which may be useful for treatment of lung inflammatory diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

PubMed

Deville, Sarah; Penjweini, Rozhin; Smisdom, Nick; Notelaers, Kristof; Nelissen, Inge; Hooyberghs, Jef; Ameloot, Marcel

2015-10-01

Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

NASA Astrophysics Data System (ADS)

Lewis, Katherine Jean Reeder

The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

The enriched fraction of Vernonia cinerea L. induces apoptosis and inhibits multi-drug resistance transporters in human epithelial cancer cells.

PubMed

Appadath Beeran, Asmy; Maliyakkal, Naseer; Rao, Chamallamudi Mallikarjuna; Udupa, Nayanabhirama

2014-12-02

Vernonia cinerea Less. (VC) of the family Asteraceaes is considered as the sacred plant; 'Dasapushpam' which is ethnopharmacologically significant to the people of Kerala in India. In fact, VC has been used in the traditional system of medicine (Ayurveda) for the treatment of various ailments including cancer. Cytotoxicity of the ethanolic extract of VC (VC-ET), petroleum ether fraction (VC-PET), dichloromethane fraction (VC-DCM), n-butyl alcohol fraction (VC-BT), and rest fraction (VC-R) was evaluated in cervical carcinoma (HeLa), lung adenocarcinoma (A549), breast cancer (MCF-7), and colon carcinoma (Caco-2) cells using Sulforhodamine B (SRB) assay. The apoptotic effects of VC-DCM were assessed in cancer cells using Annexin V assay. The effects of VC-DCM on multi-drug resistance (MDR) transporters in HeLa, A549, MCF-7, and Caco-2 cells were evaluated using flow cytometry based functional assays. Similarly, drug uptake in cancer cells and sensitization of cancer cells towards chemotherapeutic drugs in the presence of VC-DCM were studied using Daunorubicin (DNR) accumulation assay and SRB assay, respectively. Cytotoxicity assay revealed that the enriched fraction of VC (VC-DCM) possessed dose-dependent cytotoxic effects in human epithelial cancer cells (HeLa, A549, MCF-7, and Caco-2). Further, treatment of cancer cells (HeLa, A549, MCF-7, and Caco-2) with VC-DCM led to a significant increase in both early and late apoptosis, indicating the induction of apoptosis. Interestingly, VC-DCM significantly inhibited functional activity of MDR transporters (ABC-B1 and ABC-G2), enhanced DNR-uptake in cancer cells, and sensitized cancer cells towards chemotherapeutic drug-mediated cytotoxicity, thus indicating the ability of VC-DCM to reverse MDR in cancer and enhance the cytotoxic effects of anticancer drugs. A methodological investigation on the anti-cancer properties of Vernonia cinerea Less. (VC) revealed that an enriched fraction of VC (VC-DCM) possessed cytotoxic

Comparing the enhancement efficiency between liposomes and microbubbles for insulin pulmonary absorption.

PubMed

Xu, Yan-Yan; Lu, Cui-Tao; Fu, Hong-Xing; Zhao, Ying-Zheng; Yang, Wei; Li, Xing; Zhang, Lu; Li, Xiao-Kun; Zhang, Ming

2011-07-01

The present study investigated the enhancement efficiency between liposomes and microbubbles for insulin pulmonary absorption. Two types of phospholipid-based vesicle-liposomes and microbubbles-were prepared, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity test was used to evaluate their in vitro toxicity in A549 cells. Cellular uptake of insulin combined with liposomes or microbubbles was determined using A549 cells. With intratracheal insufflation of Sprague-Dawley rats, an insulin mixture with liposomes or microbubbles was administered to assess its potential for promoting drug pulmonary absorption. Both liposomes and microbubbles had a narrow and monodispersed size distribution with average diameter of 3.1 μm and 1.0 μm, respectively. From the MTT cytotoxicity test, a phospholipid-based vesicle concentration of <25% (vol/vol) in the final volume was the safe dosage range that could avoid severe cytotoxic effects. The intracellular uptake amount of insulin in the insulin-microbubble mixture was significantly higher than that in the insulin-liposome mixture. The minimum reductions of the blood glucose concentration produced by insulin-microbubble and insulin-liposome mixtures were 60.8% and 35.0% of the initial glucose levels, respectively, and their bioavailabilities relative to subcutaneous injection were 48.6% and 30.8%, respectively. Microbubbles have much better efficiency than liposomes in the rate and extent of insulin pulmonary absorption. Microbubbles might be recommended as a potential agent for enhancing protein intrapulmonary absorption.

Evidence for the involvement of cofilin in Aspergillus fumigatus internalization into type II alveolar epithelial cells.

PubMed

Bao, Zhiyao; Han, Xuelin; Chen, Fangyan; Jia, Xiaodong; Zhao, Jingya; Zhang, Changjian; Yong, Chen; Tian, Shuguang; Zhou, Xin; Han, Li

2015-08-13

The internalization of Aspergillus fumigatus into alveolar epithelial cells (AECs) is tightly controlled by host cellular actin dynamics, which require close modulation of the ADF (actin depolymerizing factor)/cofilin family. However, the role of cofilin in A. fumigatus internalization into AECs remains unclear. Here, we demonstrated that germinated A. fumigatus conidia were able to induce phosphorylation of cofilin in A549 cells during the early stage of internalization. The modulation of cofilin activity by overexpression, knockdown, or mutation of the cofilin gene in A549 cells decreased the efficacy of A. fumigatus internalization. Reducing the phosphorylation status of cofilin with BMS-5 (LIM kinase inhibitor) or overexpression of the slingshot phosphatases also impeded A. fumigatus internalization. Both the C. botulimun C3 transferase (a specific RhoA inhibitor) and Y27632 (a specific ROCK inhibitor) reduced the internalization of A. fumigatus and the level of phosphorylated cofilin. β-1,3-glucan (the major component of the conidial cell wall) and its host cell receptor dectin-1 did not seem to be associated with cofilin phosphorylation during A. fumigatus infection. These results indicated that cofilin might be involved in the modulation of A. fumigatus internalization into type II alveolar epithelial cells through the RhoA-ROCK-LIM kinase pathway.

31 CFR 549.311 - U.S. financial institution.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false U.S. financial institution. 549.311 Section 549.311 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., commodity futures or options, or procuring purchasers and sellers thereof, as principal or agent. It...

31 CFR 549.311 - U.S. financial institution.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false U.S. financial institution. 549.311 Section 549.311 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., commodity futures or options, or procuring purchasers and sellers thereof, as principal or agent. It...

31 CFR 549.311 - U.S. financial institution.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false U.S. financial institution. 549.311 Section 549.311 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., commodity futures or options, or procuring purchasers and sellers thereof, as principal or agent. It...

31 CFR 549.311 - U.S. financial institution.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false U.S. financial institution. 549.311 Section 549.311 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., commodity futures or options, or procuring purchasers and sellers thereof, as principal or agent. It...

Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy.

PubMed

Hidvegi, Tunda; Stolz, Donna B; Alcorn, John F; Yousem, Samuel A; Wang, Jieru; Leme, Adriana S; Houghton, A McGarry; Hale, Pamela; Ewing, Michael; Cai, Houming; Garchar, Evelyn Akpadock; Pastore, Nunzia; Annunziata, Patrizia; Kaminski, Naftali; Pilewski, Joseph; Shapiro, Steven D; Pak, Stephen C; Silverman, Gary A; Brunetti-Pierri, Nicola; Perlmutter, David H

2015-12-11

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Luteolin attenuates TGF-β1-induced epithelial-mesenchymal transition of lung cancer cells by interfering in the PI3K/Akt-NF-κB-Snail pathway.

PubMed

Chen, Kun-Chieh; Chen, Chiu-Yuan; Lin, Chih-Ru; Lin, Chih-Ju; Yang, Tsung-Ying; Chen, Tzu-Hsiu; Wu, Li-Chen; Wu, Chun-Chi

2013-12-05

Luteolin is a natural flavonoid that possesses a variety of pharmacological activities, such as anti-inflammatory and anti-cancer abilities. Whether luteolin regulates the transformation ability of lung cancer cells remains unclear. The current study aims to uncover the effects and underlying mechanisms of luteolin in regulation of and epithelial-mesenchymal transition of lung cancer cells. The lung adenocarcinoma A549 cells were used in this experiment; the cells were pretreated with luteolin followed by administration with TGF-β1. The expression levels of various cadherin and related upstream regulatory modules were examined. Pretreatment of luteolin prevented the morphological change and downregulation of E-cadherin of A549 cells induced by TGF-β1. In addition, the activation of PI3K-Akt-IκBa-NF-κB-Snail pathway which leads to the decline of E-cadherin induced by TGF-β1 was also attenuated under the pretreatment of luteolin. We provide the mechanisms about how luteolin attenuated the epithelial-mesenchymal transition of A549 lung cancer cells induced by TGF-β1. This finding will strengthen the anti-cancer effects of flavonoid compounds via the regulation of migration/invasion and EMT ability of various cancer cells. © 2013.

Industrial PM2.5 cause pulmonary adverse effect through RhoA/ROCK pathway.

PubMed

Yan, Junyan; Lai, Chia-Hsiang; Lung, Shih-Chun Candice; Chen, Chongjun; Wang, Wen-Cheng; Huang, Pin-I; Lin, Chia-Hua

2017-12-01

According to the Chinese Ministry of Health, industrial pollution-induced health impacts have been the leading cause of death in China. While industrial fine particulate matter (PM 2.5 ) is associated with adverse health effects, the major action mechanisms of different compositions of PM 2.5 are currently unclear. In this study, we treated normal human lung epithelial BEAS-2B cells with industrial organic and water-soluble PM 2.5 extracts under daily alveolar deposition dose to elucidate the molecular mechanisms underlying adverse pulmonary effects induced by PM 2.5 , including oxidative damage, inflammatory response, lung epithelial barrier dysfunction, and the recruitment of macrophages. We found that water-soluble PM 2.5 extracts caused more severe cytotoxic effects on BEAS-2B cells compared with that of organic extracts. Both organic and water-soluble PM 2.5 extracts induced activation of the RhoA/ROCK pathway. Inflammatory response, epithelial barrier dysfunction, and the activation of NF-кB caused by both PM 2.5 extracts were attenuated by ROCK inhibitor Y-27632. This indicated that both PM 2.5 extracts could cause damage to epithelial cells through RhoA/ROCK-dependent NF-кB activation. Furthermore, the upregulation of macrophage adhesion induced by both PM 2.5 extracts was also attenuated by Y-27632 in a co-culture model of macrophages and the epithelial cells. Therefore, our results support that industrial PM 2.5 extracts-induced activation of the RhoA/ROCK-dependent NF-кB pathway induces pulmonary adverse effect. Thus, pharmacological inhibition of ROCK activation might have therapeutic potential in preventing lung disease associated with PM 2.5 . Copyright © 2017 Elsevier B.V. All rights reserved.

Ferulic acid supresses Th2 immune response and prevents remodeling in ovalbumin-induced pulmonary allergy associated with inhibition of epithelial-derived cytokines.

PubMed

Sin Singer Brugiolo, Alessa; Carvalho Gouveia, Ana Cláudia; de Souza Alves, Caio César; de Castro E Silva, Flávia Márcia; Esteves de Oliveira, Érick; Ferreira, Ana Paula

2017-08-01

Asthma is characterized by intermittent airway obstruction and chronic inflammation, orchestrated primarily by Th2 cytokines. There is a strong rationale for developing new asthma therapies, since current treatment protocols present side effects and may not be effective in cases of difficult-to-control asthma. The purpose of this study was to evaluate the effect of ferulic acid, a phenolic acid commonly present in plants, in the ovalbumin-induced pulmonary allergy murine model. BALB/c mice were sensitized and challenged with ovalbumin, and treatments were provided by gavage. Six groups of mice (n = 6) were studied, labeled as: control, pulmonary allergy, dexamethasone, and 3 receiving ferulic acid (at 25, 50, and 100 mg/kg). Lung tissue, bronchoalveolar lavage fluid and serum were collected for analysis. Ferulic acid treatment inhibited an established allergic Th2-response by decreasing the key features of pulmonary allergy, including lung and airway inflammation, eosinophil infiltration, mucus production and serum levels of OVA-specific IgE. These results were associated with lower levels of CCL20, CCL11 and CCL5 chemokines and IL-4, IL-5, IL-13, TSLP, IL-25 and IL-33 cytokines in lung tissue homogenate. In this study it was demonstrated for the first time that ferulic acid treatment is able to suppress one of the main features of the airway remodeling, indicated by reduction of mucus production, besides the Th2 pathogenic response on ovalbumin-induced pulmonary allergy. Taken together, results shows that the immunopathological mechanism underlying these effects is linked to a reduction of the epithelial-derived chemokines and cytokines, suggesting that ferulic acid may be useful as a potential therapeutic agent for asthma. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer.

PubMed

Cai, Yong; Sheng, Zhao-Ying; Chen, Yun; Bai, Chong

2014-01-01

To explore the effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer (NSCLC). NSCNC cell line A549 was selected to explore the effect of Withaferin A on A549 cellular proliferation, apoptosis and the PI3K/Akt signal pathway capable of regulating tumor biological behavior by assessment of cellular proliferation, cellular apoptotic rates and cellular cycling as well as by immuno-blotting. Withaferin A could inhibit A549 cellular proliferation and the control rate was dosage-dependent (P<0.05), which also increased time-dependently with the same dosage of Withaferin A (P<0.05). The apoptotic indexes in A549 cells treated with 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A for 48 h were significantly different (P<0.05). In addition, the apoptotic rates of each group in both early and advanced stages were higher than those in 0 μmol·L-1 (P<0.05), which were evidently higher after 48 h than those after 24 h (P<0.05). A549 cells treated by Withaferin A for 48 h were markedly lower in Bcl-2 level and obviously higher in Bax and cleaved caspase-3 levels than those treated by 0 μmol·L-1 Withaferin A (P<0.05), and there were significant differences among 5, 10 and 20 μmol·L-1 Withaferin A (P<0.05). The ratios of A549 cells treated by Withaferin A for 48 h in G0/G1 stage were higher than those in 0 μmol·L-1 , while those in S and G2/M stages were obviously lower than those in G2/M stage, and there were significant differences in 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A (P<0.05). Additionally, p-Akt/Akt values were in reverse association with dosage, and the differences were significant (P<0.05). Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing activation of the PI3K/Akt pathways.

Aptamer based electrochemical sensor for detection of human lung adenocarcinoma A549 cells

NASA Astrophysics Data System (ADS)

Sharma, Rachna; Varun Agrawal, Ved; Sharma, Pradeep; Varshney, R.; Sinha, R. K.; Malhotra, B. D.

2012-04-01

We report results of the studies relating to development of an aptamer-based electrochemical biosensor for detection of human lung adenocarcinoma A549 cells. The aminated 85-mer DNA aptamer probe specific for the A549 cells has been covalently immobilized onto silane self assembled monolayer (SAM) onto ITO surface using glutaraldehyde as the crosslinker. The results of cyclic voltammetry and differential pulse voltammetry studies reveal that the aptamer functionalized bioelectrode can specifically detect lung cancer cells in the concentration range of 103 to 107 cells/ml with detection limit of 103 cells/ml within 60 s. The specificity studies of the bioelectrode have been carried out with control KB cells. No significant change in response is observed for control KB cells as compared to that of the A549 target cells.

6 CFR 5.49 - Prohibition on providing expert or opinion testimony.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 6 Domestic Security 1 2010-01-01 2010-01-01 false Prohibition on providing expert or opinion testimony. 5.49 Section 5.49 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY DISCLOSURE OF RECORDS AND INFORMATION Disclosure of Information in Litigation § 5.49 Prohibition on providing...

Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary insulin delivery.

PubMed

Chono, Sumio; Fukuchi, Rie; Seki, Toshinobu; Morimoto, Kazuhiro

2009-07-20

The pulmonary insulin delivery characteristics of liposomes were examined. Aerosolized liposomes containing insulin were administered into rat lungs and the enhancing effect on insulin delivery was evaluated by changes of plasma glucose levels. Liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhanced pulmonary insulin delivery in rats, however, liposomes with dilauroyl, dimyristoyl, distearoyl or dioleoyl phosphatidylcholine did not. Liposomes with DPPC also enhanced the in vitro permeation of FITC dextran (Mw 4400, FD-4) through the calu-3 cell monolayer by reducing the transepithelial electrical resistance and did not harm lung tissues in rats. These findings suggest that liposomes with DPPC enhance pulmonary insulin delivery by opening the epithelial cell space in the pulmonary mucosa not mucosal cell damage. Liposomes with DPPC could be useful as a pulmonary delivery system for peptide and protein drugs.

Fisetin inhibits the generation of inflammatory mediators in interleukin-1β-induced human lung epithelial cells by suppressing the NF-κB and ERK1/2 pathways.

PubMed

Peng, Hui-Ling; Huang, Wen-Chung; Cheng, Shu-Chen; Liou, Chian-Jiun

2018-07-01

Fisetin, a flavone that can be isolated from fruits and vegetables, has anti-tumor and anti-oxidative properties and ameliorates airway hyperresponsiveness in asthmatic mice. This study investigated whether fisetin can suppress the expression of inflammatory mediators and intercellular adhesion molecule 1 (ICAM-1) in A549 human lung epithelial cells that were stimulated with interleukin-1β (IL-1β) to induce inflammatory responses. A549 cells were treated with fisetin (3-30 μM) and then with IL-1β. Fisetin significantly inhibited COX-2 expression and reduced prostaglandin E 2 production, and it suppressed the levels of IL-8, CCL5, monocyte chemotactic protein 1, tumor necrosis factor α, and IL-6. Fisetin also significantly attenuated the expression of chemokine and inflammatory cytokine genes and decreased the expression of ICAM-1, which mediates THP-1 monocyte adhesion to inflammatory A549 cells. Fisetin decreased the translocation of nuclear transcription factor kappa-B (NF-κB) subunit p65 into the nucleus and inhibited the phosphorylation of proteins in the ERK1/2 pathway. Co-treatment of IL-1β-stimulated A549 cells with ERK1/2 inhibitors plus fisetin reduced ICAM-1 expression. Furthermore, fisetin significantly increased the effects of the protective antioxidant pathway by promoting the expression of nuclear factor erythroid-2-related factor-2 and heme oxygenase 1. Taken together, these data suggest that fisetin has anti-inflammatory effects and that it suppresses the expression of chemokines, inflammatory cytokines, and ICAM-1 by suppressing the NF-κB and ERK1/2 signaling pathways in IL-1β-stimulated human lung epithelial A549 cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells.

PubMed

Amatngalim, Gimano D; Broekman, Winifred; Daniel, Nadia M; van der Vlugt, Luciën E P M; van Schadewijk, Annemarie; Taube, Christian; Hiemstra, Pieter S

2016-01-01

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.

Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549

PubMed Central

Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

2017-01-01

To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin. PMID:28928819

Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549.

PubMed

Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

2017-09-01

To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin.

Diesel exhaust alters the response of cultured primary bronchial epithelial cells from patients with chronic obstructive pulmonary disease (COPD) to non-typeable Haemophilus influenzae.

PubMed

Zarcone, Maria C; van Schadewijk, Annemarie; Duistermaat, Evert; Hiemstra, Pieter S; Kooter, Ingeborg M

2017-01-28

Exacerbations constitute a major cause of morbidity and mortality in patients suffering from chronic obstructive pulmonary disease (COPD). Both bacterial infections, such as those with non-typeable Haemophilus influenzae (NTHi), and exposures to diesel engine emissions are known to contribute to exacerbations in COPD patients. However, the effect of diesel exhaust (DE) exposure on the epithelial response to microbial stimulation is incompletely understood, and possible differences in the response to DE of epithelial cells from COPD patients and controls have not been studied. Primary bronchial epithelial cells (PBEC) were obtained from age-matched COPD patients (n = 7) and controls (n = 5). PBEC were cultured at the air-liquid interface (ALI) to achieve mucociliary differentiation. ALI-PBECs were apically exposed for 1 h to a stream of freshly generated whole DE or air. Exposure was followed by 3 h incubation in presence or absence of UV-inactivated NTHi before analysis of epithelial gene expression. DE alone induced an increase in markers of oxidative stress (HMOX1, 50-100-fold) and of the integrated stress response (CHOP, 1.5-2-fold and GADD34, 1.5-fold) in cells from both COPD patients and controls. Exposure of COPD cultures to DE followed by NTHi caused an additive increase in GADD34 expression (up to 3-fold). Importantly, DE caused an inhibition of the NTHi-induced expression of the antimicrobial peptide S100A7, and of the chaperone protein HSP5A/BiP. Our findings show that DE exposure of differentiated primary airway epithelial cells causes activation of the gene expression of HMOX1 and markers of integrated stress response to a similar extent in cells from COPD donors and controls. Furthermore, DE further increased the NTHi-induced expression of GADD34, indicating a possible enhancement of the integrated stress response. DE reduced the NTHi-induced expression of S100A7. These data suggest that DE exposure may cause adverse health effects in part by

46 CFR 169.549 - Ring lifebuoys and water lights.

Code of Federal Regulations, 2014 CFR

2014-10-01

... chapter and be international orange in color. (2) Each water light must be approved under subpart 161.010... 46 Shipping 7 2014-10-01 2014-10-01 false Ring lifebuoys and water lights. 169.549 Section 169.549... lights. (a)(1) The minimum number of life buoys and the minimum number to which water lights must be...

46 CFR 169.549 - Ring lifebuoys and water lights.

Code of Federal Regulations, 2012 CFR

2012-10-01

... chapter and be international orange in color. (2) Each water light must be approved under subpart 161.010... 46 Shipping 7 2012-10-01 2012-10-01 false Ring lifebuoys and water lights. 169.549 Section 169.549... lights. (a)(1) The minimum number of life buoys and the minimum number to which water lights must be...

Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

PubMed

Behzad, A R; Morimoto, K; Gosselink, J; Green, J; Hogg, J C; Hayashi, S

2006-12-01

Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.

Ferrous glycinate regulates cell energy metabolism by restrictinghypoxia-induced factor-1α expression in human A549 cells.

PubMed

Kuo, Yung-Ting; Jheng, Jhong-Huei; Lo, Mei-Chen; Chen, Wei-Lu; Wang, Shyang-Guang; Lee, Horng-Mo

2018-06-04

Iron or oxygen regulates the stability of hypoxia inducible factor-1α (HIF-1α). We investigated whether ferrous glycinate would affect HIF-1α accumulation, aerobic glycolysis and mitochondrial energy metabolism in human A549 lung cancer cells. Incubation of A549 cells with ferrous glycinate decreased the protein levels of HIF-1α, which was abrogated by proteosome inhibitor, or prolyl hydroxylase inhibitor. The addition of ferrous glycinate decreased protein levels of glucose transporter-1, hexokinase-2, and lactate dehydrogenase A, and decreased pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase (PDH) phosphorylation in A549 cells. Ferrous glycinate also increased the expression of the mitochondrial transcription factor A (TFAM), and the mitochondrial protein, cytochrome c oxidase (COX-IV). Silencing of HIF-1α expression mimicked the effects of ferrous glycinate on PDK-1, PDH, TFAM and COX-IV in A549 cells. Ferrous glycinate increased mitochondrial membrane potential and ATP production in A549 cells. These results suggest that ferrous glycinate may reverse Warburg effect through down regulating HIF-1α in A549 cells.

Formulation of Biologically-Inspired Silk-Based Drug Carriers for Pulmonary Delivery Targeted for Lung Cancer

PubMed Central

Kim, Sally Yunsun; Naskar, Deboki; Kundu, Subhas C.; Bishop, David P.; Doble, Philip A.; Boddy, Alan V.; Chan, Hak-Kim; Wall, Ivan B.; Chrzanowski, Wojciech

2015-01-01

The benefits of using silk fibroin, a major protein in silk, are widely established in many biomedical applications including tissue regeneration, bioactive coating and in vitro tissue models. The properties of silk such as biocompatibility and controlled degradation are utilized in this study to formulate for the first time as carriers for pulmonary drug delivery. Silk fibroin particles are spray dried or spray-freeze-dried to enable the delivery to the airways via dry powder inhalers. The addition of excipients such as mannitol is optimized for both the stabilization of protein during the spray-freezing process as well as for efficient dispersion using an in vitro aerosolisation impactor. Cisplatin is incorporated into the silk-based formulations with or without cross-linking, which show different release profiles. The particles show high aerosolisation performance through the measurement of in vitro lung deposition, which is at the level of commercially available dry powder inhalers. The silk-based particles are shown to be cytocompatible with A549 human lung epithelial cell line. The cytotoxicity of cisplatin is demonstrated to be enhanced when delivered using the cross-linked silk-based particles. These novel inhalable silk-based drug carriers have the potential to be used as anti-cancer drug delivery systems targeted for the lungs. PMID:26234773

Combined treatment with apatinib and docetaxel in A549 xenograft mice and its cellular pharmacokinetic basis.

PubMed

Feng, Si-Qi; Wang, Guang-Ji; Zhang, Jing-Wei; Xie, Yuan; Sun, Run-Bin; Fei, Fei; Huang, Jing-Qiu; Wang, Ying; Aa, Ji-Ye; Zhou, Fang

2018-05-17

Apatinib, a small-molecule inhibitor of VEGFR-2, has attracted much attention due to its encouraging anticancer activity in third-line clinical treatment for many malignancies, including non-small cell lung cancer (NSCLC). Its usage in second-line therapy with chemotherapeutic drugs is still under exploration. In this study we investigated the antitumor effect of apatinib combined with docetaxel against NSCLC and its cellular pharmacokinetic basis. A549 xenograft nude mice were treated with apatinib (100 mg/kg every day for 20 days) combined with docetaxel (8 mg/kg, ip, every four days for 5 times). Apatinib significantly enhanced the antitumor effect of docetaxel and alleviated docetaxel-induced liver damage as well as decreased serum transaminases (ALT and AST). LC-MS/MS analysis revealed that apatinib treatment significantly increased the docetaxel concentration in tumors (up to 1.77 times) without enhancing the docetaxel concentration in the serum, heart, liver, lung and kidney. Furthermore, apatinib decreased docetaxel-induced upregulation of P-glycoprotein in tumors. The effects of apatinib on the uptake, efflux and subcellular distribution of docetaxel were investigated in A549 and A549/DTX (docetaxel-resistant) cells in vitro. A cellular pharmacokinetic study revealed that apatinib significantly increased cellular/subcellular accumulation (especially in the cytosol) and decreased the efflux of docetaxel in A549/DTX cells through P-gp, while apatinib exerted no significant effect on the cellular pharmacokinetics of docetaxel in A549 cells. Consequently, the IC 50 value of docetaxel in A549/DTX cells was more significantly decreased by apatinib than that in A549 cells. These results demonstrate that apatinib has potential for application in second-line therapy combined with docetaxel for NSCLC patients, especially for docetaxel-resistant or multidrug-resistant patients.

[HDAC1 expression and effect of TSA on proliferation and apoptosis of A549 cells].

PubMed

Huang, Hong; Zhang, Zhen-Xiang; Xu, Yong-Jian; Shao, Jing-Fang

2003-09-01

Histone deacetylase (HDAC) shows a high expression in many cancer cells and the inhibitor of HDAC1, trichostatin A (TSA), can inhibit the growth of cancer cells. Hypoxia is a common feature of malignant tumors. This paper was designed to investigate the expression of HDAC1 of A549 cell strains in hypoxia condition and the effect of TSA on their proliferation and apoptosis. The authors designed 1 normoxia group (control group) and 5 hypoxia groups (test groups): hypoxia 6h group (A), TSA + hypoxia 6h (B), hypoxia 12h group (C), hypoxia 24h group (D), TSA + hypoxia 24h (E), hypoxia 48h group (F). The expression of HDAC1 in A549 cells was examined using Western blot analysis. Proliferation, the apoptotic rates of A549 cells and the effect of TSA on them were determined using MTT method, immunohistochemistry, TUNEL method, and flow cytometry. The expression of mRNA of HDAC1 and the effect of TSA on it were determined using reverse transcription-polymerase chain reaction (RT-PCR). The A values expressed by HDAC1 in A549 cell strains were 138+/-11 in the control group, 78+/-4, 86+/-5, 124+/-3, and 120+/-9 in test groups A, C, D, and F, respectively. The A values of HDAC1mRNA versus the A values of beta-Atin mRNA were 0.68+/-0.03 in the control group, 0.46+/-0.03, 0.45+/-0.02, 0.70+/-0.03, and 0.33+/-0.02 in test groups A, C, D, and F, respectively. The A values of the expression of PCNA in A549 cell strains were 0.13+/-0.03 in the control group, 0.10+/-0.02, 0.11+/-0.02, 0.16+/-0.02, and 0.11+/-0.03 in test groups A, B, D, and E, respectively. The A values of MTT in A549 cell strains were 0.50+/-0.06 in the control group, 0.41+/-0.04, 0.45+/-0.03, 0.59+/-0.02, and 0.45+/-0.03 in test groups A, B, D, and E, respectively. The A values of positive cells of apoptosis in A549 cell strains were 0.16+/-0.04 in the control group, 0.18+/-0.02, 0.18+/-0.05, 0.20+/-0.05, and 0.23+/-0.05 in test groups A, B, D, and E, respectively. The apoptotic rates in A549 cells were 1.11% in the

Pathogenesis of Idiopathic Pulmonary Fibrosis

PubMed Central

Wolters, Paul J.; Collard, Harold R.; Jones, Kirk D.

2014-01-01

Idiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial lung disease associated with aging that is characterized by the histopathological pattern of usual interstitial pneumonia. Although an understanding of the pathogenesis of IPF is incomplete, recent advances delineating specific clinical and pathologic features of IPF have led to better definition of the molecular pathways that are pathologically activated in the disease. In this review we highlight several of these advances, with a focus on genetic predisposition to IPF and how genetic changes, which occur primarily in epithelial cells, lead to activation of profibrotic pathways in epithelial cells. We then discuss the pathologic changes within IPF fibroblasts and the extracellular matrix, and we conclude with a summary of how these profibrotic pathways may be interrelated. PMID:24050627

Spilanthol Inhibits COX-2 and ICAM-1 Expression via Suppression of NF-κB and MAPK Signaling in Interleukin-1β-Stimulated Human Lung Epithelial Cells.

PubMed

Huang, Wen-Chung; Wu, Ling-Yu; Hu, Sindy; Wu, Shu-Ju

2018-06-30

Spilanthol a phytochemical derived from the Spilanthes acmella plant has antimicrobial, antioxidant, and anti-inflammatory properties. This study evaluated its effects on the expression of intercellular adhesion molecule 1 (ICAM-1) and inflammation-related mediators in IL-1β-stimulated human lung epithelial A549 cells. Human lung epithelial A549 cells were pretreated with various concentrations of spilanthol (3-100 μM) followed by treatment with IL-1β to induce inflammation. The protein levels of pro-inflammatory cytokines, chemokines, and prostaglandin E2 (PGE2) were measured using ELISA. Cyclooxygenase-2 (COX-2), heme oxygenase (HO-1), nuclear transcription factor kappa-B (NF-κB), and mitogen-activated protein kinase (MAPK) were measured by immunoblotting. The mRNA expression levels of ICAM-1 and MUC5AC were determined by real-time polymerase chain reaction. Spilanthol decreased the expression of PGE 2 , COX-2, TNF-α, and MCP-1. It also decreased ICAM-1 expression and suppressed monocyte adhesion to IL-1β-stimulated A549 cells. Spilanthol also significantly inhibited the phosphorylation of MAPK and I-κB. These results suggest that spilanthol exerts anti-inflammatory effects by inhibiting the expression of the pro-inflammatory cytokines, COX-2, and ICAM-1 by inhibiting the NF-κB and MAPK signaling pathways. Graphical Abstract ᅟ.

Direct and indirect air particle cytotoxicity in human alveolar epithelial cells.

PubMed

Orona, N S; Astort, F; Maglione, G A; Saldiva, P H N; Yakisich, J S; Tasat, D R

2014-08-01

Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their Soluble Fractions (SF) and Conditioned Media (CM) on A549 lung epithelial cells was examined. After 24 h exposure to TP (10 and 100 μg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O₂(-)) generation by NBT and proinflammatory cytokine (TNFα, IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O₂(-) for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O₂(-) and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O₂(-) but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators. Copyright © 2014 Elsevier Ltd. All rights reserved.

28 CFR 549.11 - Program responsibility.

Code of Federal Regulations, 2011 CFR

2011-07-01

... MEDICAL SERVICES Infectious Disease Management § 549.11 Program responsibility. Each institution's Health... institution's infectious disease program in accordance with applicable laws and regulations. ...

28 CFR 549.11 - Program responsibility.

Code of Federal Regulations, 2013 CFR

2013-07-01

... MEDICAL SERVICES Infectious Disease Management § 549.11 Program responsibility. Each institution's Health... institution's infectious disease program in accordance with applicable laws and regulations. ...

28 CFR 549.11 - Program responsibility.

Code of Federal Regulations, 2014 CFR

2014-07-01

... MEDICAL SERVICES Infectious Disease Management § 549.11 Program responsibility. Each institution's Health... institution's infectious disease program in accordance with applicable laws and regulations. ...

28 CFR 549.11 - Program responsibility.

Code of Federal Regulations, 2012 CFR

2012-07-01

... MEDICAL SERVICES Infectious Disease Management § 549.11 Program responsibility. Each institution's Health... institution's infectious disease program in accordance with applicable laws and regulations. ...

28 CFR 549.11 - Program responsibility.

Code of Federal Regulations, 2010 CFR

2010-07-01

... MEDICAL SERVICES Infectious Disease Management § 549.11 Program responsibility. Each institution's Health... institution's infectious disease program in accordance with applicable laws and regulations. ...

Human bronchial epithelial cells injury and cytokine production induced by Tityus serrulatus scorpion venom: An in vitro study.

PubMed

Rigoni, Vera Lucia Silva; Kwasniewski, Fabio H; Vieira, Rodolfo Paula; Linhares, Ingrid Sestrem; da Silva, Joelmir Lucena Veiga; Nogueira-Pedro, Amanda; Zamuner, Stella Regina

2016-09-15

Tityus serrulatus is the scorpion specie responsible for the majority of scorpion sting accidents in Brazil. Symptoms of envenomation by Tityus serrulatus range from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema. Thus, this study has evaluated the participation of bronchial epithelial cells in the pulmonary effects of Tityus serrulatus scorpion venom (Tsv). Human bronchial epithelial cell line BEAS-2B were utilized as a model target and were incubated with Tsv (10 or 50 μg/mL) for 1, 3, 6 and 24 h. Effects on cellular response of venom-induce cytotoxicity were examined including cell viability, cell integrity, cell morphology, apoptosis/necrosis as well as cell activation through the release of pro-inflammatory cytokines IL-1β, IL-6 and IL-8. Tsv caused a decrease in cell viability at 10 and 50 μg/mL, which was confirmed by lactate dehydrogenase (LDH) measurement. Flow cytometry analyses revealed necrosis as the main cell death pathway caused by Tsv. Furthermore, Tsv induced the release of IL-1β, IL-6 and IL-8. Altogether, these results demonstrate that Tsv induces cytotoxic effects on bronchial epithelial cells, involving necrosis and release of pro-inflammatory cytokines, suggesting that bronchial epithelial cells may play a role in the pulmonary injury caused by Tsv. Copyright © 2016 Elsevier Ltd. All rights reserved.

28 CFR 549.62 - Initial referral.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Hunger Strikes, Inmate § 549.62 Initial referral. (a) Staff shall refer an inmate who is observed to be on a hunger strike to medical or mental health staff for evaluation and, when appropriate, for...

Effect of taxol from Pestalotiopsis mangiferae on A549 cells-In vitro study.

PubMed

Kathiravan, Govindarajan; Sureban, Sripathi M

2009-12-01

Pestalotiopsis mangiferae Coelomycete fungi were used to examine the production of taxol. The taxol isolated from this fungus is biologically active against cancer cell lines were investigated for its antiproliferative activity in human Non Small Cell Lung Cancer A549 cells. The results showed that the methylene chloride extraction of Pestalotiopsis mangiferae inhibited the proliferation of A 549 cells as measured by MTT and Trypan blue assay. Flow cytometric analysis showed that methylene chloride extraction of Pestalotiopsis mangiferae blocked cell cycle progression in G0/G1 phase. In addition fungal taxol induced A549 cell apoptosis as determined by propidium iodide staining. Further the percentage of LDH release was increased at increasing concentrations which is a measure of cell death. The levels of sialic acid levels and DNA, RNA and protein levels were decreased after treatment with methylene chloride extraction of Pestalotiopsis mangiferae. We suggests that methylene chloride extraction of Pestalotiopsis mangiferae might be considered for future therapeutic application with further studies against lung cancer.

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

PubMed

Voynow, J A; Young, L R; Wang, Y; Horger, T; Rose, M C; Fischer, B M

1999-05-01

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Per a 10 activates human derived epithelial cell line in a protease dependent manner via PAR-2.

PubMed

Kale, Sagar L; Arora, Naveen

2015-04-01

Protease activity of Per a 10 has been shown to modulate dendritic cells toward Th-2 polarization and to induce airway inflammation. To elucidate the role of serine protease activity of Per a 10 in inducing biochemical responses in epithelial cells. Per a 10 was inactivated by heat treatment (ΔPer a 10) or AEBSF (iPer a 10). A549 cells were exposed to either enzymatically active/inactive Per a 10. The supernatant was analyzed for the secretion of proinflammatory cytokines by ELISA. Ca(2+) mobilization was analyzed by flow cytometry. A PAR-2 derived synthetic peptide 28GTNRSSKGRSLIGKVDGTSHVTGKGVTC54 was incubated with Per a 10 and the resultant cleaved products were analyzed by LC-MS. PAR-2 activation was inhibited by PAR-2 cleavage inhibiting antibody. ΔPer a 10 was completely inactivated whereas iPer a 10 showed some residual activity. nPer a 10 having protease activity increased the secretion of IL-6, IL-8 and GMCSF from A549 in a dose and time dependent manner whereas iPer a 10 has reduced cytokine secretion. ΔPer a 10 and rPer a 10 were unable to activate the cells. nPer a 10 mobilized intracellular Ca(2+). nPer a 10 cleaved the PAR-2 derived peptide between arginine and serine residues (36R-S37) to expose PAR-2 ligand SLIGKV, as determined by LC-MS. Incubating with anti-PAR-2 cleavage antibody showed diminished cytokine secretion when treated with nPer a 10. Serine protease activity of Per a 10 activates A549 cells to secrete proinflammatory cytokines by PAR-2 activation and Ca(2+)mobilization and can be exploited therapeutically. Copyright © 2014 Elsevier GmbH. All rights reserved.

Novel therapeutic approaches for pulmonary fibrosis

PubMed Central

Datta, Arnab; Scotton, Chris J; Chambers, Rachel C

2011-01-01

Pulmonary fibrosis represents the end stage of a number of heterogeneous conditions and is, to a greater or lesser degree, the hallmark of the interstitial lung diseases. It is characterized by the excessive deposition of extracellular matrix proteins within the pulmonary interstitium leading to the obliteration of functional alveolar units and in many cases, respiratory failure. While a small number of interstitial lung diseases have known aetiologies, most are idiopathic in nature, and of these, idiopathic pulmonary fibrosis is the most common and carries with it an appalling prognosis – median survival from the time of diagnosis is less than 3 years. This reflects the lack of any effective therapy to modify the course of the disease, which in turn is indicative of our incomplete understanding of the pathogenesis of this condition. Current prevailing hypotheses focus on dysregulated epithelial–mesenchymal interactions promoting a cycle of continued epithelial cell injury and fibroblast activation leading to progressive fibrosis. However, it is likely that multiple abnormalities in a myriad of biological pathways affecting inflammation and wound repair – including matrix regulation, epithelial reconstitution, the coagulation cascade, neovascularization and antioxidant pathways – modulate this defective crosstalk and promote fibrogenesis. This review aims to offer a pathogenetic rationale behind current therapies, briefly outlining previous and ongoing clinical trials, but will focus on recent and exciting advancements in our understanding of the pathogenesis of idiopathic pulmonary fibrosis, which may ultimately lead to the development of novel and effective therapeutic interventions for this devastating condition. LINKED ARTICLES This article is part of a themed issue on Respiratory Pharmacology. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-1 PMID:21265830

Effects of Bisphenol A Metabolite 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on Lung Function and Type 2 Pulmonary Alveolar Epithelial Cell Growth

PubMed Central

Liu, Shing-Hwa; Su, Chin-Chuan; Lee, Kuan-I; Chen, Ya-Wen

2016-01-01

Bisphenol A (BPA) is recognized as a major pollutant worldwide. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is a major active metabolite of BPA. The epidemiological and animal studies have reported that BPA is harmful to lung function. The role of MBP in lung dysfunction after BPA exposure still remains unclear. This study investigated whether MBP would induce lung alveolar cell damage and evaluated the role of MBP in the BPA exposure-induced lung dysfunction. An in vitro type 2 alveolar epithelial cell (L2) model and an ex vivo isolated reperfused rat lung model were used to determine the effects of BPA or MBP on cell growth and lung function. MBP, but not BPA, dose-dependently increased the mean artery pressure (Pa), pulmonary capillary pressure (Pc), pulmonary capillary filtration coefficient (Kfc), and wet/dry weight ratio in isolated reperfused rat lungs. MBP significantly reduced cell viability and induced caspases-3/7 cleavage and apoptosis and increased AMP-activated protein kinas (AMPK) phosphorylation and endoplasmic reticulum (ER) stress-related molecules expression in L2 cells, which could be reversed by AMPK-siRNA transfection. These findings demonstrated for the first time that MBP exposure induced type 2 alveolar cell apoptosis and lung dysfunction through an AMPK-regulated ER stress signaling pathway. PMID:27982077

Effects of Bisphenol A Metabolite 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on Lung Function and Type 2 Pulmonary Alveolar Epithelial Cell Growth.

PubMed

Liu, Shing-Hwa; Su, Chin-Chuan; Lee, Kuan-I; Chen, Ya-Wen

2016-12-16

Bisphenol A (BPA) is recognized as a major pollutant worldwide. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is a major active metabolite of BPA. The epidemiological and animal studies have reported that BPA is harmful to lung function. The role of MBP in lung dysfunction after BPA exposure still remains unclear. This study investigated whether MBP would induce lung alveolar cell damage and evaluated the role of MBP in the BPA exposure-induced lung dysfunction. An in vitro type 2 alveolar epithelial cell (L2) model and an ex vivo isolated reperfused rat lung model were used to determine the effects of BPA or MBP on cell growth and lung function. MBP, but not BPA, dose-dependently increased the mean artery pressure (Pa), pulmonary capillary pressure (Pc), pulmonary capillary filtration coefficient (K fc ), and wet/dry weight ratio in isolated reperfused rat lungs. MBP significantly reduced cell viability and induced caspases-3/7 cleavage and apoptosis and increased AMP-activated protein kinas (AMPK) phosphorylation and endoplasmic reticulum (ER) stress-related molecules expression in L2 cells, which could be reversed by AMPK-siRNA transfection. These findings demonstrated for the first time that MBP exposure induced type 2 alveolar cell apoptosis and lung dysfunction through an AMPK-regulated ER stress signaling pathway.

Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection.

PubMed

Dapat, Clyde; Saito, Reiko; Suzuki, Hiroshi; Horigome, Tsuneyoshi

2014-01-22

The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited. In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC-MS/MS. We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host-virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development. Copyright © 2013 Elsevier B.V. All rights reserved.

Adaptive changes in global gene expression profile of lung carcinoma A549 cells acutely exposed to distinct types of AhR ligands.

PubMed

Procházková, Jiřina; Strapáčová, Simona; Svržková, Lucie; Andrysík, Zdeněk; Hýžďalová, Martina; Hrubá, Eva; Pěnčíková, Kateřina; Líbalová, Helena; Topinka, Jan; Kléma, Jiří; Espinosa, Joaquín M; Vondráček, Jan; Machala, Miroslav

2018-08-01

Exposure to persistent ligands of aryl hydrocarbon receptor (AhR) has been found to cause lung cancer in experimental animals, and lung adenocarcinomas are often associated with enhanced AhR expression and aberrant AhR activation. In order to better understand the action of toxic AhR ligands in lung epithelial cells, we performed global gene expression profiling and analyze TCDD-induced changes in A549 transcriptome, both sensitive and non-sensitive to CH223191 co-treatment. Comparison of our data with results from previously reported microarray and ChIP-seq experiments enabled us to identify candidate genes, which expression status reflects exposure of lung cancer cells to TCDD, and to predict processes, pathways (e.g. ER stress, Wnt/β-cat, IFNɣ, EGFR/Erbb1), putative TFs (e.g. STAT, AP1, E2F1, TCF4), which may be implicated in adaptive response of lung cells to TCDD-induced AhR activation. Importantly, TCDD-like expression fingerprint of selected genes was observed also in A549 cells exposed acutely to both toxic (benzo[a]pyrene, benzo[k]fluoranthene) and endogenous AhR ligands (2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester and 6-formylindolo[3,2-b]carbazole). Overall, our results suggest novel cellular candidates, which could help to improve monitoring of AhR-dependent transcriptional activity during acute exposure of lung cells to distinct types of environmental pollutants. Copyright © 2018 Elsevier B.V. All rights reserved.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis.

PubMed

Holla, Sahana; Ghorpade, Devram Sampat; Singh, Vikas; Bansal, Kushagra; Balaji, Kithiganahalli Narayanaswamy

2014-09-11

Increased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guérin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells. Cancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-α in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain- and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-α treatment. Here, we show that BCG inhibits TNF-α-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-α-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53(-/-) and MDA-MB-231 cells. Our results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells.

PubMed

Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

2017-01-14

BACKGROUND Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. MATERIAL AND METHODS We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). RESULTS We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. CONCLUSIONS Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer.

Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells

PubMed Central

Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

2017-01-01

Background Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. Material/Methods We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). Results We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. Conclusions Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer. PMID:28087861

A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis.

PubMed

Bauer, Yasmina; Tedrow, John; de Bernard, Simon; Birker-Robaczewska, Magdalena; Gibson, Kevin F; Guardela, Brenda Juan; Hess, Patrick; Klenk, Axel; Lindell, Kathleen O; Poirey, Sylvie; Renault, Bérengère; Rey, Markus; Weber, Edgar; Nayler, Oliver; Kaminski, Naftali

2015-02-01

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-β1-treated primary human lung fibroblasts and transforming growth factor-β1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.

A Novel Genomic Signature with Translational Significance for Human Idiopathic Pulmonary Fibrosis

PubMed Central

Tedrow, John; de Bernard, Simon; Birker-Robaczewska, Magdalena; Gibson, Kevin F.; Guardela, Brenda Juan; Hess, Patrick; Klenk, Axel; Lindell, Kathleen O.; Poirey, Sylvie; Renault, Bérengère; Rey, Markus; Weber, Edgar; Nayler, Oliver; Kaminski, Naftali

2015-01-01

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat–human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-β1–treated primary human lung fibroblasts and transforming growth factor-β1–treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model–human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease. PMID:25029475

4-Nitroquinoline-1-oxide effects human lung adenocarcinoma A549 cells by regulating the expression of POLD4

PubMed Central

HUANG, QIN-MIAO; ZENG, YI-MING; ZHANG, HUA-PING; LV, LIANG-CHAO; YANG, DONG-YONG; LIN, HUI-HUANG

2016-01-01

The aim of the present study was to explore the expression of POLD4 in human lung adenocarcinoma A549 cells under 4-nitroquinoline-1-oxide (4NQO) stimulation to investigate the role of POLD4 in smoking-induced lung cancer. The lung cancer A549 cell line was treated with 4NQO, with or without MG132 (an inhibitor of proteasome activity), and subsequently the POLD4 level was determined by western blot analysis. Secondly, the cell sensitivity to 4NQO and Taxol was determined when the POLD4 expression level was downregulated by siRNA. The POLD4 protein levels in the A549 cells decreased following treatment with 4NQO; however, MG132 could reverse this phenotype. Downregulation of the POLD4 expression by siRNA enhanced A549 cell sensitivity to 4NQO, but not to Taxol. In conclusion, 4NQO affects human lung adenocarcinoma A549 cells by regulating the expression of POLD4. PMID:26998273

Integrated Stress Response Mediates Epithelial Injury in Mechanical Ventilation.

PubMed

Dolinay, Tamas; Himes, Blanca E; Shumyatcher, Maya; Lawrence, Gladys Gray; Margulies, Susan S

2017-08-01

Ventilator-induced lung injury (VILI) is a severe complication of mechanical ventilation that can lead to acute respiratory distress syndrome. VILI is characterized by damage to the epithelial barrier with subsequent pulmonary edema and profound hypoxia. Available lung-protective ventilator strategies offer only a modest benefit in preventing VILI because they cannot impede alveolar overdistension and concomitant epithelial barrier dysfunction in the inflamed lung regions. There are currently no effective biochemical therapies to mitigate injury to the alveolar epithelium. We hypothesize that alveolar stretch activates the integrated stress response (ISR) pathway and that the chemical inhibition of this pathway mitigates alveolar barrier disruption during stretch and mechanical ventilation. Using our established rat primary type I-like alveolar epithelial cell monolayer stretch model and in vivo rat mechanical ventilation that mimics the alveolar overdistension seen in acute respiratory distress syndrome, we studied epithelial responses to mechanical stress. Our studies revealed that the ISR signaling pathway is a key modulator of epithelial permeability. We show that prolonged epithelial stretch and injurious mechanical ventilation activate the ISR, leading to increased alveolar permeability, cell death, and proinflammatory signaling. Chemical inhibition of protein kinase RNA-like endoplasmic reticulum kinase, an upstream regulator of the pathway, resulted in decreased injury signaling and improved barrier function after prolonged cyclic stretch and injurious mechanical ventilation. Our results provide new evidence that therapeutic targeting of the ISR can mitigate VILI.

[Overexpression of Keap1 inhibits the cell proliferation and metastasis and overcomes the drug resistance in human lung cancer A549 cells].

PubMed

Weng, X; Yan, Y Y; Tong, Y H; Fan, Y; Zeng, J M; Wang, L L; Lin, N M

2016-06-23

To investigate the effect of Keap1-Nrf2 pathway on cell proliferation, metastasis and drug resistance of human lung cancer A549 cell line. A549-Keap1 cell line, constantly expressing wild type Keap1, was established by lentiviral transfection. Real-time RT-PCR and western blot were used to determine the expression of Nrf2 and its target gene in A549 cells. Sulforhodamine B (SRB) assay, flow cytometry, colony formation assay, transwell assay, and cell wound-healing assay were performed to explore the effect of wild type Keap1 expression on the proliferation, invasion, migration and drug resistance of A549 cells. Over-expressed Keap1 decreased the expression of Nrf2 protein and the mRNA level of its downstream target genes and inhibited the ability of cell proliferation and clone formation of A549 cells. Keap1 overexpression induced G0/G1 phase arrest. The percentage of A549-Keap1 cells in G0/G1 phase was significantly higher than that of A549-GFP cells (80.2±5.9)% vs. (67.1±0.9%)(P<0.05). Compared with the invasive A549-Keap1 cells (156.33±17.37), the number of invasive A549-GFP cells was significantly higher (306.67±22.19) in a high power field. Keap1 overexpression significantly enhanced the sensitivity of A549 cells to carboplatin and gemcitabine (P<0.01). The IC50s of carboplatin in A549-Keap1 and A549-GFP cells were (52.1±3.3) μmol/L and (107.8±12.9) μmol/L, respectively. The IC50s of gemcitabine in A549-Keap1 and A549-GFP cells were (6.8±1.2) μmol/L and (9.9±0.5) μmol/L, respectively. Keap1 overexpression significantly inhibits the expression of Nrf2 and its downstream target genes, suppresses tumor cell proliferation and metastasis, and enhances the sensitivity of A549 cells to anticancer drugs.

Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection.

PubMed

Remuzgo-Martínez, Sara; Pilares-Ortega, Lilian; Alvarez-Rodríguez, Lorena; Aranzamendi-Zaldunbide, Maitane; Padilla, Daniel; Icardo, Jose Manuel; Ramos-Vivas, Jose

2013-08-01

Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

28 CFR 549.42 - Involuntary admission.

Code of Federal Regulations, 2010 CFR

2010-07-01

... SERVICES Administrative Safeguards for Psychiatric Treatment and Medication § 549.42 Involuntary admission. A court determination is necessary for involuntary hospitalization for psychiatric treatment. A sentenced inmate, not currently committed for psychiatric treatment, who is not able or willing to...

Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis.

PubMed

Luzina, Irina G; Lockatell, Virginia; Hyun, Sang W; Kopach, Pavel; Kang, Phillip H; Noor, Zahid; Liu, Anguo; Lillehoj, Erik P; Lee, Chunsik; Miranda-Ribera, Alba; Todd, Nevins W; Goldblum, Simeon E; Atamas, Sergei P

2016-05-15

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-β and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Jian, E-mail: zhangjian197011@yahoo.com; Zhang, Tao; Ti, Xinyu

2010-08-13

Research highlights: {yields} Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells {yields} Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway {yields} Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* {yields} miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities ofmore » curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.« less

XB130 translocation to microfilamentous structures mediates NNK-induced migration of human bronchial epithelial cells.

PubMed

Wu, Qifei; Nadesalingam, Jeya; Moodley, Serisha; Bai, Xiaohui; Liu, Mingyao

2015-07-20

Cigarette smoking contributes to the pathogenesis of chronic obstructive pulmonary disease and lung cancer. Nicotine-derived nitrosamine ketone (NNK) is the most potent carcinogen among cigarette smoking components, and is known to enhance migration of cancer cells. However, the effect of NNK on normal human bronchial epithelial cells is not well studied. XB130 is a member of actin filament associated protein family and is involved in cell morphology changes, cytoskeletal rearrangement and outgrowth formation, as well as cell migration. We hypothesized that XB130 mediates NNK-induced migration of normal human bronchial epithelial cells. Our results showed that, after NNK stimulation, XB130 was translocated to the cell periphery and enriched in cell motility-associated structures, such as lamellipodia, in normal human bronchial epithelial BEAS2B cells. Moreover, overexpression of XB130 significantly enhanced NNK-induced migration, which requires both the N- and C-termini of XB130. Overexpression of XB130 enhanced NNK-induced protein tyrosine phosphorylation and promoted matrix metalloproteinase-14 translocation to cell motility-associated cellular structures after NNK stimulation. XB130-mediated NNK-induced cell migration may contribute to airway epithelial repair; however, it may also be involved in cigarette smoking-related chronic obstructive pulmonary disease and lung cancer.

What do we know about pulmonary blastoma?: review of literature and clinical case report

PubMed Central

Brodowska-Kania, Dorota; Kotwica, Ewa; Paturej, Aleksandra; Sośnicki, Witold; Patera, Janusz; Giżewska, Agnieszka; Niemczyk, Stanisław

2016-01-01

ABSTRACT Pulmonary blastoma (PB) is a rare form of lung tumour and is accountable for 0.25–0.5% of primary pulmonary malignancies. Initially pulmonary blastoma was divided into three subtypes: biphasic pulmonary blastoma (BPB) consisting of an epithelial and mesenchymal component, well differentiated fetal adenocarcinoma (WDFA) built of well differentiated epithelium and a mesenchymal component and malignant pleuropulmonary blastoma (PPB). Prognosis in this type of cancer is really poor. We present a current review of literature and a clinical case report. Treatment of PB is very difficult. Data and recommendations about the treatment of pulmonary blastoma are still available therefore we should use only observations and clinical case reports. PMID:28008207

Effects of TGF-β signaling blockade on human A549 lung adenocarcinoma cell lines.

PubMed

Xu, Cheng-Cheng; Wu, Lei-Ming; Sun, Wei; Zhang, Ni; Chen, Wen-Shu; Fu, Xiang-Ning

2011-01-01

Transforming growth factor β (TGF-β) is overexpressed in a wide variety of cancer types including lung adenocarcinoma (LAC), and the TGF-β signaling pathway plays an important role in tumor development. To determine whether blockade of the TGF-β signaling pathway can inhibit the malignant biological behavior of LAC, RNA interference (RNAi) technology was used to silence the expression of TGF-β receptor, type II (TGFβRII) in the LAC cell line, A549, and its effects on cell proliferation, invasion and metastasis were examined. Three specific small interfering RNAs (siRNAs) designed for targeting human TGFβRII were transfected into A549 cells. The expression of TGFβRII was detected by Western blot analysis. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The invasion and metastasis of A549 cells were investigated using the wound healing and Matrigel invasion assays. The expression of PI3K, phosphorylated Smad2, Smad4, Akt, Erk1/2, P38 and MMPs was detected by Western blot analysis. The TGFβRII siRNA significantly reduced the expression of TGFβRII in A549 cells. The knockdown of TGFβRII in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis. In addition to the Smad-dependent pathway, independent pathways including the Erk MAPK, PI3K/Akt and p38 MAPK pathways, as well as the expression of MMPs and VEGF, were inhibited. In conclusion, TGF-β signaling is required for LAC progression. Therefore, the blockade of this signaling pathway by the down-regulation of TGFβRII using SiRNA may provide a potential gene therapy for LAC.

Rapamycin protects against paraquat-induced pulmonary fibrosis: Activation of Nrf2 signaling pathway.

PubMed

Xu, Yiheng; Tai, Wenlin; Qu, Xiaoyuan; Wu, Wenjuan; Li, ZhenKun; Deng, Shuhao; Vongphouttha, Chanthasone; Dong, Zhaoxing

2017-08-19

Paraquat (PQ) is a widely used herbicide indeveloping countries worldwide, and pulmonary fibrosis is one of the most typical features of PQ poisoning. The molecular mechanism of PQ toxicity especially how to treat PQ-induced pulmonary fibrosis is still largely unknown. In animal model of pulmonary fibrosis, we used HE staining, western blotting assay and Real-time PCR assay to analyze the effects of rapamycin on the PQ-induced epithelial mesenchymal transition (EMT). We found that PQ induced the pulmonary fibrosis using HE staining and Masson's staining, and up-regulated the activity of HYP and the mRNA expressions of Collagen I and III (COL-1and COL-3) in pulmonary tissues. We also found that rapamycin down-regulated the mesenchymal cell marker Vimentin and up-regulated the epithelial cell marker E-cadherin both in mRNA and protein levels compared with PQ group. And the EMT associated transcription factor Snail was decreased by rapamycin treatment compared with PQ group. And PQ decreased the Nrf2 expression both in mRNA and protein levels, and rapamycin inhibited these effects of PQ. SFN, a activator of Nrf2, could inhibit the EMT and the expression of Snail. And knockdowon of Nrf2 could abolish the inhibitory effects of rapamycin of PQ-induced EMT. In conclusion, rapamycin protects against paraquat-induced pulmonary fibrosis by activation of Nrf2 signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

PubMed Central

Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

2017-01-01

Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

Effect of taxol from Pestalotiopsis mangiferae on A549 cells-In vitro study

PubMed Central

Kathiravan, Govindarajan; Sureban, Sripathi M.

2009-01-01

Pestalotiopsis mangiferae Coelomycete fungi were used to examine the production of taxol. The taxol isolated from this fungus is biologically active against cancer cell lines were investigated for its antiproliferative activity in human Non Small Cell Lung Cancer A549 cells. The results showed that the methylene chloride extraction of Pestalotiopsis mangiferae inhibited the proliferation of A 549 cells as measured by MTT and Trypan blue assay. Flow cytometric analysis showed that methylene chloride extraction of Pestalotiopsis mangiferae blocked cell cycle progression in G0/G1 phase. In addition fungal taxol induced A549 cell apoptosis as determined by propidium iodide staining. Further the percentage of LDH release was increased at increasing concentrations which is a measure of cell death. The levels of sialic acid levels and DNA, RNA and protein levels were decreased after treatment with methylene chloride extraction of Pestalotiopsis mangiferae. We suggests that methylene chloride extraction of Pestalotiopsis mangiferae might be considered for future therapeutic application with further studies against lung cancer. PMID:25206246

[Effect of ginseng rare ginsenoside components combined with paclitaxel on A549 lung cancer].

PubMed

Yang, Lei; Zhang, Zhen-Hai; Jia, Xiao-Bin

2018-04-01

Traditional Chinese medicine combined with anticancer drugs is a new direction of clinical cancer therapy in recent years. In this study, the optimal ratio of ginseng rare ginsenoside components and paclitaxel was optimized by MTT method, and the proliferative, apoptotic and anti-tumor effects of lung cancer A549 cells were investigated. It was found that the inhibitory effect on the proliferation of lung cancer A549 cells was the same as that on paclitaxel when the ratio of rare ginseng rare ginsenoside components to paclitaxel was 4∶6. Further studies showed that the combined therapy significantly increased the inductive effect of apoptosis in A549 cells, and up-regulated the expression of caspase-3 protein and down-regulated the ratio of Bcl-2/Bax. The tumor-bearing mice model showed that the combination therapy of ginseng rare ginsenoside components and paclitaxel could significantly inhibit the growth of tumor and alleviate the toxic and side effects of paclitaxel on liver. A multi-component system of ginseng rare ginsenoside components-paclitaxel was established in this paper. The proliferation and growth of lung cancer A549 cells were inhibited by paclitaxel-induced apoptosis, the dosage of paclitaxel and the toxicity of paclitaxel were reduced, and the effect of anti-lung cancer was enhanced, which provided a theoretical basis for later studies and clinical application. Copyright© by the Chinese Pharmaceutical Association.

Neutral lipid trafficking regulates alveolar type II cell surfactant phospholipid and surfactant protein expression.

PubMed

Torday, John; Rehan, Virender

2011-08-01

Adipocyte differentiation-related protein (ADRP) is a critically important protein that mediates lipid uptake, and is highly expressed in lung lipofibroblasts (LIFs). Triacylglycerol secreted from the pulmonary circulation and stored in lipid storage droplets is a robust hormonal-, growth factor-, and stretch-regulated precursor for surfactant phospholipid synthesis by alveolar type II epithelial (ATII) cells. A549 lung epithelial cells rapidly take up green fluorescent protein (GFP)-ADRP fusion protein-associated lipid droplets (LDs) in a dose-dependent manner. The LDs initially localize to the perinuclear region of the cell, followed by localization in the cytoplasm. Uptake of ADRP-LDs causes a time- and dose-dependent increase in surfactant protein-B (SP-B) expression. This mechanism can be inhibited by either actinomycin D or cycloheximide, indicating that ADRP-LDs induce newly synthesized SP-B. ADRP-LDs concomitantly stimulate saturated phosphatidylcholine (satPC) synthesis by A549 cells, which is inhibited by ADRP antibody, indicating that this is a receptor-mediated mechanism. Intravenous administration of GFP-ADRP LDs to adult rats results in dose-dependent increases in lung ADRP and SP-B expression. These data indicate that lipofibroblast-derived ADRP coordinates ATII cells' synthesis of the surfactant phospholipid-protein complex by stimulating both satPC and SP-B. The authors propose, therefore, that ADRP is the physiologic determinant for the elusive coordinated, stoichiometric synthesis of surfactant phospholipid and protein by pulmonary ATII cells.

[Astaxanthin inhibits proliferation and promotes apoptosis of A549 lung cancer cells via blocking JAK1/STAT3 pathway].

PubMed

Wu, Chuntao; Zhang, Jinji; Liu, Tienan; Jiao, Guimei; Li, Changzai; Hu, Baoshan

2016-06-01

Objective To investigate the anti-tumor effects of astaxanthin on A549 lung cancer cells and the related mechanisms. Methods A549 cells were cultured with various concentrations of astaxanthin (20, 40, 60, 80, 100 μmol/L), and DMSO at the same concentrations served as vehicle controls. The viability of A549 cells was detected by CCK-8 assay; cell cycle and apoptosis were observed by flow cytometry; and the expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), signal transducers and activators of transcription 3 (STAT3), and Janus kinase 1 (JAK1) were evaluated by Western blotting. Results CCK-8 assay showed that astaxanthin decreased the proliferation of A549 cells in a dose-dependent manner. Flow cytometry showed that astaxanthin increased the number of cells in the G0/G1 phase and induced apoptosis in A549 cells. Western blotting showed that astaxanthin up-regulated the expression of Bax and down-regulated the expressions of Bcl-2, STAT3 and JAK1. Conclusion Astaxanthin functions as a potent inhibitor of A549 lung cancer cell growth by targeting JAK1/STAT3 signaling pathway.

40 CFR 63.549 - Notification requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... operating procedures manual required under § 63.545(a) and the standard operating procedures manual for... (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National Emission Standards For Hazardous Air Pollutants From Secondary Lead Smelting § 63.549 Notification requirements. (a...

40 CFR 63.549 - Notification requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... operating procedures manual required under § 63.545(a) and the standard operating procedures manual for... (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National Emission Standards For Hazardous Air Pollutants From Secondary Lead Smelting § 63.549 Notification requirements. (a...

40 CFR 63.549 - Notification requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... operating procedures manual required under § 63.545(a) and the standard operating procedures manual for... (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS FOR SOURCE CATEGORIES National Emission Standards For Hazardous Air Pollutants From Secondary Lead Smelting § 63.549 Notification requirements. (a...

A549 Cells: Lung Carcinoma Cell Line for Adenovirus | NCI Technology Transfer Center | TTC

Cancer.gov

Scientists at the National Cancer Institute have developed a cell line designated A549 that was derived from explanted cultures of human lung cancer tissue. The A549 cell line has been tested under the guidance of the United States Food and Drug Administration (FDA) so, under current Good Manufacturing Practices (GMP), these cells may be suitable for use in manufacturing constructs for use in clinical trials. The National Cancer Institute seeks parties to non-exclusively license this research material.

Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB.

PubMed

Deng, Xu-Feng; Zhou, Dong; Liu, Quan-Xing; Zheng, Hong; Ding, Yan; Xu, Wen-Yue; Min, Jia-Xin; Dai, Ji-Gang

2018-05-01

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

31 CFR 549.312 - United States person; U.S. person.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false United States person; U.S. person. 549.312 Section 549.312 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued... any United States citizen, permanent resident alien, entity organized under the laws of the United...

31 CFR 549.312 - United States person; U.S. person.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false United States person; U.S. person. 549.312 Section 549.312 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued... any United States citizen, permanent resident alien, entity organized under the laws of the United...

31 CFR 549.312 - United States person; U.S. person.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false United States person; U.S. person. 549.312 Section 549.312 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued... any United States citizen, permanent resident alien, entity organized under the laws of the United...

Aberrant epithelial differentiation by cigarette smoke dysregulates respiratory host defence.

PubMed

Amatngalim, Gimano D; Schrumpf, Jasmijn A; Dishchekenian, Fernanda; Mertens, Tinne C J; Ninaber, Dennis K; van der Linden, Abraham C; Pilette, Charles; Taube, Christian; Hiemstra, Pieter S; van der Does, Anne M

2018-04-01

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections. Copyright ©ERS 2018.

Reactive Oxygen Species/Hypoxia-Inducible Factor-1α/Platelet-Derived Growth Factor-BB Autocrine Loop Contributes to Cocaine-Mediated Alveolar Epithelial Barrier Damage

PubMed Central

Yang, Lu; Chen, Xufeng; Simet, Samantha M.; Hu, Guoku; Cai, Yu; Niu, Fang; Kook, Yeonhee

2016-01-01

Abuse of psychostimulants, such as cocaine, has been shown to be closely associated with complications of the lung, such as pulmonary hypertension, edema, increased inflammation, and infection. However, the mechanism by which cocaine mediates impairment of alveolar epithelial barrier integrity that underlies various pulmonary complications has not been well determined. Herein, we investigate the role of cocaine in disrupting the alveolar epithelial barrier function and the associated signaling cascade. Using the combinatorial electric cell–substrate impedance sensing and FITC-dextran permeability assays, we demonstrated cocaine-mediated disruption of the alveolar epithelial barrier, as evidenced by increased epithelial monolayer permeability with a concomitant loss of the tight junction protein zonula occludens-1 (Zo-1) in both mouse primary alveolar epithelial cells and the alveolar epithelial cell line, L2 cells. To dissect the signaling pathways involved in this process, we demonstrated that cocaine-mediated induction of permeability factors, platelet-derived growth factor (PDGF-BB) and vascular endothelial growth factor, involved reactive oxygen species (ROS)-dependent induction of hypoxia-inducible factor (HIF)-1α. Interestingly, we demonstrated that ROS-dependent induction of another transcription factor, nuclear factor erythroid-2–related factor-2, that did not play a role in cocaine-mediated barrier dysfunction. Importantly, this study identifies, for the first time, that ROS/HIF-1α/PDGF-BB autocrine loop contributes to cocaine-mediated barrier disruption via amplification of oxidative stress and downstream signaling. Corroboration of these cell culture findings in vivo demonstrated increased permeability of the alveolar epithelial barrier, loss of expression of Zo-1, and a concomitantly increased expression of both HIF-1α and PDGF-BB. Pharmacological blocking of HIF-1α significantly abrogated cocaine-mediated loss of Zo-1. Understanding the

28 CFR 549.50 - Purpose and scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Plastic Surgery § 549.50 Purpose and scope. The Bureau of Prisons does not ordinarily perform plastic... circumstances where plastic surgery is a component of a presently medically necessary standard of treatment (for...

28 CFR 549.51 - Approval procedures.

Code of Federal Regulations, 2013 CFR

2013-07-01

... SERVICES Plastic Surgery § 549.51 Approval procedures. The Clinical Director shall consider individually any request from an inmate or a BOP medical consultant. (a) In circumstances where plastic surgery is... forward the surgery request to the Office of Medical Designations and Transportation for approval. (b) If...

28 CFR 549.51 - Approval procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

... SERVICES Plastic Surgery § 549.51 Approval procedures. The Clinical Director shall consider individually any request from an inmate or a BOP medical consultant. (a) In circumstances where plastic surgery is... forward the surgery request to the Office of Medical Designations and Transportation for approval. (b) If...

28 CFR 549.51 - Approval procedures.

Code of Federal Regulations, 2011 CFR

2011-07-01

... SERVICES Plastic Surgery § 549.51 Approval procedures. The Clinical Director shall consider individually any request from an inmate or a BOP medical consultant. (a) In circumstances where plastic surgery is... forward the surgery request to the Office of Medical Designations and Transportation for approval. (b) If...

28 CFR 549.51 - Approval procedures.

Code of Federal Regulations, 2012 CFR

2012-07-01

... SERVICES Plastic Surgery § 549.51 Approval procedures. The Clinical Director shall consider individually any request from an inmate or a BOP medical consultant. (a) In circumstances where plastic surgery is... forward the surgery request to the Office of Medical Designations and Transportation for approval. (b) If...

28 CFR 549.51 - Approval procedures.

Code of Federal Regulations, 2010 CFR

2010-07-01

... SERVICES Plastic Surgery § 549.51 Approval procedures. The Clinical Director shall consider individually any request from an inmate or a BOP medical consultant. (a) In circumstances where plastic surgery is... the Clinical Director recommends plastic surgery for the good order and security of the institution...

46 CFR 308.549 - Application for appointment of Cargo Underwriting Agent, Form MA-319.

Code of Federal Regulations, 2011 CFR

2011-10-01

..., Form MA-319. 308.549 Section 308.549 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION EMERGENCY OPERATIONS WAR RISK INSURANCE War Risk Cargo Insurance Iv-General § 308.549 Application for... American War Risk Agency or MARAD. ...

46 CFR 308.549 - Application for appointment of Cargo Underwriting Agent, Form MA-319.

Code of Federal Regulations, 2012 CFR

2012-10-01

..., Form MA-319. 308.549 Section 308.549 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION EMERGENCY OPERATIONS WAR RISK INSURANCE War Risk Cargo Insurance Iv-General § 308.549 Application for... American War Risk Agency or MARAD. ...

46 CFR 308.549 - Application for appointment of Cargo Underwriting Agent, Form MA-319.

Code of Federal Regulations, 2010 CFR

2010-10-01

..., Form MA-319. 308.549 Section 308.549 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION EMERGENCY OPERATIONS WAR RISK INSURANCE War Risk Cargo Insurance Iv-General § 308.549 Application for... American War Risk Agency or MARAD. ...

46 CFR 308.549 - Application for appointment of Cargo Underwriting Agent, Form MA-319.

Code of Federal Regulations, 2013 CFR

2013-10-01

..., Form MA-319. 308.549 Section 308.549 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION EMERGENCY OPERATIONS WAR RISK INSURANCE War Risk Cargo Insurance Iv-General § 308.549 Application for... American War Risk Agency or MARAD. ...

Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis

PubMed Central

Naikawadi, Ram P.; Disayabutr, Supparerk; Mallavia, Benat; Donne, Matthew L.; Green, Gary; La, Janet L.; Rock, Jason R.; Looney, Mark R.; Wolters, Paul J.

2016-01-01

Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin+ mesenchymal cells, collagen deposition, and accumulation of senescence-associated β-galactosidase+ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction. PMID:27699234

Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.

PubMed

Chaudhary, Neelkamal; Datta, Kausik; Askin, Frederic B; Staab, Janet F; Marr, Kieren A

2012-02-01

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.

Matrix Remodeling in Pulmonary Fibrosis and Emphysema

PubMed Central

O’Reilly, Philip; Antony, Veena B.; Gaggar, Amit

2016-01-01

Pulmonary fibrosis and emphysema are chronic lung diseases characterized by a progressive decline in lung function, resulting in significant morbidity and mortality. A hallmark of these diseases is recurrent or persistent alveolar epithelial injury, typically caused by common environmental exposures such as cigarette smoke. We propose that critical determinants of the outcome of the injury-repair processes that result in fibrosis versus emphysema are mesenchymal cell fate and associated extracellular matrix dynamics. In this review, we explore the concept that regulation of mesenchymal cells under the influence of soluble factors, in particular transforming growth factor-β1, and the extracellular matrix determine the divergent tissue remodeling responses seen in pulmonary fibrosis and emphysema. PMID:26741177

Radical-Containing Ultrafine Particulate Matter Initiates Epithelial-to-Mesenchymal Transitions in Airway Epithelial Cells

PubMed Central

Thevenot, Paul T.; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D.; Chustz, Regina E.; Mahne, Sarah; Kelley, Matthew A.; Hebert, Valeria Y.; Dellinger, Barry; Dugas, Tammy R.; DeMayo, Francesco J.

2013-01-01

Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm2) caused substantial necrosis. At low doses (20 μg/cm2), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α–smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air–liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway.

PubMed

Tang, Xia-Li; Yan, Li; Zhu, Ling; Jiao, De-Min; Chen, Jun; Chen, Qing-Yong

2017-09-01

Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC) treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1) up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Green tea extract induces protective autophagy in A549 non-small lung cancer cell line.

PubMed

Izdebska, Magdalena; Klimaszewska-Wiśniewska, Anna; Hałas, Marta; Gagat, Maciej; Grzanka, Alina

2015-12-31

For many decades, polyphenols, including green tea extract catechins, have been reported to exert multiple anti-tumor activities. However, to date the mechanisms of their action have not been completely elucidated. Thus, the aim of this study was to assess the effect of green tea extract on non-small lung cancer A549 cells. A549 cells following treatment with GTE were analyzed using the inverted light and fluorescence microscope. In order to evaluate cell sensitivity and cell death, the MTT assay and Tali image-based cytometer were used, respectively. Ultrastructural alterations were assessed using a transmission electron microscope. The obtained data suggested that GTE, even at the highest dose employed (150 μM), was not toxic to A549 cells. Likewise, the treatment with GTE resulted in only a very small dose-dependent increase in the population of apoptotic cells. However, enhanced accumulation of vacuole-like structures in response to GTE was seen at the light and electron microscopic level. Furthermore, an increase in the acidic vesicular organelles and LC3-II puncta formation was observed under the fluorescence microscope, following GTE treatment. The analysis of the functional status of autophagy revealed that GTE-induced autophagy may provide self-protection against its own cytotoxicity, since we observed that the blockage of autophagy by bafilomycin A1 decreased the viability of A549 cells and potentiated necrotic cell death induction in response to GTE treatment. Collectively, our results revealed that A549 cells are insensitive to both low and high concentrations of the green tea extract, probably due to the induction of cytoprotective autophagy. These data suggest that a potential utility of GTE in lung cancer therapy may lie in its synergistic combinations with drugs or small molecules that target autophagy, rather than in monotherapy.

Effects of tanshinone nanoemulsion and extract on inhibition of lung cancer cells A549

NASA Astrophysics Data System (ADS)

Lee, W. D.; Liang, Y. J.; Chen, B. H.

2016-12-01

Danshen (Salvia miltiorrhiza), a Chinese medicinal herb, consists of several functional components including tanshinones responsible for prevention of several chronic diseases. This study intends to prepare tanshinone extract and nanoemulsion from danshen and determine their inhibition effect on lung cancer cells A549. A highly stable tanshinone nanoemulsion composed of Capryol 90, Tween 80, ethanol and deionized water with the mean particle size of 14.2 nm was successfully prepared. Tanshinone nanoemulsion was found to be more effective in inhibiting A549 proliferation than tanshinone extract. Both nanoemulsion and extract could penetrate into cytoplasm through endocytosis, with the former being more susceptible than the latter. A dose-dependent response in up-regulation of p-JNK, p53 and p21 and down-regulation of CDK2, cyclin D1 and cyclin E1 expressions was observed with the cell cycle arrested at G0/G1 phase. The cellular microcompartment change of A549 was also investigated. The study demonstrated that tanshinone nanoemulsion may be used as a botanic drug for treatment of lung cancer.

Identification of cellular microRNA-136 as a dual regulator of RIG-I-mediated innate immunity that antagonizes H5N1 IAV replication in A549 cells.

PubMed

Zhao, Lianzhong; Zhu, Jiping; Zhou, Hongbo; Zhao, Zongzheng; Zou, Zhong; Liu, Xiaokun; Lin, Xian; Zhang, Xue; Deng, Xuexia; Wang, Ruifang; Chen, Huanchun; Jin, Meilin

2015-10-09

H5N1 influenza A virus (IAV) causes severe respiratory diseases and high mortality rates in animals and humans. MicroRNAs are being increasingly studied to evaluate their potential as therapeutic entities to combat viral infection. However, mechanistic studies delineating the roles of microRNAs in regulating host-H5N1 virus interactions remain scarce. Here, we performed microRNA microarray analysis using A549 human lung epithelial cells infected with a highly pathogenic avian influenza virus. The microRNA expression profile of infected cells identified a small number of microRNAs being dysregulated upon H5N1 influenza A virus infection. Of the differentially expressed microRNAs, miR-136 was up-regulated 5-fold and exhibited potent antiviral activity in vitro against H5N1 influenza A virus, as well as vesicular stomatitis virus. On the one hand, 3'-untranslated region (UTR) reporter analysis revealed a miR-136 binding site in the 3' UTR of IL-6. However, on the other hand, we subsequently determined that miR-136 meanwhile acts as an immune agonist of retinoic acid-inducible gene 1 (RIG-I), thereby causing IL-6 and IFN-β accumulation in A549 cells. Overall, this study implicates the dual role of miRNA-136 in the regulation of host antiviral innate immunity and suggests an important role for the microRNA-activated pathway in viral infection via pattern recognition receptors.

Induction of ER Stress-Mediated Apoptosis by α-Lipoic Acid in A549 Cell Lines

PubMed Central

Kim, Jong In; Lee, Chang Min; Park, Eok-Sung; Kim, Ki Nyun; Kim, Hyung Chul; Lee, Hae Young

2012-01-01

Background α-Lipoic acid (α-LA) has been studied as an anticancer agent as well as a therapeutic agent for diabetes and obesity. We performed this study to evaluate the anticancer effects and mechanisms of α-LA in a lung cancer cell line, A549. Materials and Methods α-LA-induced apoptosis of A549 cells was detected by fluorescence-activated cell sorting analysis and a DNA fragmentation assay. Expression of apoptosis-related genes was analyzed by western blot and reverse transcription-polymerase chain reaction analyses. Results α-LA induced apoptosis and DNA fragmentation in A549 cells in a dose- and time-dependent manner. α-LA increased caspase activity and the degradation of poly (ADP-ribose) polymerase. It induced expression of endoplasmic reticulum (ER) stress-related genes, such as glucose-regulated protein 78, C/EBP-homologous protein, and the short form of X-box binding protein-1, and decreased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein. Reactive oxygen species (ROS) production was induced by α-LA, and the antioxidant N-acetyl-L-cysteine decreased the α-LA-induced increase in expression of apoptosis and ER stress-related proteins. Conclusion α-LA induced ER stress-mediated apoptosis in A549 cells via ROS. α-LA may therefore be clinically useful for treating lung cancer. PMID:22363901

46 CFR 308.549 - Application for appointment of Cargo Underwriting Agent, Form MA-319.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 8 2014-10-01 2014-10-01 false Application for appointment of Cargo Underwriting Agent, Form MA-319. 308.549 Section 308.549 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION EMERGENCY OPERATIONS WAR RISK INSURANCE War Risk Cargo Insurance General § 308.549 Application for...

31 CFR 549.203 - Holding of funds in interest-bearing accounts; investment and reinvestment.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Holding of funds in interest-bearing accounts; investment and reinvestment. 549.203 Section 549.203 Money and Finance: Treasury Regulations... LEBANON SANCTIONS REGULATIONS Prohibitions § 549.203 Holding of funds in interest-bearing accounts...

TXNIP mediates the differential responses of A549 cells to sodium butyrate and sodium 4-phenylbutyrate treatment.

PubMed

Jin, Xuefang; Wu, Nana; Dai, Juji; Li, Qiuxia; Xiao, XiaoQiang

2017-02-01

Sodium butyrate (NaBu) and sodium 4-phenylbutyrate (4PBA) have promising futures in cancer treatment; however, their underlying molecular mechanisms are not clearly understood. Here, we show A549 cell death induced by NaBu and 4PBA are not the same. NaBu treatment induces a significantly higher level of A549 cell death than 4PBA. A gene expression microarray identified more than 5000 transcripts that were altered (>1.5-fold) in NaBu-treated A549 cells, but fewer than 2000 transcripts that were altered in 4PBA. Moreover, more than 100 cell cycle-associated genes were greatly repressed by NaBu, but slightly repressed by 4PBA; few genes were significantly upregulated only in 4PBA-treated cells. Gene expression was further validated by other experiments. Additionally, A549 cells that were treated with these showed changes in glucose consumption, caspase 3/7 activation and histone modifications, as well as enhanced mitochondrial superoxide production. TXNIP was strongly induced by NaBu (30- to 40-fold mRNA) but was only slightly induced by 4PBA (two to fivefold) in A549 cells. TXNIP knockdown by shRNA in A549 cells significantly attenuated caspase 3/7 activation and restored cell viability, while TXNIP overexpression significantly increased caspase 3/7 activation and cell death only in NaBu-treated cells. Moreover, TXNIP also regulated NaBu- but not 4PBA-induced H4K5 acetylation and H3K4 trimethylation, possibly by increasing WDR5 expression. Finally, we demonstrated that 4PBA induced a mitochondrial superoxide-associated cell death, while NaBu did so mainly through a TXNIP-mediated pathway. The above data might benefit the future clinic application. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards.

PubMed

Gminski, Richard; Tang, Tao; Mersch-Sundermann, Volker

2010-06-16

Due to the massive reduction of air-change rates in modern, energy-saving houses and dwellings, the contribution of volatile organic compound (VOCs) emissions from wood-based materials to indoor air quality has become increasingly important. To evaluate toxicity of VOC mixtures typically emitted from pine wood and oriented strand boards (OSB) and their main constituents (selected terpenes and aldehydes), cytotoxicity and genotoxicity were investigated in human A549 lung cells. To facilitate exposure directly via gas phase, a 250 L emission chamber was combined with a Vitrocell exposure system. VOC exposure concentrations were measured by GC/MSD. Biological effects were determined after an exposure time of 1h by measuring cytotoxicity (erythrosine B staining) and genotoxicity (comet assay). Neither cytotoxic nor genotoxic effects were observed for VOC mixtures emitted from pine wood or OSB at loading factors of approximately 13 m(2)/m(3) (worst case conditions) of the panels (with maximum VOC levels of about 80 mg/m(3)) in comparison to clean air. While alpha-pinene and Delta(3)-carene did not induce toxic effects even at exposure concentrations of up to 1800 mg/m(3) and 600 mg/m(3), respectively, hexanal showed a cytotoxic effect at 2000 mg/m(3). The alpha,beta-unsaturated aldehydes 2-heptenal and 2-octenal caused genotoxic effects in concentrations exceeding 100mg/m(3) and 40 mg/m(3), respectively. In conclusion, high concentrations of VOCs and VOC mixtures emitted from pine wood and OSB did not lead to adverse effects in A549 human lung cells even at concentrations 10(2) to 10(5)-fold higher than those found in normal indoor air. Attention must be paid to mutagenic and possibly carcinogenic alpha,beta-unsaturated aldehydes. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

In vitro effects of nicotine on the non-small-cell lung cancer line A549.

PubMed

Gao, Tao; Zhou, Xue-Liang; Liu, Sheng; Rao, Chang-Xiu; Shi, Wen; Liu, Ji-Chun

2016-04-01

To investigate in vitro effects of nicotine on the non-small-cell lung cancer line A549. The case-control study was conducted at the First Affiliated Hospital of Nanchang University from 1st January to 30th June, 2014 and comprised A549 cells which were treated with a series of concentrations of nicotine (0.01 µM, 0.1 µM, 1 µM and 10 µM) for 24 hours. Control cells were incubated under the same conditions without the addition of nicotine. Cell growth was detected by monotetrazolium salt [3-(4, 5-dimethyl-2-thiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Cell apoptosis was detected by Haematoxylin and Eosin staining, immunofluorescence analysis of Filamentous actin and electron microscope observation. Nicotine had no significant effect on A549 cell growth at the dose of 0.01µM (p>0.05), but had significant growth inhibitory effects at the doses of 0.1µM, 1µM and 10µM (p< 0.05 each). A significant decrease in cell numbers was observed on staining (p< 0.05). Significant changes in the size and shape of cells and concomitant changes in cytoskeletons and organelles were observed by immunofluorescence and electron microscope observation (p< 0.05). The growth inhibitory effects of nicotine on A549 cells were found to be dose-dependent.

Maternal exposure to fine particulate air pollution induces epithelial-to-mesenchymal transition resulting in postnatal pulmonary dysfunction mediated by transforming growth factor-β/Smad3 signaling.

PubMed

Tang, Wenting; Du, Lili; Sun, Wen; Yu, Zhiqiang; He, Fang; Chen, Jingsi; Li, Xiaomei; Li, Xiuying; Yu, Lin; Chen, Dunjin

2017-02-05

Fine particles from air pollution, also called particulate matter, less than 2.5 micrometers in diameter (PM2.5), are a threat to child health. Epidemiological investigations have related maternal exposure to PM2.5 to postnatal respiratory symptoms, such as frequent wheezing, chronic cough, and lung function decrements. However, only few experimental animal studies have been performed to study the effects of PM2.5.The aim of this study was to investigate the effects of maternal exposure to PM2.5 on postnatal pulmonary dysfunction in a rat model and to examine the mechanism of PM2.5-induced morphological pulmonary changes.Timed pregnant Sprague-Dawley rats were treated with PM2.5 (0.1, 0.5, 2.5, or 7.5mg/kg) once every three days from day 0 to 18 of pregnancy. After delivery, pups were sacrificed on postnatal day (PND)1 and 28. The effects of transforming growth factor-beta (TGF-β) on epithelial-mesenchymal transition (EMT) were determined by immunohistochemistry, Western blotting, and quantitative RT-PCR. The offspring underwent pulmonary function measurements on PND28, lung tissues were histopathologically examined, and markers of oxidative stress were measured. Maternally PM2.5-exposed offspring pups displayed significant decreases in lung volume parameters, compliance, and airflow during expiration on PND28. The PM2.5-exposed group showed interstitial proliferation in lung histology, significant oxidative stress in lungs, and up-regulation of TGF-β-induced EMT via increased vimentin and α-smooth muscle actin and decreased E-cadherin levels on PND1 and PND28.These results suggest that EMT up-regulation mediated by the TGF-β/Smad3 pathway plays a role in postnatal pulmonary dysfunction associated with maternal exposure to PM2.5. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

PubMed

Tobinaga, Shuichi; Matsumoto, Keitaro; Nagayasu, Takeshi; Furukawa, Katsuro; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Koji, Takehiko

2015-06-29

Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.