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In Vitro Toxicity of Naturally Occurring Silica Nanoparticles in C1 Coal in Bronchial Epithelial Cells  [cached]
Guangjian LI,Yunchao HUANG,Yongjun LIU,Lv GUO
Chinese Journal of Lung Cancer , 2012, DOI: 10.3779/j.issn.1009-3419.2012.10.01
Abstract: Background and objective China’s Xuan Wei County in Yunnan Province have the world’s highest incidence of lung cancer in nonsmoking women-20 times higher than the rest of China. Previous studies showed, this high lung cancer incidence may be associated with the silica particles embedded in the production combustion from the C1 coal. The aim of this study is to separate the silica particles from production combustion from the C1 bituminous coal in Xuan Wei County of Yunnan Province, and study in vitro toxicity of naturally occurring silica particles on BEAS-2B. Methods ①Separating the silica particles from combustion products of C1 bituminous coal by physical method, observing the morphology by Scanning Electron Microscope, analysis elements by SEM-EDX, observed the single particle morphology by Transmission Electron Microscope, analyed its particle size distribution by Laser particle size analyzer, the surface area of silica particles were determined by BET nitrogen adsorption analysis; ②Cell viability of the experimental group (silica; naturally occurring), control group (silica; industrial produced and crystalline silica) was detected by assay used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, and the reactive oxygen species (ROS), lactate dehydrogenase (LDH) were determined after 24 h-72 h exposed to these particles. Results ①The physical method can separate silica particles from production combustion from the C1 bituminous coal, which have different size, and from 30 nm to 120 nm particles accounted for 86.8%, different morphology, irregular surface area and containing trace of aluminum, calcium and iron and other elements; ②Under the same concentration, the experiment group have higher toxicity on BEAS-2B than control groups. Conclusion ①Physical method can separate silica particles from production combustion from the C1 bituminous coal and not change the original morphology and containing trace; ②Naturally occurring silica nanoparticles have irregular morphology, surface area, and containing complex trace elements may has greater toxicity than the silica nanoparticle of industrial produced and crystalline silica.
Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells  [PDF]
Atsushi Yasukawa, Koa Hosoki, Masaaki Toda, Yasushi Miyake, Yuki Matsushima, Takahiro Matsumoto, Daniel Boveda-Ruiz, Paloma Gil-Bernabe, Mizuho Nagao, Mayumi Sugimoto, Yukiko Hiraguchi, Reiko Tokuda, Masahiro Naito, Takehiro Takagi, Corina N. D'Alessandro-Gabazza, Shigeru Suga, Tetsu Kobayashi, Takao Fujisawa, Osamu Taguchi, Esteban C. Gabazza
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0064281
Abstract: Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.
Continuous monitoring of the bronchial epithelial lining fluid by microdialysis
Stig S Tyvold, Erik Sollig?rd, Oddveig Lyng, Sigurd L Steinshamn, Sigurd Gunnes, Petter Aadahl
Respiratory Research , 2007, DOI: 10.1186/1465-9921-8-78
Abstract: Microdialysis catheters were introduced into the right main stem bronchus and into the right subclavian artery of five anesthetized and normoventilated pigs. The flowrate was 2 μl/min and the sampling interval was 60 minutes. Lactate and fluorescein-isothiocyanate-dextran 4 kDa (FD-4) infusions were performed to obtain two levels of steady-state concentrations in blood. Accuracy was defined as [bronchial-MD] divided by [arterial-MD] in percent. Data presented as mean ± 95 percent confidence interval.The accuracy of bronchial MD was calculated with and without correction by the arteriobronchial urea gradient. The arteriobronchial lactate gradient was 1.2 ± 0.1 and FD-4 gradient was 4.0 ± 1.2. Accuracy of bronchial MD with a continuous lactate infusion was mean 25.5% (range 5.7–59.6%) with a coefficient of variation (CV) of 62.6%. With correction by the arteriobronchial urea gradient accuracy was mean 79.0% (57.3–108.1%) with a CV of 17.0%.Urea as a marker of catheter functioning enhances bronchial MD and makes it useful for monitoring substantial changes in the composition of the ELF.The epithelial lining fluid of the lung is important in the understanding mechanisms in acute lung injury, inflammatory lung diseases, cardiac failure, and in pharmacokinetic studies.Today there are no established methods of direct continuous monitoring of the epithelial lining fluid of the lower respiratory tract. The epithelial lining fluid of the bronchi has been examined by bronchioalveolar lavage (BAL), direct aspiration, microsampling, and exhaled breath condensates. BAL and other bronchoscopic techniques have in common that they are invasive, may create lung injury, are based on single or intermittent samples and therefore have limited value as continuous and dynamic monitors of the epithelial lining fluid of the lung. The exhaled breath condensates technique is non-invasive and continuous, but is indirect and best suited for collection of non-volatile hydrophilic solutes[1].Micro
Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells  [PDF]
Anthony Tam, Samuel Wadsworth, Delbert Dorscheid, Shu-Fan Paul Man, Don D. Sin
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0100633
Abstract: Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.
Multipotent Capacity of Immortalized Human Bronchial Epithelial Cells  [PDF]
Oliver Delgado,Aadil A. Kaisani,Monica Spinola,Xian-Jin Xie,Kimberly G. Batten,John D. Minna,Woodring E. Wright,Jerry W. Shay
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0022023
Abstract: While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer.
Defining the Subcellular Interface of Nanoparticles by Live-Cell Imaging  [PDF]
Peter H. Hemmerich, Anna H. von Mikecz
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0062018
Abstract: Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP) experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS) indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.
Comparison of Nasal and Bronchial Epithelial Cells Obtained from Patients with COPD  [PDF]
David M. Comer, J. Stuart Elborn, Madeleine Ennis
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0032924
Abstract: For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.
Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells
Apostolos Bossios, Stelios Psarras, Dimitrios Gourgiotis, Chrysanthi L Skevaki, Andreas G Constantopoulos, Photini Saxoni-Papageorgiou, Nikolaos G Papadopoulos
Respiratory Research , 2005, DOI: 10.1186/1465-9921-6-114
Abstract: Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry.RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation.RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.The bronchial epithelium plays a unique role as a protective physical and functional barrier between external environment and underlying tissues. As a result of this role it is frequently injured and epithelial integrity is damaged. A repair process starts quickly which includes migration of the remaining basal airway epithelial cells to repopulate damaged areas, and subsequent proliferation and differentiation until epithelial integrity has been restored [1,2].Epithelial damage is a key feature of asthma. As a result of inflammation, a large portion of columnar epithelial cells shed and form Creola bodies, detected in sputum and during bronchoscopy in asthmatic patients [3]. This cycle of damage and repair has been proposed as a key mechanism leading to thickening of the airway wall, and other pathologic alterations collectively characterized as airway remodeling [4], which in turn has been associated with incompletely reversible airway narrowing, bronchial
Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation  [PDF]
Laurie-Anne Payet, Linette Kadri, Sébastien Giraud, Caroline Norez, Jean Marc Berjeaud, Christophe Jayle, Sandra Mirval, Frédéric Becq, Clarisse Vandebrouck, Thierry Ferreira
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0089044
Abstract: The F508del-CFTR mutation, responsible for Cystic Fibrosis (CF), leads to the retention of the protein in the endoplasmic reticulum (ER). The mistrafficking of this mutant form can be corrected by pharmacological chaperones, but these molecules showed limitations in clinical trials. We therefore hypothesized that important factors in CF patients may have not been considered in the in vitro assays. CF has also been associated with an altered lipid homeostasis, i. e. a decrease in polyunsaturated fatty acid levels in plasma and tissues. However, the precise fatty acyl content of membrane phospholipids from human CF bronchial epithelial cells had not been studied to date. Since the saturation level of phospholipids can modulate crucial membrane properties, with potential impacts on membrane protein folding/trafficking, we analyzed this parameter for freshly isolated bronchial epithelial cells from CF patients. Interestingly, we could show that Palmitate, a saturated fatty acid, accumulates within Phosphatidylcholine (PC) in CF freshly isolated cells, in a process that could result from hypoxia. The observed PC pattern can be recapitulated in the CFBE41o? cell line by incubation with 100 μM Palmitate. At this concentration, Palmitate induces an ER stress, impacts calcium homeostasis and leads to a decrease in the activity of the corrected F508del-CFTR. Overall, these data suggest that bronchial epithelial cells are lipointoxicated by hypoxia-related Palmitate accumulation in CF patients. We propose that this phenomenon could be an important bottleneck for F508del-CFTR trafficking correction by pharmacological agents in clinical trials.
Cytotoxicity and Induction of Inflammation by Pepsin in Acid in Bronchial Epithelial Cells  [PDF]
Erik Bathoorn,Paul Daly,Birgit Gaiser,Karl Sternad,Craig Poland,William MacNee,Ellen M. Drost
International Journal of Inflammation , 2011, DOI: 10.4061/2011/569416
Abstract: Introduction. Gastroesophageal reflux has been associated with chronic inflammatory diseases and may be a cause of airway remodelling. Aspiration of gastric fluids may cause damage to airway epithelial cells, not only because acidity is toxic to bronchial epithelial cells, but also since it contains digestive enzymes, such as pepsin. Aim. To study whether pepsin enhances cytotoxicity and inflammation in airway epithelial cells, and whether this is pH-dependent. Methods. Human bronchial epithelial cells were exposed to increasing pepsin concentrations in varying acidic milieus, and cell proliferation and cytokine release were assessed. Results. Cell survival was decreased by pepsin exposure depending on its concentration ( ) and pH level of the medium ( ) (both ). Pepsin-induced interleukin-8 release was greater at lower pH ( ; ). Interleukin-6 induction by pepsin was greater at pH 1.5 compared to pH 2.5 (mean difference 434%; ). Conclusion. Pepsin is cytotoxic to bronchial epithelial cells and induces inflammation in addition to acid alone, dependent on the level of acidity. Future studies should assess whether chronic aspiration causes airway remodelling in chronic inflammatory lung diseases. 1. Introduction Aspiration of gastric fluids damages airway epithelial cells [1] due to the toxicity of its low pH [2]. Several in vivo and in vitro models have assessed the effect of acid aspiration on lung injury and inflammation, using a hydrochloric acid solution with a pH ranging from 1 to 1.5 [2–5]. In addition, gastric particles have been found to contribute to lung injury [6]. Previous in vivo and in vitro studies have shown that acid aspiration causes an IL-6 and IL-8 mediated neutrophil influx into the lungs [2, 3, 7, 8]. A correlation between acid aspiration, increased IL-8 levels, and airway neutrophil counts has been found in asthma patients [9]. However, the acidity of gastric fluids might not be the only cause of damage and inflammatory response. Digestive enzymes such as pepsin might be an important factor as well. Pepsin is stored as inactive pepsinogen in the chief cells of the gastric mucosa. It is a protease involved in the digestion of food, and its activity is acid-dependent. The conversion of pepsinogen to pepsin in the stomach starts slowly at pH 6 and reaches optimal activity between pH 1.5 to 2.5. Above pH 6.8, pepsin becomes inactive and above pH 7.5 it is fully inactive and irreversibly denatured [10]. In human gastric fluid, the pH varies from 1.5 to 3, which agrees with pepsin’s activity optimum, and the concentration of pepsin varies
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