WorldWideScience

Sample records for human airway epithelial

  1. The human airway epithelial basal cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Neil R Hackett

    2011-05-01

    Full Text Available The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population.Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels.The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.

  2. Human airway xenograft models of epithelial cell regeneration

    Directory of Open Access Journals (Sweden)

    Puchelle Edith

    2000-10-01

    Full Text Available Abstract Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa.

  3. Detonation Nanodiamond Toxicity in Human Airway Epithelial Cells Is Modulated by Air Oxidation

    Science.gov (United States)

    Detonational nanodiamonds (DND), a nanomaterial with an increasing range of industrial and biomedical applications, have previously been shown to induce a pro-inflammatory response in cultured human airway epithelial cells (HAEC). We now show that surface modifications induced by...

  4. Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

    Science.gov (United States)

    Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

    2018-02-01

    Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

  5. Polystyrene nanoparticles activate ion transport in human airway epithelial cells

    Directory of Open Access Journals (Sweden)

    McCarthy J

    2011-06-01

    Full Text Available J McCarthy1, X Gong2, D Nahirney2, M Duszyk2, MW Radomski11School of Pharmacy and Pharmaceutical Sciences, Panoz Institute, Trinity College Dublin, Dublin, Ireland; 2Department of Physiology, University of Alberta, Edmonton, Alberta, CanadaBackground: Over the last decade, nanotechnology has provided researchers with new nanometer materials, such as nanoparticles, which have the potential to provide new therapies for many lung diseases. In this study, we investigated the acute effects of polystyrene nanoparticles on epithelial ion channel function.Methods: Human submucosal Calu-3 cells that express cystic fibrosis transmembrane conductance regulator (CFTR and baby hamster kidney cells engineered to express the wild-type CFTR gene were used to investigate the actions of negatively charged 20 nm polystyrene nanoparticles on short-circuit current in Calu-3 cells by Ussing chamber and single CFTR Cl- channels alone and in the presence of known CFTR channel activators by using baby hamster kidney cell patches.Results: Polystyrene nanoparticles caused sustained, repeatable, and concentration-dependent increases in short-circuit current. In turn, these short-circuit current responses were found to be biphasic in nature, ie, an initial peak followed by a plateau. EC50 values for peak and plateau short-circuit current responses were 1457 and 315.5 ng/mL, respectively. Short-circuit current was inhibited by diphenylamine-2-carboxylate, a CFTR Cl- channel blocker. Polystyrene nanoparticles activated basolateral K+ channels and affected Cl- and HCO3- secretion. The mechanism of short-circuit current activation by polystyrene nanoparticles was found to be largely dependent on calcium-dependent and cyclic nucleotide-dependent phosphorylation of CFTR Cl- channels. Recordings from isolated inside-out patches using baby hamster kidney cells confirmed the direct activation of CFTR Cl- channels by the nanoparticles.Conclusion: This is the first study to identify

  6. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

    Energy Technology Data Exchange (ETDEWEB)

    Lasalvia, Maria [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Castellani, Stefano [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); D’Antonio, Palma [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Perna, Giuseppe [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Carbone, Annalucia [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy); Colia, Anna Laura; Maffione, Angela Bruna [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Capozzi, Vito [Department of Clinical and Experimental Medicine, University of Foggia, Foggia (Italy); Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari (Italy); Conese, Massimo, E-mail: massimo.conese@unifg.it [Department of Medical and Surgical Sciences, University of Foggia, Foggia (Italy)

    2016-10-15

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in

  7. Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

    International Nuclear Information System (INIS)

    Lasalvia, Maria; Castellani, Stefano; D’Antonio, Palma; Perna, Giuseppe; Carbone, Annalucia; Colia, Anna Laura; Maffione, Angela Bruna; Capozzi, Vito; Conese, Massimo

    2016-01-01

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity in the

  8. Oxytetracycline Inhibits Mucus Secretion and Inflammation in Human Airway Epithelial Cells.

    Science.gov (United States)

    Shah, Said Ahmad; Ishinaga, Hajime; Takeuchi, Kazuhiko

    2017-01-01

    Oxytetracycline is a broad-spectrum antibiotic, but its nonantibacterial effects in the human respiratory tract are unknown. In this study, the effects of oxytetracycline on mucus secretion and inflammation were examined by PCR and ELISA in the human airway epithelial cell line NCI-H292. Oxytetracycline (10 μg/mL) significantly inhibited TNF-α-induced MUC5AC gene expression and MUC5AC protein levels in NCI-H292 cells. It also downregulated IL-8 and IL-1β gene expression and IL-1β protein levels. Our findings demonstrated that oxytetracycline suppressed mucus production and inflammation in human respiratory epithelial cells, providing further evidence for the usefulness of oxytetracycline for human airway inflammatory diseases. © 2017 S. Karger AG, Basel.

  9. Human airway epithelial cell cultures for modeling respiratory syncytial virus infection.

    Science.gov (United States)

    Pickles, Raymond J

    2013-01-01

    Respiratory syncytial virus (RSV) is an important human respiratory pathogen with narrow species tropism. Limited availability of human pathologic specimens during early RSV-induced lung disease and ethical restrictions for RSV challenge studies in the lower airways of human volunteers has slowed our understanding of how RSV causes airway disease and greatly limited the development of therapeutic strategies for reducing RSV disease burden. Our current knowledge of RSV infection and pathology is largely based on in vitro studies using nonpolarized epithelial cell-lines grown on plastic or in vivo studies using animal models semipermissive for RSV infection. Although these models have revealed important aspects of RSV infection, replication, and associated inflammatory responses, these models do not broadly recapitulate the early interactions and potential consequences of RSV infection of the human columnar airway epithelium in vivo. In this chapter, the pro et contra of in vitro models of human columnar airway epithelium and their usefulness in respiratory virus pathogenesis and vaccine development studies will be discussed. The use of such culture models to predict characteristics of RSV infection and the correlation of these findings to the human in vivo situation will likely accelerate our understanding of RSV pathogenesis potentially identifying novel strategies for limiting the severity of RSV-associated airway disease.

  10. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE cell model we found that both micromolar (3.9 μM and submicromolar (0.8 μM arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-. We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  11. Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells.

    Science.gov (United States)

    Wu, Qun; Jiang, Di; Minor, Maisha; Chu, Hong Wei

    2014-01-01

    The use of electronic cigarettes (e-cigarettes) is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV) infection. We examined the effects of e-cigarette liquid (e-liquid) on pro-inflammatory cytokine (e.g., IL-6) production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1) in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.

  12. Electronic cigarette liquid increases inflammation and virus infection in primary human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Qun Wu

    Full Text Available The use of electronic cigarettes (e-cigarettes is rapidly increasing in the United States, especially among young people since e-cigarettes have been perceived as a safer alternative to conventional tobacco cigarettes. However, the scientific evidence regarding the human health effects of e-cigarettes on the lung is extremely limited. The major goal of our current study is to determine if e-cigarette use alters human young subject airway epithelial functions such as inflammatory response and innate immune defense against respiratory viral (i.e., human rhinovirus, HRV infection.We examined the effects of e-cigarette liquid (e-liquid on pro-inflammatory cytokine (e.g., IL-6 production, HRV infection and host defense molecules (e.g., short palate, lung, and nasal epithelium clone 1, SPLUNC1 in primary human airway epithelial cells from young healthy non-smokers. Additionally, we examined the role of SPLUNC1 in lung defense against HRV infection using a SPLUNC1 knockout mouse model. We found that nicotine-free e-liquid promoted IL-6 production and HRV infection. Addition of nicotine into e-liquid further amplified the effects of nicotine-free e-liquid. Moreover, SPLUNC1 deficiency in mice significantly increased lung HRV loads. E-liquid inhibited SPLUNC1 expression in primary human airway epithelial cells. These findings strongly suggest the deleterious health effects of e-cigarettes in the airways of young people. Our data will guide future studies to evaluate the impact of e-cigarettes on lung health in human populations, and help inform the public about potential health risks of e-cigarettes.

  13. Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes.

    Directory of Open Access Journals (Sweden)

    David Proud

    Full Text Available Human rhinovirus (HRV infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

  14. Response of cultured human airway epithelial cells to X-rays and energetic α-particles

    International Nuclear Information System (INIS)

    Yang, T.C.; Holley, W.R.; Curtis, S.B.; Gruenert, D.C.; California Univ., San Francisco, CA

    1990-01-01

    Radon and its progeny, which emit α-particles during decay, may play an important role in inducing human lung cancer. To gain a better understanding of the biological effects of α-particles in human lung we studied the response of cultured human airway epithelial cells to X-rays and monoenergetic helium ions. Experimental results indicated that the radiation response of primary cultures was similar to that for airway epithelial cells that were transformed with a plasmid containing an origin-defective SV40 virus. The RBE for cell inactivation determined by the ratio of D 0 for X-rays to that for 8 MeV helium ions was 1.8-2.2. The cross-section for helium ions, calculated from the D 0 value, was about 24 μm 2 for cells of the primary culture. This cross-section is significantly smaller than the average geometric nuclear area (∼ 180 μm 2 ), suggesting that an average of 7.5 α-particles (8 MeV helium ions) per cell nucleus are needed to induce a lethal lesion. (author)

  15. Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells

    Science.gov (United States)

    Yadav, Umesh CS; Ramana, KV; Srivastava, SK

    2013-01-01

    Aldose reductase (AR), a glucose metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30μM) than glucose. Acrolein, a major endogenous lipid peroxidation product as well as component of environmental pollutant and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells SAECs. Exposure of SAECs to varying concentrations of acrolein caused cell-death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low (5 to 10 μM) but not high (>10 μM) concentrations of acrolein-induced SAECs cell death. AR inhibition protected SAECs from low dose (5 μM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail-moment, and annexin-V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of pro-apoptotic proteins Bax and Bad from cytosol to the mitochondria, and that of Bcl2 and BclXL from mitochondria to cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPK) such as extracellular signal-regulated kinases 1 and 2 (ERK1/2), stress-activated protein kinases/c-jun NH2-terminal kinases (SAPK/JNK) and p38MAPK, and c-jun were transiently activated in airway epithelial cells by acrolein in a concentration and time-dependent fashion, which were significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. PMID:23770200

  16. Aldose reductase regulates acrolein-induced cytotoxicity in human small airway epithelial cells.

    Science.gov (United States)

    Yadav, Umesh C S; Ramana, K V; Srivastava, Satish K

    2013-12-01

    Aldose reductase (AR), a glucose-metabolizing enzyme, reduces lipid aldehydes and their glutathione conjugates with more than 1000-fold efficiency (Km aldehydes 5-30 µM) relative to glucose. Acrolein, a major endogenous lipid peroxidation product as well as a component of environmental pollutants and cigarette smoke, is known to be involved in various pathologies including atherosclerosis, airway inflammation, COPD, and age-related disorders, but the mechanism of acrolein-induced cytotoxicity is not clearly understood. We have investigated the role of AR in acrolein-induced cytotoxicity in primary human small airway epithelial cells (SAECs). Exposure of SAECs to varying concentrations of acrolein caused cell death in a concentration- and time-dependent manner. AR inhibition by fidarestat prevented the low-dose (5-10 µM) but not the high-dose (>10 µM) acrolein-induced SAEC death. AR inhibition protected SAECs from low-dose (5 µM) acrolein-induced cellular reactive oxygen species (ROS). Inhibition of acrolein-induced apoptosis by fidarestat was confirmed by decreased condensation of nuclear chromatin, DNA fragmentation, comet tail moment, and annexin V fluorescence. Further, fidarestat inhibited acrolein-induced translocation of the proapoptotic proteins Bax and Bad from the cytosol to the mitochondria and that of Bcl2 and BclXL from the mitochondria to the cytosol. Acrolein-induced cytochrome c release from mitochondria was also prevented by AR inhibition. The mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases 1 and 2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38MAPK, and c-Jun were transiently activated in airway epithelial cells by acrolein in a concentration- and time-dependent fashion, which was significantly prevented by AR inhibition. These results suggest that AR inhibitors could prevent acrolein-induced cytotoxicity in the lung epithelial cells. Copyright © 2013 Elsevier Inc. All rights

  17. The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

    Science.gov (United States)

    Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

    2004-09-01

    Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

  18. Staphylococcus aureus α-Toxin Induces Actin Filament Remodeling in Human Airway Epithelial Model Cells.

    Science.gov (United States)

    Ziesemer, Sabine; Eiffler, Ina; Schönberg, Alfrun; Müller, Christian; Hochgräfe, Falko; Beule, Achim G; Hildebrandt, Jan-Peter

    2018-04-01

    Exposure of cultured human airway epithelial model cells (16HBE14o-, S9) to Staphylococcus aureus α-toxin (hemolysin A, Hla) induces changes in cell morphology and cell layer integrity that are due to the inability of the cells to maintain stable cell-cell or focal contacts and to properly organize their actin cytoskeletons. The aim of this study was to identify Hla-activated signaling pathways involved in regulating the phosphorylation level of the actin-depolymerizing factor cofilin. We used recombinant wild-type hemolysin A (rHla) and a variant of Hla (rHla-H35L) that is unable to form functional transmembrane pores to treat immortalized human airway epithelial cells (16HBE14o-, S9) as well as freshly isolated human nasal tissue. Our results indicate that rHla-mediated changes in cofilin phosphorylation require the formation of functional Hla pores in the host cell membrane. Formation of functional transmembrane pores induced hypophosphorylation of cofilin at Ser3, which was mediated by rHla-induced attenuation of p21-activated protein kinase and LIM kinase activities. Because dephosphorylation of pSer3-cofilin results in activation of this actin-depolymerizing factor, treatment of cells with rHla resulted in loss of actin stress fibers from the cells and destabilization of cell shape followed by the appearance of paracellular gaps in the cell layers. Activation of protein kinase A or activation of small GTPases (Rho, Rac, Cdc42) do not seem to be involved in this response.

  19. Bioaerosols from a food waste composting plant affect human airway epithelial cell remodeling genes.

    Science.gov (United States)

    Chang, Min-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-12-24

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 10(2) conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5-10 μm) having higher endotoxin levels than did fine particles (0.5-2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21 WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers.

  20. Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

    Science.gov (United States)

    Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-01-01

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

  1. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    Science.gov (United States)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-09-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO2 NPs (size range 4-33 nm), two preparations of CeO2 NPs (9-36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15-240 μg/cm2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm2, in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO2 and CeO2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured nanomaterials.

  2. Comparative effects of metal oxide nanoparticles on human airway epithelial cells and macrophages

    International Nuclear Information System (INIS)

    Rotoli, Bianca Maria; Bussolati, Ovidio; Costa, Anna Luisa; Blosi, Magda; Di Cristo, Luisana; Zanello, Pier Paolo; Bianchi, Massimiliano G.; Visigalli, Rossana; Bergamaschi, Enrico

    2012-01-01

    Among nanomaterials of industrial relevance, metal-based nanoparticles (NPs) are widely used, but their effects on airway cells are relatively poorly characterized. To compare the effects of metal NPs on cells representative of the lung-blood barrier, Calu-3 epithelial cells and Raw264.7 macrophages were incubated with three industrially relevant preparations of TiO 2 NPs (size range 4–33 nm), two preparations of CeO 2 NPs (9–36 nm) and CuO NPs (25 nm). While Raw264.7 were grown on standard plasticware, Calu-3 cells were seeded on permeable filters, where they form a high-resistance monolayer, providing an in vitro model of the airway barrier. Metal NPs, obtained from industrial sources, were characterized under the conditions adopted for the biological tests. Cytotoxicity was assessed with resazurin method in both epithelial and macrophage cells, while epithelial barrier permeability was monitored measuring the trans-epithelial electrical resistance (TEER). In macrophages, titania and ceria had no significant effect on viability in the whole range of nominal doses tested (15–240 μg/cm 2 of monolayer), while CuO NPs produced a marked viability loss. Moreover, only CuO NPs, but not the other NPs, lowered TEER of Calu-3 monolayers, pointing to the impairment of the epithelial barrier. TEER decreased by 30 % at the dose of 10 μg/cm 2 of CuO NPs, compared to untreated control, and was abolished at doses ≥80 μg/cm 2 , in strict correlation with changes in cell viability. These results indicate that (1) CuO NPs increase airway epithelium permeability even at relatively low doses and are significantly toxic for macrophages and airway epithelial cells, likely through the release of Cu ions in the medium; (2) TiO 2 and CeO 2 NPs do not affect TEER and exhibit little acute toxicity for airway epithelial cells and macrophages; and (3) TEER measurement can provide a simple method to assess the impairment of in vitro airway epithelial barrier model by manufactured

  3. Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Takashi Kawaguchi

    1999-01-01

    Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

  4. Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases

    Directory of Open Access Journals (Sweden)

    Adil Aldhahrani

    2017-03-01

    Full Text Available Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B. The immortalised human bronchial epithelial cell line (BEAS-2B was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL-8, IL-6 and granulocyte−macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L−1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L−1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B. Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo. This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

  5. Anti-Cytotoxic and Anti-Inflammatory Effects of the Macrolide Antibiotic Roxithromycin in Sulfur Mustard-Exposed Human Airway Epithelial Cells

    National Research Council Canada - National Science Library

    Gao1, Radharaman Ray2, Yan Xiao3, Peter E. Barker3 and Prab, Xiugong

    2006-01-01

    .... In this study, the anti-cytotoxic and anti-inflammatory effects of a representative macrolide antibiotic, roxithromycin, were tested in vitro using SM-exposed normal human small airway epithelial (SAE...

  6. Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles

    International Nuclear Information System (INIS)

    Tal, Tamara L.; Simmons, Steven O.; Silbajoris, Robert; Dailey, Lisa; Cho, Seung-Hyun; Ramabhadran, Ram; Linak, William; Reed, William; Bromberg, Philip A.; Samet, James M.

    2010-01-01

    Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

  7. Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts

    Directory of Open Access Journals (Sweden)

    Luketich James D

    2004-12-01

    Full Text Available Abstract Background Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR. We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers. Methods and Results At the mRNA level, human bronchial epithelial (HBE cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (α1, δ, and ε significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by α-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure. Conclusions These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine.

  8. Downregulation of SLC7A7 Triggers an Inflammatory Phenotype in Human Macrophages and Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Bianca Maria Rotoli

    2018-03-01

    Full Text Available Lysinuric protein intolerance (LPI is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA, a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

  9. THE BUFFER CAPACITY OF AIRWAY EPITHELIAL SECRETIONS

    Directory of Open Access Journals (Sweden)

    Dusik eKim

    2014-06-01

    Full Text Available The pH of airway epithelial secretions influences bacterial killing and mucus properties and is reduced by acidic pollutants, gastric reflux, and respiratory diseases such as cystic fibrosis (CF. The effect of acute acid loads depends on buffer capacity, however the buffering of airway secretions has not been well characterized. In this work we develop a method for titrating micro-scale (30 µl volumes and use it to study fluid secreted by the human airway epithelial cell line Calu-3, a widely used model for submucosal gland serous cells. Microtitration curves revealed that HCO3- is the major buffer. Peak buffer capacity (β increased from 17 to 28 mM/pH during forskolin stimulation, and was reduced by >50% in fluid secreted by cystic fibrosis transmembrane conductance regulator (CFTR-deficient Calu-3 monolayers, confirming an important role of CFTR in HCO3- secretion. Back-titration with NaOH revealed non-volatile buffer capacity due to proteins synthesized and released by the epithelial cells. Lysozyme and mucin concentrations were too low to buffer Calu-3 fluid significantly, however model titrations of porcine gastric mucins at concentrations near the sol-gel transition suggest that mucins may contribute to the buffer capacity of ASL in vivo. We conclude that CFTR-dependent HCO3- secretion and epithelially-derived proteins are the predominant buffers in Calu-3 secretions.

  10. Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Christian Schwarzer

    Full Text Available Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11 with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells. PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins and massively (10-80 fold increase, termed "swarming", but transiently (random swimming after 15 mins, to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii PA use pili to bind to epithelial cells near wounds.

  11. CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

    Science.gov (United States)

    Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

    2006-05-01

    We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

  12. Scavenger receptors in human airway epithelial cells: role in response to double-stranded RNA.

    Directory of Open Access Journals (Sweden)

    Audrey Dieudonné

    Full Text Available Scavenger receptors and Toll-like receptors (TLRs cooperate in response to danger signals to adjust the host immune response. The TLR3 agonist double stranded (dsRNA is an efficient activator of innate signalling in bronchial epithelial cells. In this study, we aimed at defining the role played by scavenger receptors expressed by bronchial epithelial cells in the control of the innate response to dsRNA both in vitro and in vivo. Expression of several scavenger receptor involved in pathogen recognition was first evaluated in human bronchial epithelial cells in steady-state and inflammatory conditions. Their implication in the uptake of dsRNA and the subsequent cell activation was evaluated in vitro by competition with ligand of scavenger receptors including maleylated ovalbumin and by RNA silencing. The capacity of maleylated ovalbumin to modulate lung inflammation induced by dsRNA was also investigated in mice. Exposure to tumor necrosis factor-α increased expression of the scavenger receptors LOX-1 and CXCL16 and the capacity to internalize maleylated ovalbumin, whereas activation by TLR ligands did not. In contrast, the expression of SR-B1 was not modulated in these conditions. Interestingly, supplementation with maleylated ovalbumin limited dsRNA uptake and inhibited subsequent activation of bronchial epithelial cells. RNA silencing of LOX-1 and SR-B1 strongly blocked the dsRNA-induced cytokine production. Finally, administration of maleylated ovalbumin in mice inhibited the dsRNA-induced infiltration and activation of inflammatory cells in bronchoalveolar spaces and lung draining lymph nodes. Together, our data characterize the function of SR-B1 and LOX-1 in bronchial epithelial cells and their implication in dsRNA-induced responses, a finding that might be relevant during respiratory viral infections.

  13. ATP Release from Human Airway Epithelial Cells Exposed to Staphylococcus aureus Alpha-Toxin

    Directory of Open Access Journals (Sweden)

    Romina Baaske

    2016-12-01

    Full Text Available Airway epithelial cells reduce cytosolic ATP content in response to treatment with S. aureus alpha-toxin (hemolysin A, Hla. This study was undertaken to investigate whether this is due to attenuated ATP generation or to release of ATP from the cytosol and extracellular ATP degradation by ecto-enzymes. Exposure of cells to rHla did result in mitochondrial calcium uptake and a moderate decline in mitochondrial membrane potential, indicating that ATP regeneration may have been attenuated. In addition, ATP may have left the cells through transmembrane pores formed by the toxin or through endogenous release channels (e.g., pannexins activated by cellular stress imposed on the cells by toxin exposure. Exposure of cells to an alpha-toxin mutant (H35L, which attaches to the host cell membrane but does not form transmembrane pores, did not induce ATP release from the cells. The Hla-mediated ATP-release was completely blocked by IB201, a cyclodextrin-inhibitor of the alpha-toxin pore, but was not at all affected by inhibitors of pannexin channels. These results indicate that, while exposure of cells to rHla may somewhat reduce ATP production and cellular ATP content, a portion of the remaining ATP is released to the extracellular space and degraded by ecto-enzymes. The release of ATP from the cells may occur directly through the transmembrane pores formed by alpha-toxin.

  14. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    Science.gov (United States)

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  15. Biodiesel exhaust-induced cytotoxicity and proinflammatory mediator production in human airway epithelial cells.

    Science.gov (United States)

    Mullins, Benjamin J; Kicic, Anthony; Ling, Kak-Ming; Mead-Hunter, Ryan; Larcombe, Alexander N

    2016-01-01

    Increasing use of biodiesel has prompted research into the potential health effects of biodiesel exhaust exposure. Few studies directly compare the health consequences of mineral diesel, biodiesel, or blend exhaust exposures. Here, we exposed human epithelial cell cultures to diluted exhaust generated by the combustion of Australian ultralow-sulfur-diesel (ULSD), unprocessed canola oil, 100% canola biodiesel (B100), and a blend of 20% canola biodiesel mixed with 80% ULSD. The physicochemical characteristics of the exhaust were assessed and we compared cellular viability, apoptosis, and levels of interleukin (IL)-6, IL-8, and Regulated on Activation, Normal T cell Expressed and Secreted (RANTES) in exposed cultured cells. Different fuel types produced significantly different amounts of exhaust gases and different particle characteristics. All exposures resulted in significant apoptosis and loss of viability when compared with control, with an increasing proportion of biodiesel being correlated with a decrease in viability. In most cases, exposure to exhaust resulted in an increase in mediator production, with the greatest increases most often in response to B100. Exposure to pure canola oil (PCO) exhaust did not increase mediator production, but resulted in a significant decrease in IL-8 and RANTES in some cases. Our results show that canola biodiesel exhaust exposure elicits inflammation and reduces viability of human epithelial cell cultures in vitro when compared with ULSD exhaust exposure. This may be related to an increase in particle surface area and number in B100 exhaust when compared with ULSD exhaust. Exposure to PCO exhaust elicited the greatest loss of cellular viability, but virtually no inflammatory response, likely due to an overall increase in average particle size. © 2014 Wiley Periodicals, Inc.

  16. Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Taissia G Popova

    Full Text Available Rift valley fever virus (RVFV infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK and downstream transcriptional factors [STAT1 (Y701, ATF2 (T69/71, MSK1 (S360 and CREB (S133]. NF-κB phosphorylation was also increased. Activation of p53 (S15, S46 correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473, along with phosphorylation of FOX 01/03 (T24/31 which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication.

  17. Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells.

    Science.gov (United States)

    Zhao, Jinming; Minami, Yoshinori; Etling, Emily; Coleman, John M; Lauder, Sarah N; Tyrrell, Victoria; Aldrovandi, Maceler; O'Donnell, Valerie; Claesson, Hans-Erik; Kagan, Valerian; Wenzel, Sally

    2017-12-01

    Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 μM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

  18. Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

    Science.gov (United States)

    Silva, Carlos A M; Danelishvili, Lia; McNamara, Michael; Berredo-Pinho, Márcia; Bildfell, Robert; Biet, Franck; Rodrigues, Luciana S; Oliveira, Albanita V; Bermudez, Luiz E; Pessolani, Maria C V

    2013-07-01

    This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

  19. Dioscorin pre-treatment protects A549 human airway epithelial cells from hydrogen peroxide-induced oxidative stress.

    Science.gov (United States)

    Hsu, Jeng-Yuan; Chu, Jao-Jia; Chou, Ming-Chih; Chen, Ya-Wen

    2013-10-01

    Hydrogen peroxide (H(2)O(2)) is a highly reactive oxygen species involved in lung and bronchial epithelium injury. Increased H(2)O(2) levels have been reported in expired breath condensates of patients with inflammatory airway diseases such as chronic obstructive pulmonary disease. Protecting airway epithelial cells from oxidative stress is an important task in the prevention and management of airway diseases. Previous studies demonstrate that yam (Dioscorea batatas Decne) has antioxidant and anti-trypsin activities. This study evaluated the validity of dioscorin in vitro. The results showed that dioscorin attenuated the alteration of H(2)O(2) on G2/M cell cycle arrest. This might be associated with the activation of IκB and subsequent inactivation of NF-κB. Furthermore, dioscorin suppressed IL-8 secretion and reduced changes of adhesion molecule expressions in H(2)O(2)-injured A549 cells. These results help in understanding the potential of traditional Chinese herbal medicine as treatment for airway inflammatory diseases.

  20. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Science.gov (United States)

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  1. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    International Nuclear Information System (INIS)

    Zaccone, Eric J.; Goldsmith, W. Travis; Shimko, Michael J.; Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A.; Fedan, Jeffrey S.

    2015-01-01

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (R t ) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na + transport, without affecting Cl − transport or Na + ,K + -pump activity. R t was unaffected. Na + transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. - Highlights: • Butter flavoring vapor effects on human cultured airway epithelium were studied. • Na transport was reduced by a 6-h exposure to 25 ppm diacetyl and 2,3-pentanedione. • Na transport recovered 18 h after exposure. • > 60 ppm transepithelial voltage and resistance were abolished; cells were damaged. • Cells metabolized diacetyl and 2,3-pentanedione into acetoin and 2-OH-3-pentanone.

  2. The effects of gas humidification with high-flow nasal cannula on cultured human airway epithelial cells.

    Science.gov (United States)

    Chidekel, Aaron; Zhu, Yan; Wang, Jordan; Mosko, John J; Rodriguez, Elena; Shaffer, Thomas H

    2012-01-01

    Humidification of inspired gas is important for patients receiving respiratory support. High-flow nasal cannula (HFNC) effectively provides temperature and humidity-controlled gas to the airway. We hypothesized that various levels of gas humidification would have differential effects on airway epithelial monolayers. Calu-3 monolayers were placed in environmental chambers at 37°C with relative humidity (RH) 90% (HFNC) for 4 and 8 hours with 10 L/min of room air. At 4 and 8 hours, cell viability and transepithelial resistance measurements were performed, apical surface fluid was collected and assayed for indices of cell inflammation and function, and cells were harvested for histology (n = 6/condition). Transepithelial resistance and cell viability decreased over time (P < 0.001) between HFNC and dry groups (P < 0.001). Total protein secretion increased at 8 hours in the dry group (P < 0.001). Secretion of interleukin (IL)-6 and IL-8 in the dry group was greater than the other groups at 8 hours (P < 0.001). Histological analysis showed increasing injury over time for the dry group. These data demonstrate that exposure to low humidity results in reduced epithelial cell function and increased inflammation.

  3. The Effects of Gas Humidification with High-Flow Nasal Cannula on Cultured Human Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Aaron Chidekel

    2012-01-01

    Full Text Available Humidification of inspired gas is important for patients receiving respiratory support. High-flow nasal cannula (HFNC effectively provides temperature and humidity-controlled gas to the airway. We hypothesized that various levels of gas humidification would have differential effects on airway epithelial monolayers. Calu-3 monolayers were placed in environmental chambers at 37°C with relative humidity (RH 90% (HFNC for 4 and 8 hours with 10 L/min of room air. At 4 and 8 hours, cell viability and transepithelial resistance measurements were performed, apical surface fluid was collected and assayed for indices of cell inflammation and function, and cells were harvested for histology (n=6/condition. Transepithelial resistance and cell viability decreased over time (P<0.001 between HFNC and dry groups (P<0.001. Total protein secretion increased at 8 hours in the dry group (P<0.001. Secretion of interleukin (IL-6 and IL-8 in the dry group was greater than the other groups at 8 hours (P<0.001. Histological analysis showed increasing injury over time for the dry group. These data demonstrate that exposure to low humidity results in reduced epithelial cell function and increased inflammation.

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

    Science.gov (United States)

    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

  5. CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways.

    Science.gov (United States)

    Shahabuddin, Syed; Ji, Rong; Wang, Ping; Brailoiu, Eugene; Dun, Na; Yang, Yi; Aksoy, Mark O; Kelsen, Steven G

    2006-07-01

    Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 +/- 88% (mean +/- SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca(2+)] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5-10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

  6. Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents

    Energy Technology Data Exchange (ETDEWEB)

    Zaccone, Eric J. [Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV (United States); Goldsmith, W. Travis [Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV (United States); Shimko, Michael J. [Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV (United States); Wells, J.R.; Schwegler-Berry, Diane; Willard, Patsy A.; Case, Shannon L.; Thompson, Janet A. [Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV (United States); Fedan, Jeffrey S. [Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV (United States); Pathology and Physiology Research Branch, National Institute for Occupational Safety and Health, Morgantown, WV (United States)

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (R{sub t}) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na{sup +} transport, without affecting Cl{sup −} transport or Na{sup +},K{sup +}-pump activity. R{sub t} was unaffected. Na{sup +} transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. - Highlights: • Butter flavoring vapor effects on human cultured airway epithelium were studied. • Na transport was reduced by a 6-h exposure to 25 ppm diacetyl and 2,3-pentanedione. • Na transport recovered 18 h after exposure. • > 60 ppm transepithelial voltage and resistance were abolished; cells were damaged. • Cells metabolized diacetyl and 2,3-pentanedione

  7. Airway epithelial NF-κB activation promotes Mycoplasma pneumoniae clearance in mice.

    Directory of Open Access Journals (Sweden)

    Di Jiang

    Full Text Available Respiratory infections including atypical bacteria Mycoplasma pneumoniae (Mp contribute to the pathobiology of asthma and chronic obstructive pulmonary disease (COPD. Mp infection mainly targets airway epithelium and activates various signaling pathways such as nuclear factor κB (NF-κB. We have shown that short palate, lung, and nasal epithelium clone 1 (SPLUNC1 serves as a novel host defense protein and is up-regulated upon Mp infection through NF-κB activation in cultured human and mouse primary airway epithelial cells. However, the in vivo role of airway epithelial NF-κB activation in host defense against Mp infection has not been investigated. In the current study, we investigated the effects of in vivo airway epithelial NF-κB activation on lung Mp clearance and its association with airway epithelial SPLUNC1 expression.Non-antimicrobial tetracycline analog 9-t-butyl doxycycline (9-TB was initially optimized in mouse primary tracheal epithelial cell culture, and then utilized to induce in vivo airway epithelial specific NF-κB activation in conditional NF-κB transgenic mice (CC10-(CAIKKβ with or without Mp infection. Lung Mp load and inflammation were evaluated, and airway epithelial SPLUNC1 protein was examined by immunohistochemistry. We found that 9-TB treatment in NF-κB transgene positive (Tg+, but not transgene negative (Tg- mice significantly reduced lung Mp load. Moreover, 9-TB increased airway epithelial SPLUNC1 protein expression in NF-κB Tg+ mice.By using the non-antimicrobial 9-TB, our study demonstrates that in vivo airway epithelial NF-κB activation promotes lung bacterial clearance, which is accompanied by increased epithelial SPLUNC1 expression.

  8. Role of airway epithelial barrier dysfunction in pathogenesis of asthma.

    Science.gov (United States)

    Gon, Yasuhiro; Hashimoto, Shu

    2018-01-01

    Bronchial asthma is characterized by persistent cough, increased sputum, and repeated wheezing. The pathophysiology underlying these symptoms is the hyper-responsiveness of the airway along with chronic airway inflammation. Repeated injury, repair, and regeneration of the airway epithelium following exposure to environmental factors and inflammation results in histological changes and functional abnormalities in the airway mucosal epithelium; such changes are believed to have a significant association with the pathophysiology of asthma. Damage to the barrier functions of the airway epithelium enhances mucosal permeability of foreign substances in the airway epithelium of patients with asthma. Thus, epithelial barrier fragility is closely involved in releasing epithelial cytokines (e.g., TSLP, IL-25, and IL-33) because of the activation of airway epithelial cells, dendritic cells, and innate group 2 innate lymphoid cells (ILC2). Functional abnormalities of the airway epithelial cells along with the activation of dendritic cells, Th2 cells, and ILC2 form a single immunopathological unit that is considered to cause allergic airway inflammation. Here we use the latest published literature to discuss the potential pathological mechanisms regarding the onset and progressive severity of asthma with regard to the disruption of the airway epithelial function. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  9. Effect of titanium dioxide nanoparticles (TiO2 NPs) on the expression of mucin genes in human airway epithelial cells.

    Science.gov (United States)

    Kim, Gui Ok; Choi, Yoon Seok; Bae, Chang Hoon; Song, Si-Youn; Kim, Yong-Dae

    2017-01-01

    Titanium dioxide nanoparticles (TiO 2 NPs) are utilized with growing frequency for a wide variety of industrial applications. Recently, acute and chronic exposures to TiO 2 NPs have been found to induce inflammatory response in the human respiratory tract. However, the effect and mechanism underlying the induction of major airway mucins by TiO 2 NPs have not been elucidated. This study was conducted to characterize the effect of TiO 2 NPs, and the mechanism involved, on the expressions of airway mucins in human airway epithelial cells. In NCI-H292 cells and primary cultures of normal nasal epithelial cells, the effects of TiO 2 NPs and signaling pathway for airway mucin genes were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassays and immunoblot analysis using several specific inhibitors and small interfering RNAs (siRNAs). TiO 2 NPs increased MUC5B expression and activated the phosphorylations of extracellular signal-related kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). U0126 (an ERK1/2 MAPK inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited TiO 2 NPs-induced MUC5B expression. And knockdown of ERK1, ERK2 and p38 MAPK using siRNAs significantly blocked TiO 2 NPs-induced MUC5B mRNA expression. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by TiO 2 NPs, and knockdown by TLR4 siRNA significantly attenuated TiO 2 NPs-induced MUC5B mRNA expression and the TiO 2 NPs-induced phosphorylations of ERK1/2 and p38 MAPK. These results demonstrate for the first time that TiO 2 NPs induce MUC5B expression via TLR4-dependent ERK1/2 and p38 MAPK signaling pathways in respiratory epithelium.

  10. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    Science.gov (United States)

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  11. Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

    NARCIS (Netherlands)

    Pyrc, Krzysztof; Sims, Amy C.; Dijkman, Ronald; Jebbink, Maarten; Long, Casey; Deming, Damon; Donaldson, Eric; Vabret, Astrid; Baric, Ralph; van der Hoek, Lia; Pickles, Raymond

    2010-01-01

    Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is

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

    Science.gov (United States)

    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

  13. Regulated Mucin Secretion from Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kenneth Bruce Adler

    2013-09-01

    Full Text Available Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3x10^6 D per monomer whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ~1 um in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among MARCKS, cysteine string protein (CSP, HSP70 and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG. Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the

  14. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    Science.gov (United States)

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  15. Regulation of cytokine production in human alveolar macrophages and airway epithelial cells in response to ambient air pollution particles: Further mechanistic studies

    International Nuclear Information System (INIS)

    Becker, Susanne; Mundandhara, Sailaja; Devlin, Robert B.; Madden, Michael

    2005-01-01

    In order to better understand how ambient air particulate matter (PM) affect lung health, the two main airway cell types likely to interact with inhaled particles, alveolar macrophages (AM) and airway epithelial cells have been exposed to particles in vitro and followed for endpoints of inflammation, and oxidant stress. Separation of Chapel Hill PM 10 into fine and coarse size particles revealed that the main proinflammatory response (TNF, IL-6, COX-2) in AM was driven by material present in the coarse PM, containing 90-95% of the stimulatory material in PM10. The particles did not affect expression of hemoxygenase-1 (HO-1), a sensitive marker of oxidant stress. Primary cultures of normal human bronchial epithelial cells (NHBE) also responded to the coarse fraction with higher levels of IL-8 and COX-2, than induced by fine or ultrafine PM. All size PM induced oxidant stress in NHBE, while fine PM induced the highest levels of HO-1 expression. The production of cytokines in AM by both coarse and fine particles was blocked by the toll like receptor 4 (TLR4) antagonist E5531 involved in the recognition of LPS and Gram negative bacteria. The NHBE were found to recognize coarse and fine PM through TLR2, a receptor with preference for recognition of Gram positive bacteria. Compared to ambient PM, diesel PM induced only a minimal cytokine response in both AM and NHBE. Instead, diesel suppressed LPS-induced TNF and IL-8 release in AM. Both coarse and fine ambient air PM were also found to inhibit LPS-induced TNF release while silica, volcanic ash or carbon black had no inhibitory effect. Diesel particles did not affect cytokine mRNA induction nor protein accumulation but interfered with the release of cytokine from the cells. Ambient coarse and fine PM, on the other hand, inhibited both mRNA induction and protein production. Exposure to coarse and fine PM decreased the expression of TLR4 in the macrophages. Particle-induced decrease in TLR4 and hyporesponsiveness to LPS

  16. In vivo models of human airway epithelium repair and regeneration

    Directory of Open Access Journals (Sweden)

    C. Coraux

    2005-12-01

    Full Text Available Despite an efficient defence system, the airway surface epithelium, in permanent contact with the external milieu, is frequently injured by inhaled pollutants, microorganisms and viruses. The response of the airway surface epithelium to an acute injury includes a succession of cellular events varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even to complete denudation of the basement membrane. The epithelium has then to repair and regenerate to restore its functions. The in vivo study of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to reconstitute a functional respiratory epithelium after several weeks. Humanised tracheal xenograft models have been developed in immunodeficient nude and severe combined immunodeficient (SCID mice in order to mimic the natural regeneration process of the human airway epithelium and to analyse the cellular and molecular events involved during the different steps of airway epithelial reconstitution. These models represent very powerful tools for analysing the modulation of the biological functions of the epithelium during its regeneration. They are also very useful for identifying stem/progenitor cells of the human airway epithelium. A better knowledge of the mechanisms involved in airway epithelium regeneration, as well as the characterisation of the epithelial stem and progenitor cells, may pave the way to regenerative therapeutics, allowing the reconstitution of a functional airway epithelium in numerous respiratory diseases, such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis and bronchiolitis.

  17. Effects of nitrogen-doped multi-walled carbon nanotubes compared to pristine multi-walled carbon nanotubes on human small airway epithelial cells.

    Science.gov (United States)

    Mihalchik, Amy L; Ding, Weiqiang; Porter, Dale W; McLoughlin, Colleen; Schwegler-Berry, Diane; Sisler, Jennifer D; Stefaniak, Aleksandr B; Snyder-Talkington, Brandi N; Cruz-Silva, Rodolfo; Terrones, Mauricio; Tsuruoka, Shuji; Endo, Morinobu; Castranova, Vincent; Qian, Yong

    2015-07-03

    Nitrogen-doped multi-walled carbon nanotubes (ND-MWCNTs) are modified multi-walled carbon nanotubes (MWCNTs) with enhanced electrical properties that are used in a variety of applications, including fuel cells and sensors; however, the mode of toxic action of ND-MWCNT has yet to be fully elucidated. In the present study, we compared the interaction of ND-MWCNT or pristine MWCNT-7 with human small airway epithelial cells (SAEC) and evaluated their subsequent bioactive effects. Transmission electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and X-ray diffraction suggested the presence of N-containing defects in the lattice of the nanotube. The ND-MWCNTs were determined to be 93.3% carbon, 3.8% oxygen, and 2.9% nitrogen. A dose-response cell proliferation assay showed that low doses of ND-MWCNT (1.2μg/ml) or MWCNT-7 (0.12μg/ml) increased cellular proliferation, while the highest dose of 120μg/ml of either material decreased proliferation. ND-MWCNT and MWCNT-7 appeared to interact with SAEC at 6h and were internalized by 24h. ROS were elevated at 6 and 24h in ND-MWCNT exposed cells, but only at 6h in MWCNT-7 exposed cells. Significant alterations to the cell cycle were observed in SAEC exposed to either 1.2μg/ml of ND-MWCNT or MWCNT-7 in a time and material-dependent manner, possibly suggesting potential damage or alterations to cell cycle machinery. Our results indicate that ND-MWCNT induce effects in SAEC over a time and dose-related manner which differ from MWCNT-7. Therefore, the physicochemical characteristics of the materials appear to alter their biological effects. Published by Elsevier Ireland Ltd.

  18. [Effects of transient receptor potential melastatin 8 cation channels on inflammatory reaction induced by cold temperatures in human airway epithelial cells].

    Science.gov (United States)

    Li, Min-chao; Perelman, Juliy M; Kolosov, Victor P; Zhou, Xiang-dong

    2011-10-01

    To explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) in cold-induced production of inflammatory factors in airway epithelial cells and related signal transduction mechanism. The 16HBE human airway epithelial cells were stimulated with cold temperature (18°C). In intervention experiments, cells were pretreated with TRPM8 channel antagonist BCTC, protein kinase C (PKC) specific inhibitor calphostin C and transfected with TRPM8 shRNA or control shRNA respectively, and thereafter cold stimulation was applied. Cells were divided into 6 groups: a control group (incubated at 37°C), a cold stimulation group, a cold stimulation + BCTC group, a cold stimulation + TRPM8 shRNA group, a cold stimulation + control shRNA group, a cold stimulation + calphostin C group. Western blot was performed to show the extent of knockdown in TRPM8 protein expression in the TRPM8 shRNA transfected cells. Dynamics of relative concentration of intracellular Ca(2+) in the former 5 groups were measured by calcium imaging techniques. Images were taken at one frame per 10 seconds. The levels of interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α mRNA and protein were detected by real-time PCR and ELISA respectively. The highest relative concentration of intracellular calcium in cold stimulation group (2.36 ± 0.24) was higher than that of control group (1.01 ± 0.02) (t = 12.52, P cold stimulation group (t = 6.69 and 9.12, all P cold stimulation group[0.66 ± 0.16, 0.77 ± 0.15, 0.73 ± 0.09 and (92 ± 13) ng/L, (125 ± 22) ng/L, (88 ± 12) ng/L ] were significantly higher than those in control group [0.37 ± 0.08, 0.32 ± 0.07, 0.48 ± 0.10 and (52 ± 8) ng/L, (50 ± 9) ng/L, (61 ± 8) ng/L] (t = 3.20 - 6.26, all P cold stimulation + BCTC group [0.42 ± 0.09, 0.52 ± 0.13, 0.52 ± 0.12 and (72 ± 8) ng/L, (92 ± 14) ng/L, (68 ± 11) ng/L], cold stimulation + TRPM8 shRNA group [0.41 ± 0.10, 0.49 ± 0.08, 0.50 ± 0.08 and (60 ± 12) ng/L, (89 ± 14) ng

  19. Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium

    International Nuclear Information System (INIS)

    Rennolds, Jessica; Malireddy, Smitha; Hassan, Fatemat; Tridandapani, Susheela; Parinandi, Narasimham; Boyaka, Prosper N.; Cormet-Boyaka, Estelle

    2012-01-01

    Highlights: ► Cadmium induces secretion of IL-6 and IL-8 by two distinct pathways. ► Cadmium increases NAPDH oxidase activity leading to Erk activation and IL-8 secretion. ► Curcumin prevents cadmium-induced secretion of both IL-6 and IL-8 by airway cells. ► 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 cadmium 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-κB 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.

  20. Curcumin regulates airway epithelial cell cytokine responses to the pollutant cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Rennolds, Jessica; Malireddy, Smitha; Hassan, Fatemat; Tridandapani, Susheela; Parinandi, Narasimham [Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, The Ohio State University, Columbus, OH 43210 (United States); Boyaka, Prosper N. [Department of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210 (United States); Cormet-Boyaka, Estelle, E-mail: Estelle.boyaka@osumc.edu [Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, The Ohio State University, Columbus, OH 43210 (United States)

    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 cadmium 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.

  1. Role of H2O2 in the Oxidative Effects of Zinc Exposure in Human Airway Epithelial Cells

    Science.gov (United States)

    Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn(2+)) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn(2+) toxicity is not fully understood. H202 and Zn(2+) hav...

  2. Interleukin (IL) 36 gamma induces mucin 5AC, oligomeric mucus/gel-forming expression via IL-36 receptor-extracellular signal regulated kinase 1 and 2, and p38-nuclear factor kappa-light-chain-enhancer of activated B cells in human airway epithelial cells.

    Science.gov (United States)

    Bae, Chang Hoon; Choi, Yoon Seok; Na, Hyung Gyun; Song, Si-Youn; Kim, Yong-Dae

    2018-03-01

    Mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) expression is significantly increased in allergic and inflammatory airway diseases. Interleukin (IL) 36 gamma is predominantly expressed in airway epithelial cells and plays an important role in innate and adaptive immune responses. IL-36 gamma is induced by many inflammatory mediators, including cytokines and bacterial and viral infections. However, the association between IL-36 gamma and mucin secretion in human airway epithelial cells has not yet been fully investigated. The objective of this study was to determine whether IL-36 gamma might play a role in the regulation of mucin secretion in airway epithelial cells. We investigated the effect and brief signaling pathway of IL-36 gamma on MUC5AC expression in human airway epithelial cells. Enzyme immunoassay, immunoblot analysis, immunofluorescence staining, reverse transcriptase-polymerase chain reaction (PCR), and real-time PCR were performed in mucin-producing human airway epithelial NCI-H292 cells and in human nasal epithelial cells after pretreatment with IL-36 gamma, several specific inhibitors, or small interfering RNAs (siRNA). IL-36 gamma induced MUC5AC expression and activated the phosphorylation of extracellular signal regulated kinase (ERK) 1 and 2, p38, and nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-kappa B). IL-36 receptor antagonist significantly attenuated these effects. The specific inhibitor and siRNA of ERK1, ERK2, p38, and NF-kappa B significantly attenuated IL-36 gamma induced MUC5AC expression. These results indicated that IL-36 gamma induced MUC5AC expression via the IL-36 receptor-mediated ERK1/2 and p38/NF-kappa B pathway in human airway epithelial cells.

  3. House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

    Directory of Open Access Journals (Sweden)

    Timmerman J André B

    2006-03-01

    Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

  4. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    International Nuclear Information System (INIS)

    Doupnik, C.A.; Leikauf, G.D.

    1990-01-01

    Injury to the airway mucosa after exposure to environmental irritants is associated with pulmonary inflammation and bronchial hyperresponsiveness. To better understand the relationships between mediator release and airway epithelial cell injury during irritant exposures, we studied the effects of acrolein, a low-molecular-weight aldehyde found in cigarette smoke, on arachidonic acid metabolism in cultured bovine tracheal epithelial cells. Confluent airway epithelial cell monolayers, prelabeled with [3H]arachidonic acid, released significant levels of 3H activity when exposed (20 min) to 100 microM acrolein. [3H]arachidonic acid products were resolved using reverse-phase high-performance liquid chromatography. Under control conditions the released 3H activity coeluted predominantly with the cyclooxygenase product, prostaglandin (PG) E2. After exposure to acrolein, significant peaks in 3H activity coeluted with the lipoxygenase products 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, as well as with PGE2, PGF2 alpha, and 6-keto-PGF1 alpha. Dose-response relationships for acrolein-induced release of immunoreactive PGF2 alpha and PGE2 from unlabeled epithelial monolayers demonstrated 30 microM acrolein as the threshold dose, with 100 microM acrolein inducing nearly a fivefold increase in both PGF2 alpha and PGE2. Cellular viability after exposure to 100 microM acrolein, determined by released lactate dehydrogenase activity, was not affected until exposure periods were greater than or equal to 2 h. These results implicate the airway epithelial cell as a possible source of eicosanoids after exposure to acrolein

  5. Bioelectric and Morphological Response of Liquid-Covered Human Airway Epithelial Calu-3 Cell Monolayer to Periodic Deposition of Colloidal 3-Mercaptopropionic-Acid Coated CdSe-CdS/ZnS Core-Multishell Quantum Dots.

    Directory of Open Access Journals (Sweden)

    Aizat Turdalieva

    Full Text Available Lung epithelial cells are extensively exposed to nanoparticles present in the modern urban environment. Nanoparticles, including colloidal quantum dots (QDs, are also considered to be potentially useful carriers for the delivery of drugs into the body. It is therefore important to understand the ways of distribution and the effects of the various types of nanoparticles in the lung epithelium. We use a model system of liquid-covered human airway epithelial Calu-3 cell cultures to study the immediate and long-term effects of repeated deposition of colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs on the lung epithelial cell surface. By live confocal microscope imaging and by QD fluorescence measurements we show that the QD permeation through the mature epithelial monolayers is very limited. At the time of QD deposition, the transepithelial electrical resistance (TEER of the epithelial monolayers transiently decreased, with the decrement being proportional to the QD dose. Repeated QD deposition, once every six days for two months, lead to accumulation of only small amounts of the QDs in the cell monolayer. However, it did not induce any noticeable changes in the long-term TEER and the molecular morphology of the cells. The colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs could therefore be potentially used for the delivery of drugs intended for the surface of the lung epithelia during limited treatment periods.

  6. Neuronal NOS localises to human airway cilia.

    Science.gov (United States)

    Jackson, Claire L; Lucas, Jane S; Walker, Woolf T; Owen, Holly; Premadeva, Irnthu; Lackie, Peter M

    2015-01-30

    Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Polarized Airway Epithelial Models for Immunological Co-Culture Studies

    DEFF Research Database (Denmark)

    Papazian, Dick; Würtzen, Peter A; Hansen, Søren Werner Karlskov

    2016-01-01

    Epithelial cells line all cavities and surfaces throughout the body and play a substantial role in maintaining tissue homeostasis. Asthma and other atopic diseases are increasing worldwide and allergic disorders are hypothesized to be a consequence of a combination of dysregulation...... of the epithelial response towards environmental antigens and genetic susceptibility, resulting in inflammation and T cell-derived immune responses. In vivo animal models have long been used to study immune homeostasis of the airways but are limited by species restriction and lack of exposure to a natural...

  8. Chronic respiratory aeroallergen exposure in mice induces epithelial-mesenchymal transition in the large airways.

    Directory of Open Access Journals (Sweden)

    Jill R Johnson

    Full Text Available Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1 levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease.

  9. TRPV1 inhibition attenuates IL-13 mediated asthma features in mice by reducing airway epithelial injury.

    Science.gov (United States)

    Rehman, Rakhshinda; Bhat, Younus Ahmad; Panda, Lipsa; Mabalirajan, Ulaganathan

    2013-03-01

    Even though neurogenic axis is well known in asthma pathogenesis much attention had not been given on this aspect. Recent studies have reported the importance of TRP channels, calcium-permeable ion channels and key molecules in neurogenic axis, in asthma therapeutics. The role of TRPV1 channels has been underestimated in chronic respiratory diseases as TRPV1 knockout mice of C57BL/6 strains did not attenuate the features of these diseases. However, this could be due to strain differences in the distribution of airway capsaicin receptors. Here, we show that TRPV1 inhibition attenuates IL-13 induced asthma features by reducing airway epithelial injury in BALB/c mice. We found that IL-13 increased not only the lung TRPV1 levels but also TRPV1 expression in bronchial epithelia in BALB/c rather than in C57BL/6 mice. TRPV1 knockdown attenuated airway hyperresponsiveness, airway inflammation, goblet cell metaplasia and subepithelial fibrosis induced by IL-13 in BALB/c mice. Further, TRPV1 siRNA treatment reduced not only the cytosolic calpain and mitochondrial calpain 10 activities in the lung but also bronchial epithelial apoptosis indicating that TRPV1 siRNA might have corrected the intracellular and intramitochondrial calcium overload and its consequent apoptosis. Knockdown of IL-13 in allergen induced asthmatic mice reduced TRPV1, cytochrome c, and activities of calpain and caspase 3 in lung cytosol. Thus, these findings suggest that induction of TRPV1 with IL-13 in bronchial epithelia could lead to epithelial injury in in vivo condition. Since TRPV1 expression is correlated with human asthma severity, TRPV1 inhibition could be beneficial in attenuating airway epithelial injury and asthma features. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Immunoregulation by airway epithelial cells (AECs against respiratory virus infection

    Directory of Open Access Journals (Sweden)

    Yan YAN

    2017-11-01

    Full Text Available The respiratory tract is primary contact site of the body and environment, and it is ventilated by 10-20 thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbes, which contain the disease-causing pathogens. Airway epithelial cells (AECs are known to have innate sensor functions, which are similar to the "professional" immune cells, such as alveolar macrophage and sub- or intra-epithelial dendritic cells (DCs. Thus AECs are able to detect invading microbial danger including different types of respiratory viruses, and mount a potent host response, for example, activating type Ⅰ interferon signaling pathway genes. To avoid chronic inflammation and maintain the immunological homeostasis, the pulmonary system has developed intrinsic mechanisms to control local immune responses. Most recently, the role of AECs in control of local immunity has gained much attention, as 1 AECs express the pattern recognition receptors (PRRs, such as Toll-like receptors, retinoic acid inducible gene Ⅰ (RIG-I-like receptor, and so on, thus AECs are equipped to participate in innate detection of microbial encounter; 2 To keep immunological homeostasis in the respiratory tract, AECs behave not only as innate immune sensors but also as immune modulators in parallel, through modulating the sensitivity of innate immune sensing of both AECs per se and sub- or intra-epithelial immune cells; 3 Loss of modularity capacity of AECs might be involved in the development of chronic airway diseases. In present review, how the AECs act will be intensively discussed in response to respiratory viruses and modulate the local immunity through cis- and trans-factors (direct and indirect factors, as well as the consequence of impairment of this control of local immunity, in the development and exacerbation of airway diseases, such as acute and chronic rhinosinusitis. DOI: 10.11855/j.issn.0577-7402.2017.10.02

  11. Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

    Science.gov (United States)

    Pirela, Sandra V.; Miousse, Isabelle R.; Lu, Xiaoyan; Castranova, Vincent; Thomas, Treye; Qian, Yong; Bello, Dhimiter; Kobzik, Lester; Koturbash, Igor; Demokritou, Philip

    2015-01-01

    Background Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives We assessed the biological responses of a panel of human cell lines to PEPs. Methods Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in

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

    NARCIS (Netherlands)

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

    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

  13. Human corneal epithelial subpopulations

    DEFF Research Database (Denmark)

    Søndergaard, Chris Bath

    2013-01-01

    Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defective or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity...... subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum...

  14. ATP7B detoxifies silver in ciliated airway epithelial cells

    International Nuclear Information System (INIS)

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-01-01

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B -/- mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag + /Cu + transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  15. Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc

    Science.gov (United States)

    Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional...

  16. Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

    Directory of Open Access Journals (Sweden)

    Llobet-Brossa Enrique

    2009-08-01

    Full Text Available Abstract Background Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. Results The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. Conclusion Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide.

  17. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice.

    Science.gov (United States)

    Park, Sin-Hye; Gong, Ju-Hyun; Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee

    2015-01-01

    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases.

  18. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses.

    Directory of Open Access Journals (Sweden)

    Isabella Eckerle

    Full Text Available Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat and Eidolon helvum (Straw-colored fruit bat, were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.

  19. Time-resolved quantitative proteome profiling of host-pathogen interactions: the response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells.

    Science.gov (United States)

    Schmidt, Frank; Scharf, Sandra S; Hildebrandt, Petra; Burian, Marc; Bernhardt, Jörg; Dhople, Vishnu; Kalinka, Julia; Gutjahr, Melanie; Hammer, Elke; Völker, Uwe

    2010-08-01

    Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.

  20. Inhibition by salmeterol and cilomilast of fluticasone-enhanced IP-10 release in airway epithelial cells.

    Science.gov (United States)

    Reddy, P J; Aksoy, Mark O; Yang, Yi; Li, Xiu Xia; Ji, Rong; Kelsen, Steven G

    2008-02-01

    The CXC chemokines, IP-10/CXCL10 and IL-8/CXCL8, play a role in obstructive lung disease by attracting Th1/Tc1 lymphocytes and neutrophils, respectively. Inhaled corticosteroids (ICS) and long acting beta 2-agonists (LABA) are widely used. However, their effect(s) on the release of IP-10 and IL-8 by airway epithelial cells are poorly understood. This study examined the effects of fluticasone, salmeterol, and agents which raise intracellular cAMP (cilomilast and db-cAMP) on the expression of IP-10 and IL-8 protein and mRNA. Studies were performed in cultured human airway epithelial cells during cytokine-stimulated IP-10 and IL-8 release. Cytokine treatment (TNF-alpha, IL-1beta and IFN-gamma) increased IP-10 and IL-8 protein and mRNA levels. Fluticasone (0.1 nM to 1 microM) increased IP-10 but reduced IL-8 protein release without changing IP-10 mRNA levels assessed by real time RT-PCR. The combination of salmeterol (1 micro M) and cilomilast (1-10 mu M) reduced IP-10 but had no effect on IL-8 protein. Salmeterol alone (1 micro M) and db-cAMP alone (1 mM) antagonised the effects of fluticasone on IP-10 but not IL-8 protein. In human airway epithelial cells, inhibition by salmeterol of fluticasone-enhanced IP-10 release may be an important therapeutic effect of the LABA/ICS combination not present when the two drugs are used separately.

  1. Differential Roles of Hydrogen Peroxide in Adaptive and Inflammatory Gene Expression Induced by Exposure of Human Airway Epithelial Cells to Zn2+

    Science.gov (United States)

    Oxidant stress is believed to play an important role in particulate matter (PM)–mediated toxicity in the respiratory tract. Zinc (Zn2+) is a ubiquitous component of PM that has been shown to induce adverse responses such as inflammatory and adaptive gene expression in airway epit...

  2. Effects of Laser Printer-Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts.

    Science.gov (United States)

    Pirela, Sandra V; Miousse, Isabelle R; Lu, Xiaoyan; Castranova, Vincent; Thomas, Treye; Qian, Yong; Bello, Dhimiter; Kobzik, Lester; Koturbash, Igor; Demokritou, Philip

    2016-02-01

    Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. We assessed the biological responses of a panel of human cell lines to PEPs. Three physiologically relevant cell lines--small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)--were exposed to PEPs at a wide range of doses (0.5-100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. The in vitro findings obtained in this study suggest that laser printer-emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders.

  3. Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Philipp Gierok

    Full Text Available Staphylococcus aureus alpha-toxin (Hla is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o- under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml of recombinant Hla (rHla in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o- cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.

  4. GENETIC INFLUENCES ON IN VTIRO PARTICULATE MATTER-INDUCED AIRWAY EPITHELIAL INJURY AND INFLAMMATORY MEDIATOR RELEASE

    Science.gov (United States)

    GENETIC INFLUENCES ON IN VITRO PARTICULATE MATTER-INDUCED AIRWAY EPITHELIAL INJURY AND INFLAMMATORY MEDIATOR RELEASE. JA Dye, JH Richards, DA Andrews, UP Kodavanti. US EPA, RTP, NC, USA.Particulate matter (PM) air pollution is capable of damaging the airway epitheli...

  5. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

    OpenAIRE

    Liu, Q.; Liu, J.; Roschmann, K.I.L.; Egmond, D. van; Golebski, K.; Fokkens, W.J.; Wang, D.; Drunen, C.M. van

    2013-01-01

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL3...

  6. Activation of calcitonin gene-related peptide receptor during ozone inhalation contributes to airway epithelial injury and repair.

    Science.gov (United States)

    Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

    2009-10-01

    The authors investigated the importance of the neuropeptide, calcitonin gene-related peptide (CGRP), in epithelial injury, repair, and neutrophil emigration after ozone exposure. Wistar rats were administered either a CGRP-receptor antagonist (CGRP(8-37)) or saline and exposed to 8 hours of 1-ppm ozone or filtered air with an 8-hour postexposure period. Immediately after exposure, ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, airway dissected lung lobes were stained for 5'-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Positive epithelial cells were quantified in specific airway generations. Rats treated with CGRP(8-37) had significantly reduced epithelial injury in terminal bronchioles and reduced epithelial proliferation in proximal airways and terminal bronchioles. Bronchoalveolar lavage and sections of terminal bronchioles showed no significant difference in the number of neutrophils emigrating into airways in CGRP(8-37)-treated rats. The airway epithelial cell line, HBE-1, showed no difference in the number of oxidant stress positive cells during exposure to hydrogen peroxide and a range of CGRP(8-37) doses, demonstrating no antioxidant effect of CGRP(8-37). We conclude that activation of CGRP receptors during ozone inhalation contributes to airway epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

  7. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

    Science.gov (United States)

    Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

    2013-04-11

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

  8. Degrees of reality: airway anatomy of high-fidelity human patient simulators and airway trainers.

    Science.gov (United States)

    Schebesta, Karl; Hüpfl, Michael; Rössler, Bernhard; Ringl, Helmut; Müller, Michael P; Kimberger, Oliver

    2012-06-01

    Human patient simulators and airway training manikins are widely used to train airway management skills to medical professionals. Furthermore, these patient simulators are employed as standardized "patients" to evaluate airway devices. However, little is known about how realistic these patient simulators and airway-training manikins really are. This trial aimed to evaluate the upper airway anatomy of four high-fidelity patient simulators and two airway trainers in comparison with actual patients by means of radiographic measurements. The volume of the pharyngeal airspace was the primary outcome parameter. Computed tomography scans of 20 adult trauma patients without head or neck injuries were compared with computed tomography scans of four high-fidelity patient simulators and two airway trainers. By using 14 predefined distances, two cross-sectional areas and three volume parameters of the upper airway, the manikins' similarity to a human patient was assessed. The pharyngeal airspace of all manikins differed significantly from the patients' pharyngeal airspace. The HPS Human Patient Simulator (METI®, Sarasota, FL) was the most realistic high-fidelity patient simulator (6/19 [32%] of all parameters were within the 95% CI of human airway measurements). The airway anatomy of four high-fidelity patient simulators and two airway trainers does not reflect the upper airway anatomy of actual patients. This finding may impact airway training and confound comparative airway device studies.

  9. deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

    Science.gov (United States)

    Arason, Ari Jon; Jonsdottir, Hulda R; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K

    2014-01-01

    The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

  10. Timothy grass pollen extract-induced gene expression and signalling pathways in airway epithelial cells

    NARCIS (Netherlands)

    Röschmann, K. I. L.; Luiten, S.; Jonker, M. J.; Breit, T. M.; Fokkens, W. J.; Petersen, A.; van Drunen, C. M.

    2011-01-01

    Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics.

  11. Timothy grass pollen extract-induced gene expression and signalling pathways in airway epithelial cells

    NARCIS (Netherlands)

    Röschmann, K.I.L.; Luiten, S.; Jonker, M.J.; Breit, T.M.; Fokkens, W.J.; Petersen, A.; van Drunen, C.M.

    2011-01-01

    Background: Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new

  12. Regional aerosol deposition in human upper airways

    Energy Technology Data Exchange (ETDEWEB)

    Swift, D.L.

    1992-11-01

    Laboratory experimental studies were carried out to investigate the factors influencing the deposition of aerosols ranging in size from 1 nm to 10 [mu]m in the human nasal, oral, pharyngeal and laryngeal airways. These experimental studies were performed in replicate upper airway physical models and in human volunteer subjects. New replicate models of the oral passage of an infant, the oral passage of an adult at two openings and the combined nasal and oral airways of an adult were constructed during the period, adding to the existing models of adult, child and infant nasal and oral airways models. Deposition studies in the adult oral and adult nasal models were performed under simulated cyclic flow conditions with 1 nm particles to compare with previously measured constant flow studies. Similar studies with inertial particles (1--10 [mu]m diameter) were performed with the adult nasal model; in both instances, results with cyclic flow were similar to constant flow results using a simple average flow rate based on inspiratory volume and time of inspiration. Human subject studies were performed with particle sizes 5--20 nm for nasal inspiration; preliminary analysis shows good agreement with model studies at several representative flow rates. Nasal inspiratory inertial deposition of 1--4 [mu]m diameter particles was measured in several adults as a function of airway dimensions; dimensional changes of the valve area by decongestion did not produce concomitant deposition changes.

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

    Science.gov (United States)

    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.

  14. Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer

    DEFF Research Database (Denmark)

    Willumsen, Niels J.; Davis, C.W.; Boucher, R.C.

    1994-01-01

    exposure (10 min) to 430 mosM luminal solution elicited no regulation of any parameter. Optical measurements revealed a reduction in the thickness of preparations only in response to luminal hypertonic solutions. We conclude that (a) airway epithelial cells exhibit asymmetric water transport properties......- secretion; and (d) cell volume loss increases the resistance of the paracellular path. We speculate that these properties configure human nasal epithelium to behave as an osmotic sensor, transducing information about luminal solutions to the airway wall....

  15. Patient-specific three-dimensional explant spheroids derived from human nasal airway epithelium

    DEFF Research Database (Denmark)

    Marthin, June Kehlet; Stevens, Elizabeth Munkebjerg; Larsen, Lars Allan

    2017-01-01

    BACKGROUND: Three-dimensional explant spheroid formation is an ex vivo technique previously used in studies of airway epithelial ion and water transport. Explanted cells and sheets of nasal epithelium form fully differentiated spheroids enclosing a partly fluid-filled lumen with the ciliated apical...... surface facing the outside and accessible for analysis of ciliary function. METHODS: We performed a two-group comparison study of ciliary beat pattern and ciliary beat frequency in spheroids derived from nasal airway epithelium in patients with primary ciliary dyskinesia (PCD) and in healthy controls...... in the investigation of pathophysiological aspects and drug effects in human nasal airway epithelium....

  16. Responses of well-differentiated nasal epithelial cells exposed to particles: Role of the epithelium in airway inflammation

    International Nuclear Information System (INIS)

    Auger, Floriane; Gendron, Marie-Claude; Chamot, Christophe; Marano, Francelyne; Dazy, Anne-Catherine

    2006-01-01

    Numerous epidemiological studies support the contention that ambient air pollution particles can adversely affect human health. To explain the acute inflammatory process in airways exposed to particles, a number of in vitro studies have been performed on cells grown submerged on plastic and poorly differentiated, and on cell lines, the physiology of which is somewhat different from that of well-differentiated cells. In order to obtain results using a model system in which epithelial cells are similar to those of the human airway in vivo, apical membranes of well-differentiated human nasal epithelial (HNE) cells cultured in an air-liquid interface (ALI) were exposed for 24 h to diesel exhaust particles (DEP) and Paris urban air particles (PM 2.5 ). DEP and PM 2.5 (10-80 μg/cm 2 ) stimulated both IL-8 and amphiregulin (ligand of EGFR) secretion exclusively towards the basal compartment. In contrast, there was no IL-1β secretion and only weak non-reproducible secretion of TNF-α. IL-6 and GM-CSF were consistently stimulated towards the apical compartment and only when cells were exposed to PM 2.5 . ICAM-1 protein expression on cell surfaces remained low after particle exposure, although it increased after TNF-α treatment. Internalization of particles, which is believed to initiate oxidative stress and proinflammatory cytokine expression, was restricted to small nanoparticles (≤ 40 nm). Production of reactive oxygen species (ROS) was detected, and DEP were more efficient than PM 2.5 . Collectively, our results suggest that airway epithelial cells exposed to particles augment the local inflammatory response in the lung but cannot alone initiate a systemic inflammatory response

  17. Water permeability in human airway epithelium

    DEFF Research Database (Denmark)

    Pedersen, Peter Steen; Procida, Kristina; Larsen, Per Leganger

    2005-01-01

    Osmotic water permeability (P(f)) was studied in spheroid-shaped human airway epithelia explants derived from nasal polyps by the use of a new improved tissue collection and isolation procedure. The fluid-filled spheroids were lined with a single cell layer with the ciliated apical cell membrane ...

  18. Simvastatin inhibits smoke-induced airway epithelial injury: implications for COPD therapy.

    Science.gov (United States)

    Davis, Benjamin B; Zeki, Amir A; Bratt, Jennifer M; Wang, Lei; Filosto, Simone; Walby, William F; Kenyon, Nicholas J; Goldkorn, Tzipora; Schelegle, Edward S; Pinkerton, Kent E

    2013-08-01

    Chronic obstructive pulmonary disease (COPD) is the third leading cause of death. The statin drugs may have therapeutic potential in respiratory diseases such as COPD, but whether they prevent bronchial epithelial injury is unknown. We hypothesised that simvastatin attenuates acute tobacco smoke-induced neutrophilic lung inflammation and airway epithelial injury. Spontaneously hypertensive rats were given simvastatin (20 mg·kg(-1) i.p.) daily for either 7 days prior to tobacco smoke exposure and during 3 days of smoke exposure, or only during tobacco smoke exposure. Pretreatment with simvastatin prior to and continued throughout smoke exposure reduced the total influx of leukocytes, neutrophils and macrophages into the lung and airways. Simvastatin attenuated tobacco smoke-induced cellular infiltration into lung parenchymal and airway subepithelial and interstitial spaces. 1 week of simvastatin pretreatment almost completely prevented smoke-induced denudation of the airway epithelial layer, while simvastatin given only concurrently with the smoke exposure had no effect. Simvastatin may be a novel adjunctive therapy for smoke-induced lung diseases, such as COPD. Given the need for statin pretreatment there may be a critical process of conditioning that is necessary for statins' anti-inflammatory effects. Future work is needed to elucidate the mechanisms of this statin protective effect.

  19. Ozone Enhances Diesel Exhaust Particles (DEP-Induced Interleukin-8 (IL-8 Gene Expression in Human Airway Epithelial Cells through Activation of Nuclear Factors- κB (NF-κB and IL-6 (NF-IL6

    Directory of Open Access Journals (Sweden)

    James Kelley

    2005-12-01

    Full Text Available Ozone, a highly reactive oxidant gas is a major component of photochemical smog. As an inhaled toxicant, ozone induces its adverse effects mainly on the lung. Inhalation of particulate matter has been reported to cause airway inflammation in humans and animals. Furthermore, epidemiological evidence has indicated that exposure to particulate matter (PM2.5-10, including diesel exhaust particles (DEP has been correlated with increased acute and chronic respiratory morbidity and exacerbation of asthma. Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems. Ozone and particulate matter may be chemically coupled in the ambient air. In the present study we determined whether ozone exposure enhances DEP effect on interleukin-8 (IL-8 gene expression in human airway epithelial cells. We report that ozone exposure (0.5 ppm x 1 hr significantly increased DEP-induced IL-8 gene expression in A549 cells (117 ± 19 pg/ml, n = 6, p < 0.05 as compared to cultures treated with DEP (100 μg/ml x 4 hr alone (31 ± 3 pg/ml, n = 6, or cultures exposed to purified air (24 ± 6 pg/ml, n = 6. The increased DEP-induced IL-8 gene expression following ozone exposure was attributed to ozone-induced increase in the activity of the transcription factors NF-κB and NF-IL6. The results of the present study indicate that ozone exposure enhances the toxicity of DEP in human airway epithelial cells by augmenting IL-8 gene expression, a potent chemoattractant of neutrophils in the lung.

  20. Modulation of epithelial sodium channel in human alveolar epithelial ...

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng1,2, Li-Wei Pan2, Sheng-Rong Zhang3, Bin-Yu Ying2, Ben-Ji. Wang2, Guo-Liang Lin2 and Shi-Fang Ding1*. 1Department of Critical Care Medicine, Qilu Hospital of Shandong ...

  1. Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

    Science.gov (United States)

    Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

    2014-03-01

    Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction.

  2. Airway skills training using a human patient simulator

    African Journals Online (AJOL)

    Thesegan Moodley

    2016-04-11

    Apr 11, 2016 ... Airway management problems may be particularly challenging to junior doctors.1 ... They respond to real-time, real-life clinical ... Keywords: human patient simulator, simulation, airway management, psychomotor skills.

  3. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon; Heo, Won Il; Kim, Kyung Won; Kim, Kyu Earn [Department of Pediatrics and Institute of Allergy, Severance Medical Research Institute, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of); Sohn, Myung Hyun, E-mail: mhsohn@yuhs.ac [Department of Pediatrics and Institute of Allergy, Severance Medical Research Institute, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally

  4. Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β.

    Science.gov (United States)

    Ogawa, Hirohisa; Ledford, Julie G; Mukherjee, Sambuddho; Aono, Yoshinori; Nishioka, Yasuhiko; Lee, James J; Izumi, Keisuke; Hollingsworth, John W

    2014-11-29

    Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease. C57BL/6 wild-type (WT) and SP-D-/- mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D. Dp-challenged SP-D-/- mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D-/- mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D-/- mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D-/- mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D-/- mice. These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

  5. Involvement of Syk kinase in TNF-induced nitric oxide production by airway epithelial cells

    International Nuclear Information System (INIS)

    Ulanova, Marina; Marcet-Palacios, Marcelo; Munoz, Samira; Asfaha, Samuel; Kim, Moo-Kyung; Schreiber, Alan D.; Befus, A. Dean

    2006-01-01

    We have recently found that Syk is widely expressed in lung epithelial cells (EC) and participates in β1 integrin signaling. In this study, we assessed the role of Syk in regulation of NO production. Stimulation of human bronchial EC line HS-24 by TNF caused an increased expression of inducible nitric oxide synthase (iNOS). Inhibition of Syk using siRNA or piceatannol down-regulated the iNOS expression and reduced NO production. This effect occurred in EC simultaneously stimulated via β1 integrins, suggesting that TNF and β1 integrins provide co-stimulatory signals. Inhibition of Syk down-regulated TNF-induced p38 and p44/42 MAPK phosphorylation and nuclear translocation of p65 NF-κB. Thus, TNF-induced activation of pro-inflammatory signaling in EC leading to enhanced expression of iNOS and NO production was dependent on Syk. Syk-mediated signaling regulates NO production at least partly via activating the MAPK cascade. Understanding the role of Syk in airway EC may help in developing new therapeutic tools for inflammatory lung disorders

  6. Baicalin Inhibits Lipopolysaccharide-Induced Inflammation Through Signaling NF-κB Pathway in HBE16 Airway Epithelial Cells.

    Science.gov (United States)

    Dong, Shou-jin; Zhong, Yun-qing; Lu, Wen-ting; Li, Guan-hong; Jiang, Hong-li; Mao, Bing

    2015-08-01

    Baicalin, a flavonoid monomer derived from Scutellaria baicalensis called Huangqin in mandarin, is the main active ingredient contributing to S. baicalensis' efficacy. It is known in China that baicalin has potential therapeutic effects on inflammatory diseases. However, its anti-inflammatory mechanism has still not been fully interpreted. We aim to investigate the anti-inflammatory effect of baicalin on lipopolysaccharide (LPS)-induced inflammation in HBE16 airway epithelial cells and also to explore the underlying signaling mechanisms. The anti-inflammatory action of baicalin was evaluated in human airway epithelial cells HBE16 treated with LPS. Airway epithelial cells HBE16 were pretreated with a range of concentrations of baicalin for 30 min and then stimulated with 10 μg/ml LPS. The secretions of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α) in cell culture supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). The messenger RNA (mRNA) expressions of IL-6, IL-8, and TNF-α were tested by quantitative real-time polymerase chain reaction (real-time RT-PCR). Furthermore, Western blotting was used to determine whether the signaling pathway NF-κB was involved in the anti-inflammatory action of baicalin. The inflammatory cell model was successfully built with 10 μg/ml LPS for 24 h in our in vitro experiments. Both the secretions and the mRNA expressions of IL-6, IL-8, and TNF-α were significantly inhibited by baicalin. Moreover, the expression levels of phospho-IKKα/β and phospho-NF-κB p65 were downregulated, and the phospho-IκB-α level was upregulated by baicalin. These findings suggest that the anti-inflammatory properties of baicalin may be resulted from the inhibition of IL-6, IL-8, and TNF-α expression via preventing signaling NF-κB pathway in HBE16 airway epithelial cells. In addition, this study provides evidence to understand the therapeutic effects of baicalin on inflammatory diseases in

  7. Human airway organoid engineering as a step toward lung regeneration and disease modeling.

    Science.gov (United States)

    Tan, Qi; Choi, Kyoung Moo; Sicard, Delphine; Tschumperlin, Daniel J

    2017-01-01

    Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-β1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Development of a realistic human airway model.

    Science.gov (United States)

    Lizal, Frantisek; Elcner, Jakub; Hopke, Philip K; Jedelsky, Jan; Jicha, Miroslav

    2012-03-01

    Numerous models of human lungs with various levels of idealization have been reported in the literature; consequently, results acquired using these models are difficult to compare to in vivo measurements. We have developed a set of model components based on realistic geometries, which permits the analysis of the effects of subsequent model simplification. A realistic digital upper airway geometry except for the lack of an oral cavity has been created which proved suitable both for computational fluid dynamics (CFD) simulations and for the fabrication of physical models. Subsequently, an oral cavity was added to the tracheobronchial geometry. The airway geometry including the oral cavity was adjusted to enable fabrication of a semi-realistic model. Five physical models were created based on these three digital geometries. Two optically transparent models, one with and one without the oral cavity, were constructed for flow velocity measurements, two realistic segmented models, one with and one without the oral cavity, were constructed for particle deposition measurements, and a semi-realistic model with glass cylindrical airways was developed for optical measurements of flow velocity and in situ particle size measurements. One-dimensional phase doppler anemometry measurements were made and compared to the CFD calculations for this model and good agreement was obtained.

  9. Intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells

    International Nuclear Information System (INIS)

    Verhaeghe, Catherine; Tabruyn, Sebastien P.; Oury, Cecile; Bours, Vincent; Griffioen, Arjan W.

    2007-01-01

    Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications

  10. The role of Sox2 on lung epithelial airway epithelial differentiation

    NARCIS (Netherlands)

    J.K. Ochieng (Joshua)

    2014-01-01

    markdownabstract__Abstract__ The foregut is crucial for development of respiratory organs including the lungs. Foregut morphogenesis starts around embryonic day 8.0 in mouse when the endoderm epithelial sheet folds ventrally during gastrulation [1,2]. At embryonic day 9.0, the ventral folding

  11. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  12. Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology

    Directory of Open Access Journals (Sweden)

    McKiernan PJ

    2013-10-01

    Full Text Available Paul J McKiernan,2 Orla Cunninghamm,1,2 Catherine M Greenem,2 Sally-Ann Cryan1,31School of Pharmacy, Royal College of Surgeons in Ireland, 2Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Beaumont Hospital, 3Trinity Centre for Bioengineering, Trinity College Dublin, Dublin, IrelandAbstract: Cystic fibrosis (CF is an inherited disorder characterized by chronic airway inflammation. microRNAs (miRNAs are endogenous small RNAs which act on messenger (mRNA at a post transcriptional level, and there is a growing understanding that altered expression of miRNA is involved in the CF phenotype. Modulation of miRNA by replacement using miRNA mimics (premiRs presents a new therapeutic paradigm for CF, but effective and safe methods of delivery to the CF epithelium are limiting clinical translation. Herein, polymeric nanoparticles are investigated for delivery of miRNA mimics into CF airway epithelial cells, using miR-126 as a proof-of-concept premiR cargo to determine efficiency. Two polymers, polyethyleneimine (PEI and chitosan, were used to prepare miRNA nanomedicines, characterized for their size, surface (zeta potential, and RNA complexation efficiency, and screened for delivery and cytotoxicity in CFBE41o- (human F508del cystic fibrosis transmembrane conductance regulator bronchial epithelial cells using a novel high content analysis method. RNA extraction was carried out 24 hours post transfection, and miR-126 and TOM1 (target of Myb1 expression (a validated miR-126 target was assessed. Manufacture was optimized to produce small nanoparticles that effectively complexed miRNA. Using high content analysis, PEI-based nanoparticles were more effective than chitosan-based nanoparticles in facilitating uptake of miRNA into CFBE41o- cells and this was confirmed in miR-126 assays. PEI-premiR-126 nanoparticles at low nitrogen/phosphate (N/P ratios resulted in significant knockdown of

  13. Regional aerosol deposition in human upper airways

    International Nuclear Information System (INIS)

    Swift, D.L.

    1989-01-01

    During the report period significant progress on the quantitative understanding of regional upper airway deposition of airborne particle has been realized. Replicate models of the human upper airways obtained from post-mortem casting of the nasal, oral, pharyngeal, laryngeal and upper tracheal regions and in vivo magnetic resonance imaging (MRI) of the same regions of adults and children have been employed to determine the overall and local deposition characteristics of aerosols in the ultrafine (1--100 μm diameter) and fine (0.8--12 μm diameter) region. Studies have been carried out for both nasal and oral breathing during inspiratory and expiratory flow at constant flow rates representative of rest and states of exercise. The results of these investigations indicate that particles in the size range of ''unattached'' radon progeny (1--3 nm) are deposited in both the nasal and oral passages with high efficiency (60--80%) for both inspiration and expiration, with the nasal deposition being somewhat greater (5--10%) than oral deposition. The effect of flow rate on upper airway deposition for both pathways is not great; data analysis indicates that the deposition for all flow rates from 4--50 liters/minute can be grouped by plotting deposition vs Q- 1/8 , where Q is flow rate, a far weaker dependency than observed for inertial deposition. Diffusional transport is the primary mechanism of deposition, and size dependence can be accounted for by plotting, deposition percent vs D n where D is particle diffusion coefficient and n ranges from 0.5--0.66. 2 refs

  14. Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery.

    Directory of Open Access Journals (Sweden)

    Monica Valencia-Gattas

    Full Text Available Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases.

  15. IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17

    Directory of Open Access Journals (Sweden)

    Sandifer Tracy

    2007-07-01

    Full Text Available Abstract Background The pleiotrophic cytokine interleukin (IL-13 features prominently in allergic and inflammatory diseases. In allergic asthma, IL-13 is well established as an inducer of airway inflammation and tissue remodeling. We demonstrated previously that IL-13 induces release of transforming growth factor-α (TGFα from human bronchial epithelial cells, with proliferation of these cells mediated by the autocrine/paracrine action of this growth factor. TGFα exists as an integral membrane protein and requires proteolytic processing to its mature form, with a disintegrin and metalloproteinase (ADAM17 responsible for this processing in a variety of tissues. Methods In this study, normal human bronchial epithelial (NHBE cells grown in air/liquid interface (ALI culture were used to examine the mechanisms whereby IL-13 induces release of TGFα and cellular proliferation. Inhibitors and antisense RNA were used to examine the role of ADAM17 in these processes, while IL-13-induced changes in the intracellular expression of TGFα and ADAM17 were visualized by confocal microscopy. Results IL-13 was found to induce proliferation of NHBE cells, and release of TGFα, in an ADAM17-dependent manner; however, this IL-13-induced proliferation did not appear to result solely from ADAM17 activation. Rather, IL-13 induced a change in the location of TGFα expression from intracellular to apical regions of the NHBE cells. The apical region was also found to be a site of significant ADAM17 expression, even prior to IL-13 stimulation. Conclusion Results from this study indicate that ADAM17 mediates IL-13-induced proliferation and TGFα shedding in NHBE cells. Furthermore, they provide the first example wherein a cytokine (IL-13 induces a change in the intracellular expression pattern of a growth factor, apparently inducing redistribution of intracellular stores of TGFα to the apical region of NHBE cells where expression of ADAM17 is prominent. Thus, IL-13

  16. Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR.

    Science.gov (United States)

    Danyal, Karamatullah; de Jong, Willem; O'Brien, Edmund; Bauer, Robert A; Heppner, David E; Little, Andrew C; Hristova, Milena; Habibovic, Aida; van der Vliet, Albert

    2016-11-01

    Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca 2+ -dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens. Copyright © 2016 the American Physiological Society.

  17. TGF-β1 induced epithelial to mesenchymal transition (EMT in human bronchial epithelial cells is enhanced by IL-1β but not abrogated by corticosteroids

    Directory of Open Access Journals (Sweden)

    Zuraw Bruce L

    2009-10-01

    Full Text Available Abstract Background Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFβ1 stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1β. Methods BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFβ1 and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1β. Results were analyzed using non-parametric statistical tests. Results Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFβ1 significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFβ1 then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and α-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFβ1 induced transition but IL-1β enhanced the transition. Conclusion Our results indicate, that TGFβ1 can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFβ1 in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.

  18. Human glomerular epithelial cell proteoglycans

    International Nuclear Information System (INIS)

    Thomas, G.J.; Jenner, L.; Mason, R.M.; Davies, M.

    1990-01-01

    Proteoglycans synthesized by cultures of human glomerular epithelial cells have been isolated and characterized. Three types of heparan sulfate were detected. Heparan sulfate proteoglycan I (HSPG-I; Kav 6B 0.04) was found in the cell layer and medium and accounted for 12% of the total proteoglycans synthesized. HSPG-II (Kav 6B 0.25) accounted for 18% of the proteoglycans and was located in the medium and cell layer. A third population (9% of the proteoglycan population), heparan sulfate glycosaminoglycan (HS-GAG; Kav 6B 0.4-0.8), had properties consistent with single glycosaminoglycan chains or their fragments and was found only in the cell layer. HSPG-I and HSPG-II from the cell layer had hydrophobic properties; they were released from the cell layer by mild trypsin treatment. HS-GAG lacked these properties, consisted of low-molecular-mass heparan sulfate oligosaccharides, and were intracellular. HSPG-I and -II released to the medium lacked hydrophobic properties. The cells also produced three distinct types of chondroitin sulfates. The major species, chondroitin sulfate proteoglycan I (CSPG-I) eluted in the excluded volume of a Sepharose CL-6B column, accounted for 30% of the proteoglycans detected, and was found in both the cell layer and medium. Cell layer CSPG-I bound to octyl-Sepharose. It was released from the cell layer by mild trypsin treatment. CSPG-II (Kav 6B 0.1-0.23) accounted for 10% of the total 35S-labeled macromolecules and was found predominantly in the culture medium. A small amount of CS-GAG (Kav 6B 0.25-0.6) is present in the cell extract and like HS-GAG is intracellular. Pulse-chase experiments indicated that HSPG-I and -II and CSPG-I and -II are lost from the cell layer either by direct release into the medium or by internalization where they are metabolized to single glycosaminoglycan chains and subsequently to inorganic sulfate

  19. Characterising the mechanism of airway smooth muscle β2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    David Van Ly

    Full Text Available Rhinovirus (RV infections account for approximately two thirds of all virus-induced asthma exacerbations and often result in an impaired response to β2 agonist therapy. Using an in vitro model of RV infection, we investigated the mechanisms underlying RV-induced β2 adrenoceptor desensitization in primary human airway smooth muscle cells (ASMC. RV infection of primary human bronchial epithelial cells (HBEC for 24 hours produced conditioned medium that caused β2 adrenoceptor desensitization on ASMCs without an effect on ASMCs viability. Less than 3 kDa size fractionation together with trypsin digestion of RV-induced conditioned medium did not prevent β2 adrenoceptor desensitization, suggesting it could potentially be mediated by a small peptide or lipid. RV infection of BECs, ASMCs and fibroblasts produced prostaglandins, of which PGE2, PGF2α and PGI2 had the ability to cause β2 adrenoceptor desensitization on ASMCs. RV-induced conditioned medium from HBECs depleted of PGE2 did not prevent ASMC β2 adrenoceptor desensitization; however this medium induced PGE2 from ASMCs, suggesting that autocrine prostaglandin production may be responsible. Using inhibitors of cyclooxygenase and prostaglandin receptor antagonists, we found that β2 adrenoceptor desensitization was mediated through ASMC derived COX-2 induced prostaglandins. Since ASMC prostaglandin production is unlikely to be caused by RV-induced epithelial derived proteins or lipids we next investigated activation of toll-like receptors (TLR by viral RNA. The combination of TLR agonists poly I:C and imiquimod induced PGE2 and β2 adrenoceptor desensitization on ASMC as did the RNA extracted from RV-induced conditioned medium. Viral RNA but not epithelial RNA caused β2 adrenoceptor desensitization confirming that viral RNA and not endogenous human RNA was responsible. It was deduced that the mechanism by which β2 adrenoceptor desensitization occurs was by pattern recognition receptor

  20. Interactions between epithelial cells and dendritic cells in airway immune responses: lessons from allergic airway disease

    NARCIS (Netherlands)

    van Tongeren, J.; Reinartz, S. M.; Fokkens, W. J.; de Jong, E. C.; van Drunen, C. M.

    2008-01-01

    Micro-organisms constantly invade the human body and may form a threat to our health. Traditionally, concepts of defence mechanisms have included a protective outer layer of epithelia and a vigilant immune system searching for areas where the integrity of the outer layer may be compromised. Instead

  1. Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

    OpenAIRE

    Scull, Margaret A.; Gillim-Ross, Laura; Santos, Celia; Roberts, Kim L.; Bordonali, Elena; Subbarao, Kanta; Barclay, Wendy S.; Pickles, Raymond J.

    2009-01-01

    Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37 degrees C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human p...

  2. Mediators on human airway smooth muscle.

    Science.gov (United States)

    Armour, C; Johnson, P; Anticevich, S; Ammit, A; McKay, K; Hughes, M; Black, J

    1997-01-01

    1. Bronchial hyperresponsiveness in asthma may be due to several abnormalities, but must include alterations in the airway smooth muscle responsiveness and/or volume. 2. Increased responsiveness of airway smooth muscle in vitro can be induced by certain inflammatory cell products and by induction of sensitization (atopy). 3. Increased airway smooth muscle growth can also be induced by inflammatory cell products and atopic serum. 4. Mast cell numbers are increased in the airways of asthmatics and, in our studies, in airway smooth muscle that is sensitized and hyperresponsive. 5. We propose that there is a relationship between mast cells and airway smooth muscle cells which, once an allergic process has been initiated, results in the development of critical features in the lungs in asthma.

  3. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  4. Airway skills training using a human patient simulator | Moodley ...

    African Journals Online (AJOL)

    ... of airway management skills using the simulator. Participant satisfaction was much better in the simulator group. The importance of psychomotor reinforcement should be borne in mind when designing simulation courses. Keywords: human patient simulator, simulation, airway management, psychomotor skills ...

  5. Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Daqing [Department of Respiration, Xi’an Children’s Hospital, Xi’an 710003 (China); Wang, Jing [Department of Neonatology, Xi’an Children’s Hospital, Xi’an 710003 (China); Yang, Niandi [Outpatient Department, School of Aerospace Engineering, Air Force Engineering University, Xi’an 710038 (China); Ma, Haixin, E-mail: drhaixinma@163.com [Department of Quality Control, Xi’an Children’s Hospital, Xi’an 710003 (China)

    2016-08-12

    Matrine has been demonstrated to attenuate allergic airway inflammation. Elevated suppressor of cytokine signaling 3 (SOCS3) was correlated with the severity of asthma. The aim of this study was to investigate the effect of matrine on SOCS3 expression in airway inflammation. In this study, we found that matrine significantly inhibited OVA-induced AHR, inflammatory cell infiltration, goblet cell differentiation, and mucous production in a dose-dependent manner in mice. Matrine also abrogated the level of interleukin (IL)-4 and IL-13, but enhanced interferon (IFN)-γ expression, both in BALF and in lung homogenates. Furthermore, matrine impeded TNF-α-induced the expression of IL-6 and adhesion molecules in airway epithelial cells (BEAS-2B and MLE-12). Additionally, we found that matrine inhibited SOCS3 expression, both in asthmatic mice and TNF-α-stimulated epithelial cells via suppression of the NF-κB signaling pathway by using pcDNA3.1-SOCS3 plasmid, SOCS3 siRNA, or nuclear factor kappa-B (NF-κB) inhibitor PDTC. Conclusions: Matrine suppresses airway inflammation by downregulating SOCS3 expression via inhibition of NF-κB signaling in airway epithelial cells and asthmatic mice. - Highlights: • Matrine attenuates asthmatic symptoms and regulates Th1/Th2 balance in vivo. • Matrine suppresses inflammation responses in vitro. • Matrine decreases SOCS3 expression both in vivo and in vitro. • Matrine inhibits SOCS3 expression by suppressing NF-κB signaling.

  6. Beneficial effects of ursodeoxycholic acid via inhibition of airway remodelling, apoptosis of airway epithelial cells, and Th2 immune response in murine model of chronic asthma.

    Science.gov (United States)

    Işık, S; Karaman, M; Çilaker Micili, S; Çağlayan-Sözmen, Ş; Bağrıyanık, H Alper; Arıkan-Ayyıldız, Z; Uzuner, N; Karaman, Ö

    In previous studies, anti-inflammatory, anti-apoptotic and immunomodulatory effects of ursodeoxycholic acid (UDCA) on liver diseases have been shown. In this study, we aimed to investigate the effects of UDCA on airway remodelling, epithelial apoptosis, and T Helper (Th)-2 derived cytokine levels in a murine model of chronic asthma. Twenty-seven BALB/c mice were divided into five groups; PBS-Control, OVA-Placebo, OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone. Mice in groups OVA-50mg/kg UDCA, OVA-150mg/kg UDCA, OVA-Dexamethasone received the UDCA (50mg/kg), UDCA (150mg/kg), and dexamethasone, respectively. Epithelium thickness, sub-epithelial smooth muscle thickness, number of mast and goblet cells of samples isolated from the lung were measured. Immunohistochemical scorings of the lung tissue for matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEG-F), transforming growth factor-beta (TGF-β), terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) and cysteine-dependent aspartate-specific proteases (caspase)-3 were determined. IL-4, IL-5, IL-13, Nitric oxide, ovalbumin-specific immunoglobulin (Ig) E levels were quantified. The dose of 150mg/kg UDCA treatment led to lower epithelial thickness, sub-epithelial smooth muscle thickness, goblet and mast cell numbers compared to placebo. Except for MMP-9 and TUNEL all immunohistochemical scores were similar in both UDCA treated groups and the placebo. All cytokine levels were significantly lower in group IV compared to the placebo. These findings suggested that the dose of 150mg/kg UDCA improved all histopathological changes of airway remodelling and its beneficial effects might be related to modulating Th-2 derived cytokines and the inhibition of apoptosis of airway epithelial cells. Copyright © 2017 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

  7. Epigenetic dysregulation of interleukin 8 (CXCL8) hypersecretion in cystic fibrosis airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Poghosyan, Anna, E-mail: pannagos@yahoo.com; Patel, Jamie K.; Clifford, Rachel L.; Knox, Alan J., E-mail: alan.knox@nottingham.ac.uk

    2016-08-05

    Airway epithelial cells in cystic fibrosis (CF) overexpress Interleukin 8 (CXCL8) through poorly defined mechanisms. CXCL8 transcription is dependent on coordinated binding of CCAAT/enhancer binding protein (C/EBP)β, nuclear factor (NF)-κB, and activator protein (AP)-1 to the promoter. Here we show abnormal epigenetic regulation is responsible for CXCL8 overexpression in CF cells. Under basal conditions CF cells had increased bromodomain (Brd)3 and Brd4 recruitment and enhanced NF-κB and C/EBPβ binding to the CXCL8 promoter compared to non-CF cells due to trimethylation of histone H3 at lysine 4 (H3K4me3) and DNA hypomethylation at CpG6. IL-1β increased NF-κB, C/EBPβ and Brd4 binding. Furthermore, inhibitors of bromodomain and extra-terminal domain family (BET) proteins reduced CXCL8 production in CF cells suggesting a therapeutic target for the BET pathway. -- Highlights: •A regulatory mechanism of CXCL8 transcriptional control in CF airways is proposed. •There was an increased binding of NF-κB and C/EBPβ transcription factors. •There was enhanced recruitment of BET proteins to the CXCL8 promoter. •Epigenetic modifications are responsible for the aberrant CXCL8 transcription.

  8. Epigenetic dysregulation of interleukin 8 (CXCL8) hypersecretion in cystic fibrosis airway epithelial cells

    International Nuclear Information System (INIS)

    Poghosyan, Anna; Patel, Jamie K.; Clifford, Rachel L.; Knox, Alan J.

    2016-01-01

    Airway epithelial cells in cystic fibrosis (CF) overexpress Interleukin 8 (CXCL8) through poorly defined mechanisms. CXCL8 transcription is dependent on coordinated binding of CCAAT/enhancer binding protein (C/EBP)β, nuclear factor (NF)-κB, and activator protein (AP)-1 to the promoter. Here we show abnormal epigenetic regulation is responsible for CXCL8 overexpression in CF cells. Under basal conditions CF cells had increased bromodomain (Brd)3 and Brd4 recruitment and enhanced NF-κB and C/EBPβ binding to the CXCL8 promoter compared to non-CF cells due to trimethylation of histone H3 at lysine 4 (H3K4me3) and DNA hypomethylation at CpG6. IL-1β increased NF-κB, C/EBPβ and Brd4 binding. Furthermore, inhibitors of bromodomain and extra-terminal domain family (BET) proteins reduced CXCL8 production in CF cells suggesting a therapeutic target for the BET pathway. -- Highlights: •A regulatory mechanism of CXCL8 transcriptional control in CF airways is proposed. •There was an increased binding of NF-κB and C/EBPβ transcription factors. •There was enhanced recruitment of BET proteins to the CXCL8 promoter. •Epigenetic modifications are responsible for the aberrant CXCL8 transcription.

  9. Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo

    Directory of Open Access Journals (Sweden)

    Hu Jim

    2006-02-01

    Full Text Available Abstract Background The cationic lipid Genzyme lipid (GL 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Methods Anti-lacZ and ENaC (epithelial sodium channel siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. Results In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. Conclusion This study suggests that although siRNAs and asODNs can be developed to inhibit

  10. Recombinant human DNase in children with airway malacia and lower respiratory tract infection.

    NARCIS (Netherlands)

    Boogaard, R.; Jongste, J.C. de; Vaessen-Verberne, A.A.; Hop, W.C.J.; Merkus, P.J.F.M.

    2009-01-01

    BACKGROUND: Children with airway malacia often have protracted courses of airway infections, because dynamic airway collapse during coughing results in impaired mucociliary clearance. The aim of this study was to determine the effect of the mucolytic drug recombinant human deoxyribonuclease

  11. Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

    International Nuclear Information System (INIS)

    Tapparel, Caroline; Sobo, Komla; Constant, Samuel; Huang, Song; Van Belle, Sandra; Kaiser, Laurent

    2013-01-01

    New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent. - Highlights: • A 3D human upper airway epithelia reconstituted in vitro supports HRV-C growth. • HRV-Cs enter and exit preferentially at the apical side of this ALI culture system. • HRV-Cs are acid sensitive. • Temperature sensitivity may be type-dependent for HRV-Cs

  12. Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tapparel, Caroline, E-mail: Caroline.Tapparel@hcuge.ch [Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14 (Switzerland); Sobo, Komla [Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14 (Switzerland); Constant, Samuel; Huang, Song [Epithelix sárl, 14 Chemin des Aulx, 1228 Plan les Ouates, Geneva (Switzerland); Van Belle, Sandra; Kaiser, Laurent [Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14 (Switzerland)

    2013-11-15

    New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent. - Highlights: • A 3D human upper airway epithelia reconstituted in vitro supports HRV-C growth. • HRV-Cs enter and exit preferentially at the apical side of this ALI culture system. • HRV-Cs are acid sensitive. • Temperature sensitivity may be type-dependent for HRV-Cs.

  13. Pore-forming virulence factors of Staphylococcus aureus destabilize epithelial barriers-effects of alpha-toxin in the early phases of airway infection

    Directory of Open Access Journals (Sweden)

    Jan-Peter Hildebrandt

    2015-09-01

    Full Text Available Staphylococcus aureus (S. aureus is a human commensal and an opportunistic pathogen that may affect the gastrointestinal tract, the heart, bones, skin or the respiratory tract. S. aureus is frequently involved in hospital- or community-acquired lung infections. The pathogenic potential is associated with its ability to secrete highly effective virulence factors. Among these, the pore-forming toxins Panton-Valentine leukocidin (PVL and hemolysin A (Hla are the important virulence factors determining the prognosis of pneumonia cases. This review focuses on the structure and the functions of S. aureus hemolysin A and its sub-lethal effects on airway epithelial cells. The hypothesis is developed that Hla may not just be a tissue-destructive agent providing the bacteria with host-derived nutrients, but may also play complex roles in the very early stages of interactions of bacteria with healthy airways, possibly paving the way for establishing acute infections.

  14. Specific immune responses against airway epithelial cells in a transgenic mouse-trachea transplantation model for obliterative airway disease

    NARCIS (Netherlands)

    Qu, N; de Haan, A; Harmsen, MC; Kroese, FGM; de Leij, LFMH; Prop, J

    2003-01-01

    Background. Immune injury to airway epithelium is suggested to play a central role in the pathogenesis of obliterative bronchiolitis (OB) after clinical lung transplantation. In several studies, a rejection model of murine trachea transplants is used, resulting in obliterative airway disease (OAD)

  15. PKC activation induces inflammatory response and cell death in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hyunhee Kim

    Full Text Available A variety of airborne pathogens can induce inflammatory responses in airway epithelial cells, which is a crucial component of host defence. However, excessive inflammatory responses and chronic inflammation also contribute to different diseases of the respiratory system. We hypothesized that the activation of protein kinase C (PKC is one of the essential mechanisms of inflammatory response in airway epithelial cells. In the present study, we stimulated human bronchial lung epithelial (BEAS-2B cells with the phorbol ester Phorbol 12, 13-dibutyrate (PDBu, and examined gene expression profile using microarrays. Microarray analysis suggests that PKC activation induced dramatic changes in gene expression related to multiple cellular functions. The top two interaction networks generated from these changes were centered on NFκB and TNF-α, which are two commonly known pathways for cell death and inflammation. Subsequent tests confirmed the decrease in cell viability and an increase in the production of various cytokines. Interestingly, each of the increased cytokines was differentially regulated at mRNA and/or protein levels by different sub-classes of PKC isozymes. We conclude that pathological cell death and cytokine production in airway epithelial cells in various situations may be mediated through PKC related signaling pathways. These findings suggest that PKCs can be new targets for treatment of lung diseases.

  16. Interleukin-13-induced MUC5AC is regulated by 15-lipoxygenase 1 pathway in human bronchial epithelial cells.

    Science.gov (United States)

    Zhao, Jinming; Maskrey, Ben; Balzar, Silvana; Chibana, Kazuyuki; Mustovich, Anthony; Hu, Haizhen; Trudeau, John B; O'Donnell, Valerie; Wenzel, Sally E

    2009-05-01

    15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown. To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13-induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro. Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air-liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot. Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression. Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma.

  17. Mechanical compression attenuates normal human bronchial epithelial wound healing

    Directory of Open Access Journals (Sweden)

    Malavia Nikita

    2009-02-01

    Full Text Available Abstract Background Airway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer. Methods Accordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1 unperturbed control cells, 2 single scrape wound only, 3 static compression (6 hours of 30 cmH2O, and 4 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled. Results We found that mechanical compression and scrape injury increase TGF-β2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF. Conclusion Our results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

  18. Interleukin-17A induces bicarbonate secretion in normal human bronchial epithelial cells

    Science.gov (United States)

    Kreindler, James L.; Bertrand, Carol A.; Lee, Robert J.; Karasic, Thomas; Aujla, Shean; Pilewski, Joseph M.; Frizzell, Raymond A.; Kolls, Jay K.

    2009-01-01

    The innate immune functions of human airways include mucociliary clearance and antimicrobial peptide activity. Both functions may be affected by changes in epithelial ion transport. Interleukin-17A (IL-17A), which has a receptor at the basolateral membrane of airway epithelia, is a T cell cytokine that has been shown to increase mucus secretion and antimicrobial peptide production by human bronchial epithelial (HBE) cells. Furthermore, IL-17A levels are increased in sputum from patients during pulmonary exacerbations of cystic fibrosis. Therefore, we investigated the effects of IL-17A on basal, amiloride-sensitive, and forskolin-stimulated ion transport in mature, well-differentiated HBE cells. Exposure of HBE monolayers to IL-17A for 48 h induced a novel forskolin-stimulated bicarbonate secretion in addition to forskolin-stimulated chloride secretion and resulted in alkalinization of liquid on the mucosal surface of polarized cells. IL-17A-induced bicarbonate secretion was cystic fibrosis transmembrane conductance regulator (CFTR)-dependent, mucosal chloride-dependent, partially Na+-dependent, and sensitive to serosal, but not mucosal, stilbene inhibition. These data suggest that IL-17A modulates epithelial bicarbonate secretion and implicate a mechanism by which airway surface liquid pH changes may be abnormal in cystic fibrosis. PMID:19074559

  19. Mechanics of airflow in the human nasal airways.

    Science.gov (United States)

    Doorly, D J; Taylor, D J; Schroter, R C

    2008-11-30

    The mechanics of airflow in the human nasal airways is reviewed, drawing on the findings of experimental and computational model studies. Modelling inevitably requires simplifications and assumptions, particularly given the complexity of the nasal airways. The processes entailed in modelling the nasal airways (from defining the model, to its production and, finally, validating the results) is critically examined, both for physical models and for computational simulations. Uncertainty still surrounds the appropriateness of the various assumptions made in modelling, particularly with regard to the nature of flow. New results are presented in which high-speed particle image velocimetry (PIV) and direct numerical simulation are applied to investigate the development of flow instability in the nasal cavity. These illustrate some of the improved capabilities afforded by technological developments for future model studies. The need for further improvements in characterising airway geometry and flow together with promising new methods are briefly discussed.

  20. Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine.

    Science.gov (United States)

    Sherwood, Cara L; Boitano, Scott

    2016-05-17

    The potential for adverse respiratory effects following exposure to electronic (e-) cigarette liquid (e-liquid) flavorings remains largely unexplored. Given the multitude of flavor permutations on the market, identification of those flavor constituents that negatively impact the respiratory tract is a daunting task. In this study we examined the impact of common e-liquid flavoring chemicals on the airway epithelium, the cellular monolayer that provides the first line of defense against inhaled particulates, pathogens, and toxicants. We used the xCELLigence real-time cell analyzer (RTCA) as a primary high-capacity screening tool to assess cytotoxicity thresholds and physiological effects of common e-liquid flavoring chemicals on immortalized human bronchial epithelial cells (16HBE14o-). The RTCA was used secondarily to assess the capability of 16HBE14o- cells to respond to cellular signaling agonists following a 24 h exposure to select flavoring chemicals. Finally, we conducted biophysical measurements of well-differentiated primary mouse tracheal epithelial (MTE) cells with an Ussing chamber to measure the effects of e-cigarette flavoring constituents on barrier function and ion conductance. In our high-capacity screens five of the seven flavoring chemicals displayed changes in cellular impedance consistent with cell death at concentrations found in e-liquid. Vanillin and the chocolate flavoring 2,5-dimethylpyrazine caused alterations in cellular physiology indicative of a cellular signaling event. At subcytotoxic levels, 24 h exposure to 2,5-dimethylpyrazine compromised the ability of airway epithelial cells to respond to signaling agonists important in salt and water balance at the airway surface. Biophysical measurements of 2,5-dimethylpyrazine on primary MTE cells revealed alterations in ion conductance consistent with an efflux at the apical airway surface that was accompanied by a transient loss in transepithelial resistance. Mechanistic studies confirmed

  1. Primary Paediatric Bronchial Airway Epithelial Cell in Vitro Responses to Environmental Exposures

    Directory of Open Access Journals (Sweden)

    Neil McInnes

    2016-03-01

    Full Text Available The bronchial airway epithelial cell (BAEC is the site for initial encounters between inhaled environmental factors and the lower respiratory system. Our hypothesis was that release of pro inflammatory interleukins (IL-6 and IL-8 from primary BAEC cultured from children will be increased after in vitro exposure to common environmental factors. Primary BAEC were obtained from children undergoing clinically indicated routine general anaesthetic procedures. Cells were exposed to three different concentrations of lipopolysaccharide (LPS or house dust mite allergen (HDM or particulates extracted from side stream cigarette smoke (SSCS. BAEC were obtained from 24 children (mean age 7.0 years and exposed to stimuli. Compared with the negative control, there was an increase in IL-6 and IL-8 release after exposure to HDM (p ≤ 0.001 for both comparisons. There was reduced IL-6 after higher compared to lower SSCS exposure (p = 0.023. There was no change in BAEC release of IL-6 or IL-8 after LPS exposure. BAEC from children are able to recognise and respond in vitro with enhanced pro inflammatory mediator secretion to some inhaled exposures.

  2. Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Youngwoo Choi

    2017-01-01

    Full Text Available The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA. However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs, cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE or myeloperoxidase (MPO attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC, nonsevere asthma (NSA, and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

  3. Secreted osteopontin is highly polymerized in human airways and fragmented in asthmatic airway secretions.

    Directory of Open Access Journals (Sweden)

    Mehrdad Arjomandi

    Full Text Available Osteopontin (OPN is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING family and a cytokine with diverse biologic roles. OPN undergoes extensive post-translational modifications, including polymerization and proteolytic fragmentation, which alters its biologic activity. Recent studies suggest that OPN may contribute to the pathogenesis of asthma.To determine whether secreted OPN (sOPN is polymerized in human airways and whether it is qualitatively different in asthma, we used immunoblotting to examine sOPN in bronchoalveolar lavage (BAL fluid samples from 12 healthy and 21 asthmatic subjects (and in sputum samples from 27 healthy and 21 asthmatic subjects. All asthmatic subjects had mild to moderate asthma and abstained from corticosteroids during the study. Furthermore, we examined the relationship between airway sOPN and cellular inflammation.We found that sOPN in BAL fluid and sputum exists in polymeric, monomeric, and cleaved forms, with most of it in polymeric form. Compared to healthy subjects, asthmatic subjects had proportionately less polymeric sOPN and more monomeric and cleaved sOPN. Polymeric sOPN in BAL fluid was associated with increased alveolar macrophage counts in airways in all subjects.These results suggest that sOPN in human airways (1 undergoes extensive post-translational modification by polymerization and proteolytic fragmentation, (2 is more fragmented and less polymerized in subjects with mild to moderate asthma, and (3 may contribute to recruitment or survival of alveolar macrophages.

  4. Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

    Science.gov (United States)

    Gingerich, Aaron; Pang, Lan; Hanson, Jarod; Dlugolenski, Daniel; Streich, Rebecca; Lafontaine, Eric R; Nagy, Tamás; Tripp, Ralph A; Rada, Balázs

    2016-01-01

    Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.

  5. Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

    Directory of Open Access Journals (Sweden)

    Matthew J Kesic

    Full Text Available Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs human airway trypsin-like protease (HAT and transmembrane protease, serine 2 (TMPRSS2, whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI. Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

  6. Human influenza is more effective than avian influenza at antiviral suppression in airway cells.

    Science.gov (United States)

    Hsu, Alan Chen-Yu; Barr, Ian; Hansbro, Philip M; Wark, Peter A

    2011-06-01

    Airway epithelial cells are the initial site of infection with influenza viruses. The innate immune responses of airway epithelial cells to infection are important in limiting virus replication and spread. However, relatively little is known about the importance of this innate antiviral response to infection. Avian influenza viruses are a potential source of future pandemics; therefore, it is critical to examine the effectiveness of the host antiviral system to different influenza viruses. We used a human influenza (H3N2) and a low-pathogenic avian influenza (H11N9) to assess and compare the antiviral responses of Calu-3 cells. After infection, H3N2 replicated more effectively than the H11N9 in Calu-3 cells. This was not due to differential expression of sialic acid residues on Calu-3 cells, but was attributed to the interference of host antiviral responses by H3N2. H3N2 induced a delayed antiviral signaling and impaired type I and type III IFN induction compared with the H11N9. The gene encoding for nonstructural (NS) 1 protein was transfected into the bronchial epithelial cells (BECs), and the H3N2 NS1 induced a greater inhibition of antiviral responses compared with the H11N9 NS1. Although the low-pathogenic avian influenza virus was capable of infecting BECs, the human influenza virus replicated more effectively than avian influenza virus in BECs, and this was due to a differential ability of the two NS1 proteins to inhibit antiviral responses. This suggests that the subversion of human antiviral responses may be an important requirement for influenza viruses to adapt to the human host and cause disease.

  7. Airway basement membrane perimeter in human airways is not a constant; potential implications for airway remodeling in asthma.

    Science.gov (United States)

    McParland, Brent E; Paré, Peter D; Johnson, Peter R A; Armour, Carol L; Black, Judith L

    2004-08-01

    Many studies that demonstrate an increase in airway smooth muscle in asthmatic patients rely on the assumption that bronchial internal perimeter (P(i)) or basement membrane perimeter (P(bm)) is a constant, i.e., not affected by fixation pressure or the degree of smooth muscle shortening. Because it is the basement membrane that has been purported to be the indistensible structure, this study examines the assumption that P(bm) is not affected by fixation pressure. P(bm) was determined for the same human airway segment (n = 12) fixed at distending pressures of 0 cmH(2)O and 21 cmH(2)O in the absence of smooth muscle tone. P(bm) for the segment fixed at 0 cmH(2)O was determined morphometrically, and the P(bm) for the same segment, had the segment been fixed at 21 cmH(2)O, was predicted from knowing the luminal volume and length of the airway when distended to 21 cmH(2)O (organ bath-derived P(i)). To ensure an accurate transformation of the organ bath-derived P(i) value to a morphometry-derived P(bm) value, had the segment been fixed at 21 cmH(2)O, the relationship between organ bath-derived P(i) and morphometry-derived P(bm) was determined for five different bronchial segments distended to 21 cmH(2)O and fixed at 21 cmH(2)O (r(2) = 0.99, P < 0.0001). Mean P(bm) for bronchial segments fixed at 0 cmH(2)O was 9.4 +/- 0.4 mm, whereas mean predicted P(bm), had the segments been fixed at 21 cmH(2)O, was 14.1 +/- 0.5 mm (P < 0.0001). This indicates that P(bm) is not a constant when isolated airway segments without smooth muscle tone are fixed distended to 21 cmH(2)O. The implication of these results is that the increase in smooth muscle mass in asthma may have been overestimated in some previous studies. Therefore, further studies are required to examine the potential artifact using whole lungs with and without abolition of airway smooth muscle tone and/or inflation.

  8. Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.

    Science.gov (United States)

    Moreau-Marquis, Sophie; Coutermarsh, Bonita; Stanton, Bruce A

    2015-01-01

    Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells. P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting. ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms. Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI(®) (tobramycin) or Cayston(®) (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  9. Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

    Science.gov (United States)

    Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

    2015-01-01

    ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

  10. Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

    Science.gov (United States)

    Mao, Suifang; Shah, Alok S; Moninger, Thomas O; Ostedgaard, Lynda S; Lu, Lin; Tang, Xiao Xiao; Thornell, Ian M; Reznikov, Leah R; Ernst, Sarah E; Karp, Philip H; Tan, Ping; Keshavjee, Shaf; Abou Alaiwa, Mahmoud H; Welsh, Michael J

    2018-02-06

    Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gα i and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. Copyright © 2018 the Author(s). Published by PNAS.

  11. Characterizing adult human nasal airway dimensions

    International Nuclear Information System (INIS)

    Guilmette, R.A.; Griffith, W.C.

    1994-01-01

    Respiratory tract models used in calculating radiation dose from exposure to inhaled radioactive aerosols have only recently focused attention on the importance of the nasal airways (NAs). Because the NAs are the first tissues of the respiratory tract available for aerosol deposition in normally nose-breathing people, any deposition of aerosol in this anatomical structure will reduce the amounts available to be deposited in the remainder of the respiratory tract. Thus, uncertainties in estimating the deposition fractions in the NAs will propagate throughout the remainder of the respiratory tract, creating errors in the calculated dose estimates. Additionally, there is evidence that the NAs are also at risk for induction of cancer from exposure to certain occupational aerosols such as wood dust, leather dust, chromium, and nickel. The purpose of this investigation was to conduct an anatomical study to assess the variabilities in NA dimensions

  12. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  13. Autoradiographic detection of radionuclides on the epithelial surfaces of pulmonary airways

    International Nuclear Information System (INIS)

    Pappin, J.L.; Filipy, R.E.; Madison, R.M.

    1979-01-01

    We are developing an autoradiographic method for detection of radionuclide deposition sites on the internal surfaces of pulmonary airways. The method is expected to generate information on the distribution as well as on the quantity of radionuclides deposited in pulmonary airways

  14. miR-126 is downregulated in cystic fibrosis airway epithelial cells and regulates TOM1 expression.

    LENUS (Irish Health Repository)

    Oglesby, Irene K

    2010-02-15

    Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL-1beta and TNF-alpha-induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre-miR-126 inhibited luciferase activity in a reporter system containing the full length 3\\'-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1beta, overexpression of TOM1 was found to downregulate NF-kappaB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-kappaB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2\\/4 signaling pathways and the first to describe microRNA involvement in CF.

  15. induced acute cytotoxicity in human cervical epithelial carcinoma cells

    African Journals Online (AJOL)

    Molecular basis of arsenite (As +3 )-induced acute cytotoxicity in human cervical epithelial carcinoma cells. ... Libyan Journal of Medicine ... Methods: After performing cytotoxic assays on a human epithelial carcinoma cell line, expression analysis was done by quantitative polymerase chain reaction, western blotting, and ...

  16. Transport and deposition of cohesive pharmaceutical powders in human airway

    Directory of Open Access Journals (Sweden)

    Wang Yuan

    2017-01-01

    Full Text Available Pharmaceutical powders used in inhalation therapy are in the size range of 1-5 microns and are usually cohesive. Understanding the cohesive behaviour of pharmaceutical powders during their transportation in human airway is significant in optimising aerosol drug delivery and targeting. In this study, the transport and deposition of cohesive pharmaceutical powders in a human airway model is simulated by a well-established numerical model which combines computational fluid dynamics (CFD and discrete element method (DEM. The van der Waals force, as the dominant cohesive force, is simulated and its influence on particle transport and deposition behaviour is discussed. It is observed that even for dilute particle flow, the local particle concentration in the oral to trachea region can be high and particle aggregation happens due to the van der Waals force of attraction. It is concluded that the deposition mechanism for cohesive pharmaceutical powders, on one hand, is dominated by particle inertial impaction, as proven by previous studies; on the other hand, is significantly affected by particle aggregation induced by van der Waals force. To maximum respiratory drug delivery efficiency, efforts should be made to avoid pharmaceutical powder aggregation in human oral-to-trachea airway.

  17. Transport and deposition of cohesive pharmaceutical powders in human airway

    Science.gov (United States)

    Wang, Yuan; Chu, Kaiwei; Yu, Aibing

    2017-06-01

    Pharmaceutical powders used in inhalation therapy are in the size range of 1-5 microns and are usually cohesive. Understanding the cohesive behaviour of pharmaceutical powders during their transportation in human airway is significant in optimising aerosol drug delivery and targeting. In this study, the transport and deposition of cohesive pharmaceutical powders in a human airway model is simulated by a well-established numerical model which combines computational fluid dynamics (CFD) and discrete element method (DEM). The van der Waals force, as the dominant cohesive force, is simulated and its influence on particle transport and deposition behaviour is discussed. It is observed that even for dilute particle flow, the local particle concentration in the oral to trachea region can be high and particle aggregation happens due to the van der Waals force of attraction. It is concluded that the deposition mechanism for cohesive pharmaceutical powders, on one hand, is dominated by particle inertial impaction, as proven by previous studies; on the other hand, is significantly affected by particle aggregation induced by van der Waals force. To maximum respiratory drug delivery efficiency, efforts should be made to avoid pharmaceutical powder aggregation in human oral-to-trachea airway.

  18. Response of Differentiated Human Airway Epithelia to Alcohol Exposure and Klebsiella pneumoniae Challenge

    Directory of Open Access Journals (Sweden)

    Sammeta V. Raju

    2013-07-01

    Full Text Available Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 h did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae, the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.

  19. Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing.

    Science.gov (United States)

    Lendeckel, Derik; Eymann, Christine; Emicke, Philipp; Daeschlein, Georg; Darm, Katrin; O'Neil, Serena; Beule, Achim G; von Woedtke, Thomas; Völker, Uwe; Weltmann, Klaus-Dieter; Jünger, Michael; Hosemann, Werner; Scharf, Christian

    2015-01-01

    The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine.

  20. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP) and CCL11/eotaxin-1 in human asthmatic airways.

    Science.gov (United States)

    Nino, Gustavo; Huseni, Shehlanoor; Perez, Geovanny F; Pancham, Krishna; Mubeen, Humaira; Abbasi, Aleeza; Wang, Justin; Eng, Stephen; Colberg-Poley, Anamaris M; Pillai, Dinesh K; Rose, Mary C

    2014-01-01

    Thymic stromal lymphoproetin (TSLP) is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral) and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state. Primary human bronchial epithelial cells (HBEC) from control (n = 3) and asthmatic (n = 3) donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI) conditions and treated apically with dsRNA (viral surrogate) or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC) from normal (n = 3) and asthmatic (n = 3) donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20) vs. non-asthmatic uninfected controls (n = 20). Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay. Our data demonstrate that: 1) Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2) TSLP exposure induces unidirectional (apical) secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3) Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1. There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  1. Directional secretory response of double stranded RNA-induced thymic stromal lymphopoetin (TSLP and CCL11/eotaxin-1 in human asthmatic airways.

    Directory of Open Access Journals (Sweden)

    Gustavo Nino

    Full Text Available Thymic stromal lymphoproetin (TSLP is a cytokine secreted by the airway epithelium in response to respiratory viruses and it is known to promote allergic Th2 responses in asthma. This study investigated whether virally-induced secretion of TSLP is directional in nature (apical vs. basolateral and/or if there are TSLP-mediated effects occurring at both sides of the bronchial epithelial barrier in the asthmatic state.Primary human bronchial epithelial cells (HBEC from control (n = 3 and asthmatic (n = 3 donors were differentiated into polarized respiratory tract epithelium under air-liquid interface (ALI conditions and treated apically with dsRNA (viral surrogate or TSLP. Sub-epithelial effects of TSLP were examined in human airway smooth muscle cells (HASMC from normal (n = 3 and asthmatic (n = 3 donors. Clinical experiments examined nasal airway secretions obtained from asthmatic children during naturally occurring rhinovirus-induced exacerbations (n = 20 vs. non-asthmatic uninfected controls (n = 20. Protein levels of TSLP, CCL11/eotaxin-1, CCL17/TARC, CCL22/MDC, TNF-α and CXCL8 were determined with a multiplex magnetic bead assay.Our data demonstrate that: 1 Asthmatic HBEC exhibit an exaggerated apical, but not basal, secretion of TSLP after dsRNA exposure; 2 TSLP exposure induces unidirectional (apical secretion of CCL11/eotaxin-1 in asthmatic HBEC and enhanced CCL11/eotaxin-1 secretion in asthmatic HASMC; 3 Rhinovirus-induced asthma exacerbations in children are associated with in vivo airway secretion of TSLP and CCL11/eotaxin-1.There are virally-induced TSLP-driven secretory immune responses at both sides of the bronchial epithelial barrier characterized by enhanced CCL11/eotaxin-1 secretion in asthmatic airways. These results suggest a new model of TSLP-mediated eosinophilic responses in the asthmatic airway during viral-induced exacerbations.

  2. Proteomic profiling of acrolein adducts in human lung epithelial cells

    Science.gov (United States)

    Spiess, Page C.; Deng, Bin; Hondal, Robert J.; Matthews, Dwight E.; van der Vliet, Albert

    2011-01-01

    Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology. PMID:21704744

  3. Vectorial transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: upregulation by IFN-gamma

    NARCIS (Netherlands)

    Loman, S.; Radl, J.; Jansen, H. M.; Out, T. A.; Lutter, R.

    1997-01-01

    We have developed an in vitro airway epithelial cell model for dimeric immunoglobulin (Ig) A (dIgA) transcytosis that allows the assessment of polymeric Ig receptor (pIgR) gene expression and actual dIgA transport. Tight monolayers of human lung-derived Calu-3 adenocarcinoma cells grown on permeable

  4. Airway surface irregularities promote particle diffusion in the human lung

    International Nuclear Information System (INIS)

    Martonen, T.; North Carolina Univ., Chapel Hill, NC; Zhang, Z.; Yang, Y.; Bottei, G.

    1995-01-01

    Current NCRP and ICRP particle deposition models employed in risk assessment analyses treat the airways of the human lung as smooth-walled tubes. However, the upper airways of the tracheobronchial (TB) tree are line with cartilaginous rings. Recent supercomputer simulations of in vivo conditions (cited herein), where cartilaginous ring morphologies were based upon fibre-optic bronchoscope examinations, have clearly demonstrated their profound effects upon fluid dynamics. A physiologically based analytical model of fluid dynamics is presented, focusing upon applications to particle diffusion within the TB tree. The new model is the first to describe particle motion while simultaneously simulating effects of wall irregularities, entrance conditions and tube curvatures. This study may explain the enhanced deposition by particle diffusion detected in replica case experiments and have salient implications for the clinically observed preferential distributions of bronchogenic carcinomas associated with inhaled radionuclides. (author)

  5. Human eosinophil–airway smooth muscle cell interactions

    Directory of Open Access Journals (Sweden)

    J. Margaret Hughes

    2000-01-01

    Full Text Available Eosinophils are present throughout the airway wall of asthmatics. The nature of the interaction between human airway smooth muscle cells (ASMC and eosinophils was investigated in this study. We demonstrated, using light microscopy, that freshly isolated eosinophils from healthy donors rapidly attach to ASMC in vitro. Numbers of attached eosinophils were highest at 2 h, falling to 50% of maximum by 20 h. Eosinophil attachment at 2 h was reduced to 72% of control by anti-VCAM-1, and to 74% at 20 h by anti-ICAM-1. Pre-treatment of ASMC for 24 h with TNF-α, 10 nM, significantly increased eosinophil adhesion to 149 and 157% of control after 2 and 20 h. These results provide evidence that eosinophil interactions with ASMC involve VCAM-1 and ICAM-1 and are modulated by TNF-α.

  6. Interleukin-13–induced MUC5AC Is Regulated by 15-Lipoxygenase 1 Pathway in Human Bronchial Epithelial Cells

    Science.gov (United States)

    Zhao, Jinming; Maskrey, Ben; Balzar, Silvana; Chibana, Kazuyuki; Mustovich, Anthony; Hu, Haizhen; Trudeau, John B.; O'Donnell, Valerie; Wenzel, Sally E.

    2009-01-01

    Rationale: 15-Lipoxygenase-1 (15LO1) and MUC5AC are highly expressed in asthmatic epithelial cells. IL-13 is known to induce 15LO1 and MUC5AC in human airway epithelial cells in vitro. Whether 15LO1 and/or its product 15-HETE modulate MUC5AC expression is unknown. Objectives: To determine the expression of 15LO1 in freshly harvested epithelial cells from subjects with asthma and normal control subjects and to determine whether IL-13–induced 15LO1 expression and activation regulate MUC5AC expression in human bronchial epithelial cells in vitro. Methods: Human airway epithelial cells from subjects with asthma and normal subjects were evaluated ex vivo for 15LO1 and MUC5AC expression. The impact of 15LO1 on MUC5AC expression in vitro was analyzed by inhibiting 15LO1 through pharmacologic (PD146176) and siRNA approaches in human bronchial epithelial cells cultured under air–liquid interface. We analyzed 15 hydroxyeicosatetraenoic acid (15-HETE) by liquid chromatography/UV/mass spectrometry. MUC5AC and 15LO1 were analyzed by real-time RT-PCR, immunofluoresence, and Western blot. Measurements and Main Results: Epithelial 15LO1 expression increased with asthma severity (P < 0.0001). 15LO1 significantly correlated with MUC5AC ex vivo and in vitro. IL-13 increased 15LO1 expression and stimulated formation of two molecular species of 15-HETE esterified to phosphotidylethanolamine (15-HETE-PE). Inhibition of 15LO1 suppressed 15-HETE-PE and decreased MUC5AC expression in the presence of IL-13 stimulation. The addition of exogenous 15-HETE partially restored MUC5AC expression. Conclusions: Epithelial 15LO1 expression increases with increasing asthma severity. IL-13 induction of 15-HETE-PE enhances MUC5AC expression in human airway epithelial cells. High levels of 15LO1 activity could contribute to the increases of MUC5AC observed in asthma. PMID:19218191

  7. TLR-dependent human mucosal epithelial cell responses to microbial pathogens.

    Directory of Open Access Journals (Sweden)

    Paola eMassari

    2014-08-01

    Full Text Available AbstractToll-Like Receptor (TLR signaling represents one of the best studied pathways to implement defense mechanisms against invading microbes in humans as well as in animals. TLRs respond to specific microbial ligands and to danger signals produced by the host during infection, and initiate downstream cascades that activate both innate and adaptive immunity. TLRs are expressed by professional immune cells and by the large majority of non-hematopoietic cells, including epithelial cells. In epithelial tissues, TLR functions are particularly important because these sites are constantly exposed to microorganisms, due to their location at the host interface with the environment. While at these sites, specific defense mechanisms and inflammatory responses are initiated via TLR signaling against pathogens, suppression or lack of TLR activation is also observed in response to the commensal microbiota. The mechanisms by which TLR signaling is regulated in mucosal epithelial cells include differential expression and levels of TLRs (and their signaling partners, their cellular localization and positioning within the tissue in a fashion that favors responses to pathogens while dampening responses to commensals and maintaining tissue homeostasis in physiologic conditions. In this review, the expression and activation of TLRs in mucosal epithelial cells of several sites of the human body are examined. Specifically, the oral cavity, the ear canal and eye, the airways, the gut and the reproductive tract are discussed, along with how site-specific host defense mechanisms are implemented via TLR signaling.

  8. Regional aerosol deposition in human upper airways

    Energy Technology Data Exchange (ETDEWEB)

    Swift, D.L.

    1991-11-01

    During the current report experimental studies of upper respiratory deposition of radon progeny aerosols and stimulant aerosols were carried out in replicate casts of nasal and oral passages of adults and children. Additionally, preliminary studies of nasal passage deposition of unattached Po{sup 218} particles was carried out in four human subjects. Data on nasal inspiratory deposition in replicate models of adults and infants from three collaborating laboratories were compared and a best-fit curve of deposition efficiency for both attached and unattached particles was obtained, showing excellent inter-laboratory agreement. This curve demonstrates that nasal inspiratory deposition of radon progeny is weakly dependent upon flow rate over physiologically realistic ranges of flow, does not show a significant age effect, and is relatively independent of nasal passage dimensions for a given age range. Improved replicate models of the human adult oral passage extending to the mid-trachea were constructed for medium and higher flow mouth breathing states; these models were used to assess the deposition of unattached Po{sup 218} particles during oronasal breathing in the oral passage and demonstrated lower deposition efficiency than the nasal passage. Measurements of both Po{sup 218} particle and attached fraction particle size deposition were performed in replicate nasal passage of a four week old infant. 5 refs., 1 fig.

  9. Regional aerosol deposition in human upper airways

    International Nuclear Information System (INIS)

    Swift, D.L.

    1991-01-01

    During the current report experimental studies of upper respiratory deposition of radon progeny aerosols and stimulant aerosols were carried out in replicate casts of nasal and oral passages of adults and children. Additionally, preliminary studies of nasal passage deposition of unattached Po 218 particles was carried out in four human subjects. Data on nasal inspiratory deposition in replicate models of adults and infants from three collaborating laboratories were compared and a best-fit curve of deposition efficiency for both attached and unattached particles was obtained, showing excellent inter-laboratory agreement. This curve demonstrates that nasal inspiratory deposition of radon progeny is weakly dependent upon flow rate over physiologically realistic ranges of flow, does not show a significant age effect, and is relatively independent of nasal passage dimensions for a given age range. Improved replicate models of the human adult oral passage extending to the mid-trachea were constructed for medium and higher flow mouth breathing states; these models were used to assess the deposition of unattached Po 218 particles during oronasal breathing in the oral passage and demonstrated lower deposition efficiency than the nasal passage. Measurements of both Po 218 particle and attached fraction particle size deposition were performed in replicate nasal passage of a four week old infant. 5 refs., 1 fig

  10. Growth of airway epithelial cells at an air-liquid interface changes both the response to particle exposure and iron homeostasis

    Science.gov (United States)

    We tested the hypothesis that 1) relative to submerged cells, airway epithelial cells grown at an air-liquid interface and allowed to differentiate would have an altered response to particle exposure and 2) that these differences would be associated with indices of iron homeostas...

  11. A model of human nasal epithelial cells adapted for direct and repeated exposure to airborne pollutants.

    Science.gov (United States)

    Bardet, Gaëlle; Achard, Sophie; Loret, Thomas; Desauziers, Valérie; Momas, Isabelle; Seta, Nathalie

    2014-08-17

    Airway epithelium lining the nasal cavity plays a pivotal role in respiratory tract defense and protection mechanisms. Air pollution induces alterations linked to airway diseases such as asthma. Only very few in vitro studies to date have succeeded in reproducing physiological conditions relevant to cellular type and chronic atmospheric pollution exposure. We therefore, set up an in vitro model of human Airway Epithelial Cells of Nasal origin (hAECN) close to real human cell functionality, specifically adapted to study the biological effects of exposure to indoor gaseous pollution at the environmental level. hAECN were exposed under air-liquid interface, one, two, or three-times at 24 h intervals for 1 h, to air or formaldehyde (200 μg/m(3)), an indoor air gaseous pollutant. All experiments were ended at day 4, when both cellular viability and cytokine production were assessed. Optimal adherence and confluence of cells were obtained 96 h after cell seeding onto collagen IV-precoated insert. Direct and repeated exposure to formaldehyde did not produce any cellular damage or IL-6 production change, although weak lower IL-8 production was observed only after the third exposure. Our model is significantly better than previous ones due to cell type and the repeated exposure protocol. Copyright © 2014. Published by Elsevier Ireland Ltd.

  12. Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

    DEFF Research Database (Denmark)

    Nassini, Romina; Pedretti, Pamela; Moretto, Nadia

    2012-01-01

    The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic...... inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1.By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express...... functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells...

  13. In vitro assessment of human airway toxicity from major aldehydes in automotive emissions

    Energy Technology Data Exchange (ETDEWEB)

    Grafstroem, R.C. [Karolinska Inst., Stockholm (Sweden). Inst. of Environmental Medicine

    1997-09-01

    Automotive exhausts can significantly contribute to the levels of reactive aldehydes, including formaldehyde, acetaldehyde and acrolein, in urban air. The use of alcohols as an alternative fuel for gasoline or diesel may further increase these emissions. Since it is unclear if aldehyde inhalation may induce pathological states, including cancer, in human airways, the toxic properties of the above-mentioned aldehydes were studied in cultured target cell types. Each aldehyde modified vital cellular functions in a dose-dependent manner, and invariably inhibited growth and induced abnormal terminal differentiation. Decreases of cellular thiols and increases of intracellular Ca{sup 2+} were observed, and moreover, variable types and amounts of short-lived or persistent genetic damage were induced. The concentrations required for specified levels of a particular type of injury varied up to 10000-fold among the aldehydes. Overall, distinctive patterns of cytopathological activity were observed, which differed both qualitatively and quantitatively among the aldehydes. Finally, aldehydes inhibited DNA repair processes and increased cytotoxicity and mutagenesis in synergy with other known toxicants, indicating that aldehydes may also enhance damage by other constituents in automotive exhausts. In summary, the aldehydes, notably {sup m}u{sup M}-mM formaldehyde, caused pathological effects and induced mechanisms that relate to acute toxicity and cancer development in airway epithelial cells. Since `no-effect` levels may not exist for carcinogenic agents, the overall results support a need for elimination of aldehydes in automotive exhausts. 41 refs

  14. Interleukin-33 induces mucin gene expression and goblet cell hyperplasia in human nasal epithelial cells.

    Science.gov (United States)

    Ishinaga, Hajime; Kitano, Masako; Toda, Masaaki; D'Alessandro-Gabazza, Corina N; Gabazza, Esteban C; Shah, Said Ahmad; Takeuchi, Kazuhiko

    2017-02-01

    We investigated whether IL-33 is involved in mucus overproduction and goblet cell hyperplasia in eosinophilic chronic rhinosinusitis (ECRS). IL-33 mRNA was significantly higher in the eosinophilic CRS group than in the non-eosinophilic CRS group from human nasal polyps. IL-33 induced MUC5AC mRNA and MUC5AC protein, and also goblet cell hyperplasia at air liquid interface culture in human nasal epithelial cells. In addition to that, IL-33 induced MUC5B and FOXA3, and reduces FOXJmRNA. In conclusion, our present study demonstrated that the direct evidence of IL-33 which lead to increase mucin gene and protein expression, as well as goblet cell hyperplasia. This study provides novel insights into the role of IL-33 on mucus overproduction in eosinophilic inflammation of human airways. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs

    Directory of Open Access Journals (Sweden)

    Shingo Suzuki

    2016-01-01

    Full Text Available Cystic fibrosis (CF is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ≃100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

  16. Epithelial cells as alternative human biomatrices for comet assay.

    Science.gov (United States)

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  17. Lung disease phenotypes caused by overexpression of combinations of α-, β-, and γ-subunits of the epithelial sodium channel in mouse airways.

    Science.gov (United States)

    Livraghi-Butrico, Alessandra; Wilkinson, Kristen J; Volmer, Allison S; Gilmore, Rodney C; Rogers, Troy D; Caldwell, Ray A; Burns, Kimberlie A; Esther, Charles R; Mall, Marcus A; Boucher, Richard C; O'Neal, Wanda K; Grubb, Barbara R

    2018-02-01

    The epithelial Na + channel (ENaC) regulates airway surface hydration. In mouse airways, ENaC is composed of three subunits, α, β, and γ, which are differentially expressed (α > β > γ). Airway-targeted overexpression of the β subunit results in Na + hyperabsorption, causing airway surface dehydration, hyperconcentrated mucus with delayed clearance, lung inflammation, and perinatal mortality. Notably, mice overexpressing the α- or γ-subunit do not exhibit airway Na + hyperabsorption or lung pathology. To test whether overexpression of multiple ENaC subunits produced Na + transport and disease severity exceeding that of βENaC-Tg mice, we generated double (αβ, αγ, βγ) and triple (αβγ) transgenic mice and characterized their lung phenotypes. Double αγENaC-Tg mice were indistinguishable from WT littermates. In contrast, double βγENaC-Tg mice exhibited airway Na + absorption greater than that of βENaC-Tg mice, which was paralleled by worse survival, decreased mucociliary clearance, and more severe lung pathology. Double αβENaC-Tg mice exhibited Na + transport rates comparable to those of βENaC-Tg littermates. However, αβENaC-Tg mice had poorer survival and developed severe parenchymal consolidation. In situ hybridization (RNAscope) analysis revealed both alveolar and airway αENaC-Tg overexpression. Triple αβγENaC-Tg mice were born in Mendelian proportions but died within the first day of life, and the small sample size prevented analyses of cause(s) of death. Cumulatively, these results indicate that overexpression of βENaC is rate limiting for generation of pathological airway surface dehydration. Notably, airway co-overexpression of β- and γENaC had additive effects on Na + transport and disease severity, suggesting dose dependency of these two variables.

  18. Airway Clearance Techniques (ACTs)

    Medline Plus

    Full Text Available ... D Structure Consortium CFTR Folding Consortium Epithelial Stem Cell Consortium Mucociliary Clearance Consortium SUCCESS WITH THERAPIES RESEARCH ... clapping) or vibration to loosen mucus from airway walls. See how different airway clearance techniques work to ...

  19. Avian Influenza virus glycoproteins restrict virus replication and spread through human airway epithelium at temperatures of the proximal airways.

    Directory of Open Access Journals (Sweden)

    Margaret A Scull

    2009-05-01

    Full Text Available Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE, we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37 degrees C, avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32 degrees C. These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40 degrees C, rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32 degrees C and 37 degrees C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32 degrees C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2 or A/PR/8/34 (H1N1 genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA and neuraminidase (NA from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and

  20. Polarization Affects Airway Epithelial Conditioning of Monocyte-Derived Dendritic Cells

    DEFF Research Database (Denmark)

    Papazian, Dick; Chhoden, Tashi; Arge, Maria

    2015-01-01

    were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs....... In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens....

  1. Human papillomavirus: cause of epithelial lacrimal sac neoplasia?

    DEFF Research Database (Denmark)

    Sjö, Nicolai Christian; von Buchwald, Christian; Cassonnet, Patricia

    2007-01-01

    PURPOSE: Epithelial tumours of the lacrimal sac are rare but important entities that may carry grave prognoses. In this study the prevalence and possible role of human papillomavirus (HPV) infection in epithelial tumours of the lacrimal sac were evaluated. METHODS: Five papillomas and six...... 11 RNA was demonstrated in two papillomas. CONCLUSIONS: By analysing 11 epithelial lacrimal sac papillomas and carcinomas using PCR, DNA ISH and RNA ISH, we found HPV DNA in all investigated transitional epithelium tumours of the lacrimal sac. HPV RNA was present in two of eight epithelial lacrimal...... sac tumours positive for HPV DNA. As RNA degrades fast in paraffin-embedded tissue, only a small fraction of DNA-positive tumours can be expected to be RNA-positive. We therefore suggest that HPV infection is associated with the development of lacrimal sac papillomas and carcinomas....

  2. Gene expression patterns induced at different stages of rhinovirus infection in human alveolar epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Etemadi

    Full Text Available Human rhinovirus (HRV is the common virus that causes acute respiratory infection (ARI and is frequently associated with lower respiratory tract infections (LRTIs. We aimed to investigate whether HRV infection induces a specific gene expression pattern in airway epithelial cells. Alveolar epithelial cell monolayers were infected with HRV species B (HRV-B. RNA was extracted from both supernatants and infected monolayer cells at 6, 12, 24 and 48 hours post infection (hpi and transcriptional profile was analyzed using Affymetrix GeneChip and the results were subsequently validated using quantitative Real-time PCR method. HRV-B infects alveolar epithelial cells which supports implication of the virus with LRTIs. In total 991 genes were found differentially expressed during the course of infection. Of these, 459 genes were up-regulated whereas 532 genes were down-regulated. Differential gene expression at 6 hpi (187 genes up-regulated vs. 156 down-regulated were significantly represented by gene ontologies related to the chemokines and inflammatory molecules indicating characteristic of viral infection. The 75 up-regulated genes surpassed the down-regulated genes (35 at 12 hpi and their enriched ontologies fell into discrete functional entities such as regulation of apoptosis, anti-apoptosis, and wound healing. At later time points of 24 and 48 hpi, predominated down-regulated genes were enriched for extracellular matrix proteins and airway remodeling events. Our data provides a comprehensive image of host response to HRV infection. The study suggests the underlying molecular regulatory networks genes which might be involved in pathogenicity of the HRV-B and potential targets for further validations and development of effective treatment.

  3. Dual Function of Novel Pollen Coat (Surface) Proteins: IgE-binding Capacity and Proteolytic Activity Disrupting the Airway Epithelial Barrier

    Science.gov (United States)

    Bashir, Mohamed Elfatih H.; Ward, Jason M.; Cummings, Matthew; Karrar, Eltayeb E.; Root, Michael; Mohamed, Abu Bekr A.; Naclerio, Robert M.; Preuss, Daphne

    2013-01-01

    Background The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored. Methodology/Principal Findings Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. Conclusions/Significance Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is

  4. Dual function of novel pollen coat (surface proteins: IgE-binding capacity and proteolytic activity disrupting the airway epithelial barrier.

    Directory of Open Access Journals (Sweden)

    Mohamed Elfatih H Bashir

    Full Text Available BACKGROUND: The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., "de-fatted", and, as a result, their involvement in allergy has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass pollen (BGP by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP and endoxylanase (EXY. The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. CONCLUSIONS/SIGNIFICANCE: Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic

  5. Postnatal remodeling of the neural components of the epithelial-mesenchymal trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen.

    Science.gov (United States)

    Larson, Shawnessy D; Schelegle, Edward S; Walby, William F; Gershwin, Laural J; Fanuccihi, Michelle V; Evans, Michael J; Joad, Jesse P; Tarkington, Brian K; Hyde, Dallas M; Plopper, Charles G

    2004-02-01

    Nerves and neuroendocrine cells located within the airway epithelium are ideally situated to sample a changing airway environment, to transmit that information to the central nervous system, and to promote trophic interactions between epithelial and mesenchymal cellular and acellular components. We tested the hypothesis that the environmental stresses of ozone (O(3)) and house dust mite allergen (HDMA) in atopic infant rhesus monkeys alter the distribution of airway nerves. Midlevel bronchi and bronchioles from 6-month-old infant monkeys that inhaled filtered air (FA), house dust mite allergen HDMA, O(3), or HDMA + O(3) for 11 episodes (5 days each, 0.5 ppm O(3), 8 h/day followed by 9 days recovery) were examined using immunohistochemistry for the presence of Protein gene product 9.5 (PGP 9.5), a nonspecific neural indicator, and calcitonin gene-related peptide (CGRP). Along the axial path between the sixth and the seventh intrapulmonary airway generations, there were small significant (P < 0.05) decrements in the density of epithelial nerves in monkeys exposed to HDMA or O(3), while in monkeys exposed to HDMA + O(3) there was a greater significant (P < 0.05) reduction in epithelial innervation. In animals exposed to O(3) or HDMA + O(3) there was a significant increase in the number of PGP 9.5 positive/CGRP negative cells that were anchored to the basal lamina and emitted projections in primarily the lateral plain and often intertwined with projections and cell bodies of other similar cells. We conclude that repeated cycles of acute injury and repair associated with the episodic pattern of ozone and allergen exposure alter the normal development of neural innervation of the epithelial compartment and the appearance of a new population of undefined PGP 9.5 positive cells within the epithelium.

  6. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  7. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Emily F A van 't Wout

    2015-06-01

    Full Text Available Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA. Efficient functioning of the endoplasmic reticulum (ER is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR. Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.

  8. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells

    Science.gov (United States)

    van ‘t Wout, Emily F. A.; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E.; Clarke, Hanna J.; Tommassen, Jan; Marciniak, Stefan J.; Hiemstra, Pieter S.

    2015-01-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to “ER stress” and activation of the “unfolded protein response” (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  9. Nasal airway epithelial cell IL-6 and FKBP51 gene expression and steroid sensitivity in asthmatic children.

    Directory of Open Access Journals (Sweden)

    Michael Fayon

    Full Text Available Many asthmatic patients exhibit uncontrolled asthma despite high-dose inhaled corticosteroids (ICS. Airway epithelial cells (AEC have distinct activation profiles that can influence ICS response.A pilot study to identify gene expression markers of AEC dysfunction and markers of corticosteroid sensitivity in asthmatic and non-asthmatic control children, for comparison with published reports in adults.AEC were obtained by nasal brushings and primary submerged cultures, and incubated in control conditions or in the presence of 10 ng/ml TNFalpha, 10-8M dexamethasone, or both. RT-PCR-based expression of FKBP51 (a steroid hormone receptor signalling regulator, NF-kB, IL-6, LIF (an IL-6 family neurotrophic cytokine, serpinB2 (which inhibits plasminogen activation and promotes fibrin deposition and porin (a marker of mitochondrial mass were determined.6 patients without asthma (median age 11yr; min-max: 7-13, 8 with controlled asthma (11yr, 7-13; median daily fluticasone dose = 100 μg, and 4 with uncontrolled asthma (12yr, 7-14; 1000 μg fluticasone daily were included. Baseline expression of LIF mRNA was significantly increased in uncontrolled vs controlled asthmatic children. TNFalpha significantly increased LIF expression in uncontrolled asthma. A similar trend was observed regarding IL-6. Dexamethasone significantly upregulated FKBP51 expression in all groups but the response was blunted in asthmatic children. No significant upregulation was identified regarding NF-kB, serpinB2 and porin.LIF and FKBP51 expression in epithelial cells were the most interesting markers of AEC dysfunction/response to corticosteroid treatment.

  10. Unsteady flow characteristics through a human nasal airway.

    Science.gov (United States)

    Lee, Jong-Hoon; Na, Yang; Kim, Sung-Kyun; Chung, Seung-Kyu

    2010-07-31

    Time-dependent characteristics of the flow in a human nasal airway constructed from the CT image of a healthy volunteer were investigated using a computational fluid dynamics (CFD) technique. To capture the time-varying nature of the flow as well as pressure and temperature fields, the large eddy simulation (LES) technique instead of the RANS (Reynolds Averaged Navier-Stokes) approach was adopted. To make the present analysis more relevant to a real human breathing cycle, the flow was designed to be induced by the pressure difference and the time-varying pressure at the end of trachea was described to reproduce the flow rate data from the measurement. Comparison of the present results with those of typical steady simulations showed that the difference in flow characteristics is magnified in the expiration phase. This fact may suggest that the inertial effect associated with unsteady flow is more important during the expiration period. Also, the fact that the distribution of the flow rate in a given cross-section of the airway changes significantly with time implies the importance of unsteady data for clinical decision. The wall shear stress was found to have relatively high values at the locations near nasopharynx and larynx but the magnitude changes with time during the whole respiratory cycle. Analysis of the temperature field showed that most of the temperature change occurs in the nasal cavity when the air is incoming and thus, the nasal cavity acts as a very efficient heat exchanger during an inspiration period. Copyright 2010 Elsevier B.V. All rights reserved.

  11. Inhibition of Toll-like receptor 2-mediated interleukin-8 production in Cystic Fibrosis airway epithelial cells via the alpha7-nicotinic acetylcholine receptor.

    LENUS (Irish Health Repository)

    Greene, Catherine M

    2010-01-01

    Cystic Fibrosis (CF) is an inherited disorder characterised by chronic inflammation of the airways. The lung manifestations of CF include colonization with Pseudomonas aeruginosa and Staphylococcus aureus leading to neutrophil-dominated airway inflammation and tissue damage. Inflammation in the CF lung is initiated by microbial components which activate the innate immune response via Toll-like receptors (TLRs), increasing airway epithelial cell production of proinflammatory mediators such as the neutrophil chemokine interleukin-8 (IL-8). Thus modulation of TLR function represents a therapeutic approach for CF. Nicotine is a naturally occurring plant alkaloid. Although it is negatively associated with cigarette smoking and cardiovascular damage, nicotine also has anti-inflammatory properties. Here we investigate the inhibitory capacity of nicotine against TLR2- and TLR4-induced IL-8 production by CFTE29o- airway epithelial cells, determine the role of alpha7-nAChR (nicotinic acetylcholine receptor) in these events, and provide data to support the potential use of safe nicotine analogues as anti-inflammatories for CF.

  12. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  13. Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice

    DEFF Research Database (Denmark)

    Yao, Xianglan; Dai, Cuilian; Fredriksson, Karin

    2012-01-01

    Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (e2, e3, and e4) reflecting single ...

  14. Influence of horse stable environment on human airways.

    Science.gov (United States)

    Elfman, Lena; Riihimäki, Miia; Pringle, John; Wålinder, Robert

    2009-05-25

    Many people spend considerable amount of time each day in equine stable environments either as employees in the care and training of horses or in leisure activity. However, there are few studies available on how the stable environment affects human airways. This study examined in one horse stable qualitative differences in indoor air during winter and late summer conditions and assessed whether air quality was associated with clinically detectable respiratory signs or alterations to selected biomarkers of inflammation and lung function in stable personnel. The horse stable environment and stable-workers (n = 13) in one stable were investigated three times; first in the winter, second in the interjacent late summer and the third time in the following winter stabling period. The stable measurements included levels of ammonia, hydrogen sulphide, total and respirable dust, airborne horse allergen, microorganisms, endotoxin and glucan. The stable-workers completed a questionnaire on respiratory symptoms, underwent nasal lavage with subsequent analysis of inflammation markers, and performed repeated measurements of pulmonary function. Measurements in the horse stable showed low organic dust levels and high horse allergen levels. Increased viable level of fungi in the air indicated a growing source in the stable. Air particle load as well as 1,3-beta-glucan was higher at the two winter time-points, whereas endotoxin levels were higher at the summer time-point. Two stable-workers showed signs of bronchial obstruction with increased PEF-variability, increased inflammation biomarkers relating to reported allergy, cold or smoking and reported partly work-related symptoms. Furthermore, two other stable-workers reported work-related airway symptoms, of which one had doctor's diagnosed asthma which was well treated. Biomarkers involved in the development of airway diseases have been studied in relation to environmental exposure levels in equine stables. Respirable dust and 1

  15. Influence of horse stable environment on human airways

    Directory of Open Access Journals (Sweden)

    Pringle John

    2009-05-01

    Full Text Available Abstract Background Many people spend considerable amount of time each day in equine stable environments either as employees in the care and training of horses or in leisure activity. However, there are few studies available on how the stable environment affects human airways. This study examined in one horse stable qualitative differences in indoor air during winter and late summer conditions and assessed whether air quality was associated with clinically detectable respiratory signs or alterations to selected biomarkers of inflammation and lung function in stable personnel. Methods The horse stable environment and stable-workers (n = 13 in one stable were investigated three times; first in the winter, second in the interjacent late summer and the third time in the following winter stabling period. The stable measurements included levels of ammonia, hydrogen sulphide, total and respirable dust, airborne horse allergen, microorganisms, endotoxin and glucan. The stable-workers completed a questionnaire on respiratory symptoms, underwent nasal lavage with subsequent analysis of inflammation markers, and performed repeated measurements of pulmonary function. Results Measurements in the horse stable showed low organic dust levels and high horse allergen levels. Increased viable level of fungi in the air indicated a growing source in the stable. Air particle load as well as 1,3-β-glucan was higher at the two winter time-points, whereas endotoxin levels were higher at the summer time-point. Two stable-workers showed signs of bronchial obstruction with increased PEF-variability, increased inflammation biomarkers relating to reported allergy, cold or smoking and reported partly work-related symptoms. Furthermore, two other stable-workers reported work-related airway symptoms, of which one had doctor's diagnosed asthma which was well treated. Conclusion Biomarkers involved in the development of airway diseases have been studied in relation to

  16. A confocal microscopic study of solitary pulmonary neuroendocrine cells in human airway epithelium

    Directory of Open Access Journals (Sweden)

    Sparrow Malcolm P

    2005-10-01

    Full Text Available Abstract Background Pulmonary neuroendocrine cells (PNEC are specialized epithelial cells that are thought to play important roles in lung development and airway function. PNEC occur either singly or in clusters called neuroepithelial bodies. Our aim was to characterize the three dimensional morphology of PNEC, their distribution, and their relationship to the epithelial nerves in whole mounts of adult human bronchi using confocal microscopy. Methods Bronchi were resected from non-diseased portions of a lobe of human lung obtained from 8 thoracotomy patients (Table 1 undergoing surgery for the removal of lung tumors. Whole mounts were stained with antibodies to reveal all nerves (PGP 9.5, sensory nerves (calcitonin gene related peptide, CGRP, and PNEC (PGP 9.5, CGRP and gastrin releasing peptide, GRP. The analysis and rendition of the resulting three-dimensional data sets, including side-projections, was performed using NIH-Image software. Images were colorized and super-imposed using Adobe Photoshop. Results PNEC were abundant but not homogenously distributed within the epithelium, with densities ranging from 65/mm2 to denser patches of 250/mm2, depending on the individual wholemount. Rotation of 3-D images revealed a complex morphology; flask-like with the cell body near the basement membrane and a thick stem extending to the lumen. Long processes issued laterally from its base, some lumenal and others with feet-like processes. Calcitonin gene-related peptide (CGRP was present in about 20% of PNEC, mainly in the processes. CGRP-positive nerves were sparse, with some associated with the apical part of the PNEC. Conclusion Our 3D-data demonstrates that PNEC are numerous and exhibit a heterogeneous peptide content suggesting an active and diverse PNEC population.

  17. Effects of diesel exhaust particles on human lung epithelial cells: an in vitro study.

    Science.gov (United States)

    Mazzarella, G; Ferraraccio, F; Prati, M V; Annunziata, S; Bianco, A; Mezzogiorno, A; Liguori, G; Angelillo, I F; Cazzola, M

    2007-06-01

    Atmospheric particulate matter (PM), an ingredient of urban pollution matter, is a mixture of solid and liquid particles differing in origin, dimension and composition. There is big concern about inhaled PM in urban areas, especially due to its adverse effects on the respiratory system. Diesel exhaust particulate (DEP), which constitutes the major part of PM, is characterized by a carbonic mixture composed of approximately 18,000 different high-molecular-weight organic compounds. Diesel engines release 10 times the amount of NO(2) aldehydes and breathable PM compared to unleaded gasoline engines and more than 100 times that produced by catalysed gasoline engines; these data gain great significance when taken into account the fact that diesel-powered vehicles are becoming more and more popular. DEP polyaromatic hydrocarbons (PAH), once deposited on airways mucous surfaces easily pass through epithelial cells (ECs) membranes, bind themselves to cytosolic receptors and then affect cell growth and differentiation. Human lung epithelial cells and macrophages engulf DEP, this resulting in increased proinflammatory cytokines release (IL-6, IL-8 and GM-CSF). We investigated the biological effects of DEP-PM on the human lung EC line A549. Light microscopy analysis suggested the presence of cell wall alterations, and provided evidence of PM internalization and cytoplasmic vacuolization. Following PM stimulation, nuclei also were seen undergo clear gross morphological modifications. Immunocytochemistry was used to detect intracytoplasmic IL-6 and IL-8 expression.

  18. DEPOSITION DISTRICUTION AMONG THE PARALLEL PATHWAYS IN THE HUMAN LUNG CONDUCTING AIRWAY STRUCTURE.

    Science.gov (United States)

    DEPOSITION DISTRIBUTION AMONG THE PARALLEL PATHWAYS IN THE HUMAN LUNG CONDUCTING AIRWAY STRUCTURE. Chong S. Kim*, USEPA National Health and Environmental Effects Research Lab. RTP, NC 27711; Z. Zhang and C. Kleinstreuer, Department of Mechanical and Aerospace Engineering, North C...

  19. Expression and function of human hemokinin-1 in human and guinea pig airways.

    Science.gov (United States)

    Grassin-Delyle, Stanislas; Naline, Emmanuel; Buenestado, Amparo; Risse, Paul-André; Sage, Edouard; Advenier, Charles; Devillier, Philippe

    2010-10-07

    Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

  20. Expression and function of human hemokinin-1 in human and guinea pig airways

    Directory of Open Access Journals (Sweden)

    Sage Edouard

    2010-10-01

    Full Text Available Abstract Background Human hemokinin-1 (hHK-1 and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. Methods RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. Results In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. Conclusions We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

  1. Comparison of vectorial ion transport in primary murine airway and human sinonasal air-liquid interface cultures, models for studies of cystic fibrosis, and other airway diseases.

    Science.gov (United States)

    Zhang, Shaoyan; Fortenberry, James A; Cohen, Noam A; Sorscher, Eric J; Woodworth, Bradford A

    2009-01-01

    The purpose of this study was to compare vectorial ion transport within murine trachea, murine nasal septa, and human sinonasal cultured epithelium. Our hypothesis is that murine septal epithelium, rather than trachea, will more closely mimic the electrophysiology properties of human sinonasal epithelium. Epithelium from murine trachea, murine septa, and human sinonasal tissue were cultured at an air-liquid interface to confluence and full differentiation. A limited number of homozygous dF508 epithelia were also cultured. Monolayers were mounted in modified Ussing chambers to investigate pharmacologic manipulation of ion transport. The change in forskolin-stimulated current (delta-I(SC), expressed as micro-A/cm(2)) in murine septal (n = 19; 16.84 +/- 2.09) and human sinonasal (n = 18; 12.15 +/- 1.93) cultures was significantly increased over murine tracheal cultures (n = 15; 6.75 +/- 1.35; p = 0.035 and 0.0005, respectively). Forskolin-stimulated I(SC) was inhibited by the specific cystic fibrosis transmembrane regulator (CFTR) inhibitor INH-172 (5 microM). No forskolin-stimulated I(SC) was shown in cultures of dF508 homozygous murine septal epithelium (n = 3). Murine septal I(SC) was largely inhibited by amiloride (12.03 +/- 0.66), whereas human sinonasal cultures had a very limited response (0.70 +/- 0.47; p < 0.0001). The contribution of CFTR to stimulated chloride current as measured by INH-172 was highly significantly different between all groups (murine septa, 19.51 +/- 1.28; human sinonasal, 11.12 +/- 1.58; murine trachea, 4.85 +/- 0.49; p < 0.0001). Human sinonasal and murine septal epithelial cultures represent a useful model for studying CFTR activity and may provide significant advantages over lower airway tissues for investigating upper and lower respiratory pathophysiology.

  2. [Characterization of epithelial primary culture from human conjunctiva].

    Science.gov (United States)

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  3. Studies in human skin epithelial cell carcinogenesis

    International Nuclear Information System (INIS)

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo[a]pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and 32 P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the 32 P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts

  4. Levcromakalim- and isoprenaline-induced relaxation of human isolated airways--role of the epithelium and of K+ channel activation.

    Science.gov (United States)

    Black, J L; Johnson, P R; McKay, K O; Carey, D; Armour, C L

    1994-06-01

    In this study we have investigated the mechanism of action of levcromakalim and isoprenaline in human isolated airways with respect to the K+ channels they activate and the possibility that these smooth muscle relaxants activate K+ channels on the airway epithelium. Mechanical removal of the epithelial layer (mean percentage of epithelium present 20 +/- 3%, n = 20 tissues) did not affect the relaxation responses to levcromakalim or isoprenaline, either in terms of maximal relaxation or sensitivity. Whilst having no effect on isoprenaline-induced relaxation, studied from basal tone, the ATP-sensitive K+ channel blocker BRL 31660 (10, 30 and 50 microM) reduced relaxation responses induced (from basal tone) by levcromakalim from 74 +/- 6% (of the maximal response to isoprenaline) to 48 +/- 12% (n = 7), 9 +/- 9% (n = 4) and 0 (n = 4), respectively. Charybdotoxin, a blocker of high conductance Ca(2+)-activated K+ channels, at concentrations of 30 and 100 nM, had no effect on either levcromakalim- or or isoprenaline-induced relaxation responses and yet charybdotoxin was active at KCa channels in outside-out patches of hippocampal granule cells. Moreover, tetraethylammonium (10 mM) inhibited neither isoprenaline- nor levcromakalim-induced relaxation. This study has demonstrated that the relaxation responses elicited in human bronchus to isoprenaline and levcromakalim are likely to be the result of direct effects on the smooth muscle with no contribution from epithelial receptors or K+ channels. The actions of levcromakalim appear to be mediated only via activation of KATP channels. Further, we have made the important observation that, under the experimental conditions of our study, isoprenaline does not activate the KCa channel to produce relaxation in human bronchus.

  5. Engineered human broncho-epithelial tissue-like assemblies

    Science.gov (United States)

    Goodwin, Thomas J. (Inventor)

    2012-01-01

    Three-dimensional human broncho-epithelial tissue-like assemblies (TLAs) are produced in a rotating wall vessel (RWV) with microcarriers by coculturing mesenchymal bronchial-tracheal cells (BTC) and bronchial epithelium cells (BEC). These TLAs display structural characteristics and express markers of in vivo respiratory epithelia. TLAs are useful for screening compounds active in lung tissues such as antiviral compounds, cystic fibrosis treatments, allergens, and cytotoxic compounds.

  6. Postnatal remodeling of the neural components of the epithelial-mesenchymal trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen

    International Nuclear Information System (INIS)

    Larson, Shawnessy D.; Schelegle, Edward S.; Walby, William F.; Gershwin, Laural J.; Fanuccihi, Michelle V.; Evans, Michael J.; Joad, Jesse P.; Tarkington, Brian K.; Hyde, Dallas M.; Plopper, Charles G.

    2004-01-01

    Nerves and neuroendocrine cells located within the airway epithelium are ideally situated to sample a changing airway environment, to transmit that information to the central nervous system, and to promote trophic interactions between epithelial and mesenchymal cellular and acellular components. We tested the hypothesis that the environmental stresses of ozone (O 3 ) and house dust mite allergen (HDMA) in atopic infant rhesus monkeys alter the distribution of airway nerves. Midlevel bronchi and bronchioles from 6-month-old infant monkeys that inhaled filtered air (FA), house dust mite allergen HDMA, O 3 , or HDMA + O 3 for 11 episodes (5 days each, 0.5 ppm O 3 , 8 h/day followed by 9 days recovery) were examined using immunohistochemistry for the presence of Protein gene product 9.5 (PGP 9.5), a nonspecific neural indicator, and calcitonin gene-related peptide (CGRP). Along the axial path between the sixth and the seventh intrapulmonary airway generations, there were small significant (P 3 , while in monkeys exposed to HDMA + O 3 there was a greater significant (P 3 or HDMA + O 3 there was a significant increase in the number of PGP 9.5 positive/CGRP negative cells that were anchored to the basal lamina and emitted projections in primarily the lateral plain and often intertwined with projections and cell bodies of other similar cells. We conclude that repeated cycles of acute injury and repair associated with the episodic pattern of ozone and allergen exposure alter the normal development of neural innervation of the epithelial compartment and the appearance of a new population of undefined PGP 9.5 positive cells within the epithelium

  7. Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells

    International Nuclear Information System (INIS)

    Perkins, Timothy N.; Dentener, Mieke A.; Stassen, Frank R.; Rohde, Gernot G.; Mossman, Brooke T.; Wouters, Emiel F.M.; Reynaert, Niki L.

    2016-01-01

    Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT

  8. Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Perkins, Timothy N.; Dentener, Mieke A. [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Stassen, Frank R. [Department of Medical Microbiology, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Rohde, Gernot G. [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Mossman, Brooke T. [Department of Pathology, University of Vermont College of Medicine, Burlington, VT (United States); Wouters, Emiel F.M. [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands); Reynaert, Niki L., E-mail: n.reynaert@maastrichtuniversity.nl [Department of Respiratory Medicine, Maastricht University Medical Centre +, Maastricht University Maastricht (Netherlands)

    2016-06-15

    Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT

  9. Morphology and Three-Dimensional Inhalation Flow in Human Airways in Healthy and Diseased Subjects

    Science.gov (United States)

    Van de Moortele, Tristan

    We investigate experimentally the relation between anatomical structure and respiratory function in healthy and diseased airways. Computed Tomography (CT) scans of human lungs are analyzed from the data base of a large multi-institution clinical study on Chronic Obstructive Pulmonary Disease (COPD). Through segmentation, the 3D volumes of the airways are determined at total lung capacity. A geometric analysis provides data on the morphometry of the airways, including the length and diameter of branches, the child-to-parent diameter ratio, and branching angles. While several geometric parameters are confirmed to match past studies for healthy subjects, previously unreported trends are reported on the length of branches. Specifically, in most dichotomous airway bifurcation, the branch of smaller diameter tends to be significantly longer than the one of larger diameter. Additionally, the branch diameter tends to be smaller in diseased airways than in healthy airways up to the 7th generation of bronchial branching. 3D fractal analysis is also performed on the airway volume. Fractal dimensions of 1.89 and 1.83 are found for healthy non-smokers and declining COPD subjects, respectively, furthering the belief that COPD (and lung disease in general) significantly affects the morphometry of the airways already in early stages of the disease. To investigate the inspiratory flow, 3D flow models of the airways are generated using Computer Aided Design (CAD) software and 3D printed. Using Magnetic Resonance Velocimetry (MRV), 3-component 3D flow fields are acquired for steady inhalation at Reynolds number Re 2000 defined at the trachea. Analysis of the flow data reveals that diseased subjects may experience greater secondary flow strength in their conducting airways, especially in deeper generations.

  10. Computational Fluid Dynamics Modeling of Bacillus anthracis Spore Deposition in Rabbit and Human Respiratory Airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, Senthil; Suffield, Sarah R.; Recknagle, Kurtis P.; Jacob, Rick E.; Einstein, Daniel R.; Kuprat, Andrew P.; Carson, James P.; Colby, Sean M.; Saunders, James H.; Hines, Stephanie; Teeguarden, Justin G.; Straub, Tim M.; Moe, M.; Taft, Sarah; Corley, Richard A.

    2016-09-30

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived from computed tomography (CT) or µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation-exhalation breathing conditions using average species-specific minute volumes. The highest exposure concentration was modeled in the rabbit based upon prior acute inhalation studies. For comparison, human simulation was also conducted at the same concentration. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the upper conducting airways compared to the human at the same air concentration of anthrax spores. As a result, higher particle deposition was predicted in the conducting airways and deep lung of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology.

  11. Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses

    Science.gov (United States)

    Jazaeri Farsani, Seyed Mohammad; Deijs, Martin; Dijkman, Ronald; Molenkamp, Richard; Jeeninga, Rienk E; Ieven, Margareta; Goossens, Herman; van der Hoek, Lia

    2015-01-01

    Background Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. Method Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. Results Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). Conclusion We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates. PMID:25482367

  12. IL-17A induces Pendrin expression and chloride-bicarbonate exchange in human bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Kelly M Adams

    Full Text Available The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A, which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4 as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.

  13. Clarifying CB2 receptor-dependent and independent effects of THC on human lung epithelial cells

    International Nuclear Information System (INIS)

    Sarafian, Theodore; Montes, Cindy; Harui, Airi; Beedanagari, Sudheer R.; Kiertscher, Sylvia; Stripecke, Renata; Hossepian, Derik; Kitchen, Christina; Kern, Rita; Belperio, John; Roth, Michael D.

    2008-01-01

    Marijuana smoking is associated with a number of abnormal findings in the lungs of habitual smokers. Previous studies revealed that Δ 9 -tetrahydrocannabinol (THC) caused mitochondrial injury in primary lung epithelial cells and in the cell line, A549 [Sarafian, T. A., Kouyoumjian, S., Khoshaghideh, F., Tashkin, D. P., and Roth, M. D. (2003). Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics. Am J Physiol Lung Cell Mol Physiol 284, L298-306; Sarafian, T., Habib, N., Mao, J. T., Tsu, I. H., Yamamoto, M. L., Hsu, E., Tashkin, D. P., and Roth, M. D. (2005). Gene expression changes in human small airway epithelial cells exposed to Delta9-tetrahydrocannabinol. Toxicol Lett 158, 95-107]. The role of cannabinoid receptors in this injury was unclear, as was the potential impact on cell function. In order to investigate these questions, A549 cells were engineered to over-express the type 2 cannabinoid receptor (CB2R) using a self-inactivating lentiviral vector. This transduction resulted in a 60-fold increase in CB2R mRNA relative to cells transduced with a control vector. Transduced cell lines were used to study the effects of THC on chemotactic activity and mitochondrial function. Chemotaxis in response to a 10% serum gradient was suppressed in a concentration-dependent manner by exposure to THC. CB2R-transduced cells exhibited less intrinsic chemotactic activity (p m ) in both control and CB2R-transduced cells. However, these decreases did not play a significant role in chemotaxis inhibition since cyclosporine A, which protected against ATP loss, did not increase cell migration. Moreover, CB2R-transduced cells displayed higher Ψ m than did control cells. Since both Ψ m and chemotaxis are regulated by intracellular signaling, we investigated the effects of THC on the activation of multiple signaling pathways. Serum exposure activated several signaling events of which phosphorylation of IκB-α and JNK was regulated in a CB2R- and THC

  14. Stochastic model of radon daughter deposition and clearance in human bronchial airways

    International Nuclear Information System (INIS)

    Hofmann, W.

    1996-01-01

    Morphometric measurements of human airway casts have repealed that the human bronchial tree is an asymmetrically dividing network, exhibiting individual variations of airway dimensions within a given airway generation Thus, a statistical correlation exists between the linear dimensions of a given parent airway and those of the asymmetrically dividing daughter branches. This statistical relationship, however, is constrained by correlations among various geometrical parameters. i.e., the human lung is not a fully stochastic system. From a statistical analysis of these morphometric data, the following geometrical parameters could be obtained for each airway generation or bifurcation: (i) probability distributions for diameters and lengths, ratios of parent cross section to the combined cross section of both daughters, ratios of major to minor daughter diameters, and branching angles for major and minor branches; (ii) correlations of diameters and lengths; and (iii) the probability of terminating the bronchial region as a function of both diameter and generation number. Since the original lung morphometry refers to total lung capacity, airway diameters and lengths were scaled down to functional residual capacity. (author)

  15. Paramyxovirus Infection Mimics In Vivo Cellular Dynamics in Three-Demensional Human Bronchio-Epithelial Tissue-Like Assemblies

    Science.gov (United States)

    Deatly, Anne M.; Lin, Yen-Huei; McCarthy, Maureen; Chen, Wei; Miller, Lynn Z.; Quiroz, Jorge; Nowak, Becky M.; Lerch, Robert A.; Udem, Stephen A.; Goodwin, Thomas J.

    2012-01-01

    recapitulated by mimicking the structural organization of the HRE. In vivo, airway epithelial cells play a significant and dynamic role in host defense by blocking paracellular permeability and modulating airway function through cellular interactions or tight junctions. As regulators of the innate immune response, epithelial cells constitutively express cytokines, chemokines, and colony stimulating factors including RANTES, IL-8, IL-6, GM-CSF, and G-CSF for proactive host defense. In response to viral infection, epithelial cells induce potent immuno-modulatory and pro-inflammatory cytokines that recruit phagocytic and inflammatory cells to clear the virus and enhance protection. Although disease pathogenesis is classically attributed to the cytopathic effects of the pathogen, severe disease states associated with RSV and PIV3 are attributed to the inflammatory response, especially in infants. RSV is a potent inducer of cytokines and pro-inflammatory mediators in epithelial cells in vivo. A differentiated human epithelial model independent of the complete functional immune system will help elucidate the role of epithelial cells in respiratory disease. We reported here, virus and host cell interactions in 3D HBE TLAs are similar to that in vivo. Because the epithelial cell organization of the TLAs impacts not only the expression of airway epithelial characteristics, but also cellular communication, the TLAs represent a more physiologically relevant model of the HRE than BEAS-2B or other non-tumour monolayer models of respiratory disease. As a result, wild type respiratory viruses have a clear growth advantage over attenuated viruses in TLAs unlike traditional monolayers. In addition, the TLAs respond to wild type virus infection by secreting pro-inflammatory mediators characteristic of infected HRE. TLAs expressing microbial defense mechanisms provide an excellent model to study the interactions of respiratory pathogens with their host and to identify the innate immunity

  16. Computational fluid dynamics modeling of Bacillus anthracis spore deposition in rabbit and human respiratory airways

    Energy Technology Data Exchange (ETDEWEB)

    Kabilan, S.; Suffield, S. R.; Recknagle, K. P.; Jacob, R. E.; Einstein, D. R.; Kuprat, A. P.; Carson, J. P.; Colby, S. M.; Saunders, J. H.; Hines, S. A.; Teeguarden, J. G.; Straub, T. M.; Moe, M.; Taft, S. C.; Corley, R. A.

    2016-09-01

    Three-dimensional computational fluid dynamics and Lagrangian particle deposition models were developed to compare the deposition of aerosolized Bacillus anthracis spores in the respiratory airways of a human with that of the rabbit, a species commonly used in the study of anthrax disease. The respiratory airway geometries for each species were derived respectively from computed tomography (CT) and µCT images. Both models encompassed airways that extended from the external nose to the lung with a total of 272 outlets in the human model and 2878 outlets in the rabbit model. All simulations of spore deposition were conducted under transient, inhalation–exhalation breathing conditions using average species-specific minute volumes. Two different exposure scenarios were modeled in the rabbit based upon experimental inhalation studies. For comparison, human simulations were conducted at the highest exposure concentration used during the rabbit experimental exposures. Results demonstrated that regional spore deposition patterns were sensitive to airway geometry and ventilation profiles. Due to the complex airway geometries in the rabbit nose, higher spore deposition efficiency was predicted in the nasal sinus compared to the human at the same air concentration of anthrax spores. In contrast, higher spore deposition was predicted in the lower conducting airways of the human compared to the rabbit lung due to differences in airway branching pattern. This information can be used to refine published and ongoing biokinetic models of inhalation anthrax spore exposures, which currently estimate deposited spore concentrations based solely upon exposure concentrations and inhaled doses that do not factor in species-specific anatomy and physiology for deposition.

  17. The fate of epithelial cells in the human large intestine.

    Science.gov (United States)

    Barkla, D H; Gibson, P R

    1999-08-01

    One hundred and forty biopsies of the colon and rectum, collected during routine colonoscopies of 51 patients aged 19 to 74 years, were examined using light microscopy and transmission and scanning electron microscopy. The results indicated that surface epithelial cells undergo apoptosis, passing through fenestrations in the basement membrane to where they enter the lamina propria and are taken up by macrophages; and it is hypothesized that apoptotic cells are carried through the fenestrations on a current of fluid. The study also found that epithelial cells positioned over the crypts are better attached and more robust than those more distant from the crypt opening; and it is further hypothesized that, after reaching the top of the crypts, some goblet cells cease secreting mucus and pass onto the surface compartment of absorptive cells. An unexpected finding was that the lower regions of the crypts commonly contain isolated necrotic colonocytes. Apoptotic cells were rarely observed in the crypt epithelium. The findings of this study support the "recycling" model of epithelial cell death in the surface compartment of the human colon.

  18. Invasion of Human Oral Epithelial Cells by Prevotella intermedia

    Science.gov (United States)

    Dorn, Brian R.; Leung, K.-P.; Progulske-Fox, Ann

    1998-01-01

    Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion. PMID:9826397

  19. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

    NARCIS (Netherlands)

    Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

    2013-01-01

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely

  20. Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

    Science.gov (United States)

    Tagalakis, Aristides D; Munye, Mustafa M; Ivanova, Rositsa; Chen, Hanpeng; Smith, Claire M; Aldossary, Ahmad M; Rosa, Luca Z; Moulding, Dale; Barnes, Josephine L; Kafetzis, Konstantinos N; Jones, Stuart A; Baines, Deborah L; Moss, Guy W J; O'Callaghan, Christopher; McAnulty, Robin J; Hart, Stephen L

    2018-05-10

    Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (V t ), short circuit current (I sc ), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced V t , the amiloride-sensitive I sc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  1. Cigarette Smoke and Estrogen Signaling in Human Airway Smooth Muscle

    Directory of Open Access Journals (Sweden)

    Venkatachalem Sathish

    2015-06-01

    Full Text Available Aims: Cigarette smoke (CS in active smokers and second-hand smoke exposure exacerbate respiratory disorders such as asthma and chronic bronchitis. While women are known to experience a more asthmatic response to CS than emphysema in men, there is limited information on the mechanisms of CS-induced airway dysfunction. We hypothesize that CS interferes with a normal (protective bronchodilatory role of estrogens, thus worsening airway contractility. Methods: We tested effects of cigarette smoke extract (CSE on 17β-estradiol (E2 signaling in enzymatically-dissociated bronchial airway smooth muscle (ASM obtained from lung samples of non-smoking female patients undergoing thoracic surgery. Results: In fura-2 loaded ASM cells, CSE increased intracellular calcium ([Ca2+]i responses to 10µM histamine. Acute exposure to physiological concentrations of E2 decreased [Ca2+]i responses. However, in 24h exposed CSE cells, although expression of estrogen receptors was increased, the effect of E2 on [Ca2+]i was blunted. Acute E2 exposure also decreased store-operated Ca2+ entry and inhibited stromal interaction molecule 1 (STIM1 phosphorylation: effects blunted by CSE. Acute exposure to E2 increased cAMP, but less so in 24h CSE-exposed cells. 24h CSE exposure increased S-nitrosylation of ERα. Furthermore, 24h CSE-exposed bronchial rings showed increased bronchoconstrictor agonist responses that were not reduced as effectively by E2 compared to non-CSE controls. Conclusion: These data suggest that CS induces dysregulation of estrogen signaling in ASM, which could contribute to increased airway contractility in women exposed to CS.

  2. Effect of acrolein, a hazardous air pollutant in smoke, on human middle ear epithelial cells.

    Science.gov (United States)

    Song, Jae-Jun; Lee, Jong Dae; Lee, Byung Don; Chae, Sung Won; Park, Moo Kyun

    2013-10-01

    Acrolein is a hazardous air pollutant. Tobacco smoke and indoor air pollution are the main causes of human exposure. Acrolein has been shown to cause cytotoxicity in the airways and induce inflammation and mucin production in pulmonary cells. We investigated whether acrolein caused cytotoxicity, induced inflammation or increased expression of mucin in immortalized human middle ear epithelial cell lines (HMEECs). Cytotoxicity following acrolein treatment was investigated using the MTT assay, flow cytometry, and Hoechst 33342 staining of HMEECs. We measured expression of inflammatory cytokines tumor necrosis factor (TNF)-α and cyclo-oxygenase (COX)-2 and the mucin gene MUC5AC using semi-quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Exposure to >50 μg/mL acrolein caused a decrease in cell viability. Acrolein induced apoptosis and necrosis at 50 μg/mL. Acrolein at 5-50 μg/mL increased expression of TNF-α and COX-2, as shown by RT-PCR and Western blotting. Acrolein exposure at 5-50 μg/mL for 2-24h increased MUC5AC expression, as determined by RT-PCR. Acrolein decreased cell viability, induced an inflammatory response, and increased mucin gene expression in HMEECs. These findings support the hypothesis that acrolein, a hazardous air pollutant in tobacco smoke and ambient air, is a risk factor for otitis media. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Twite, Nicolas [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Andrei, Graciela [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Kummert, Caroline [ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium); Donner, Catherine [Department of Obstetrics and Gynecology, Erasme Hospital, Route de Lennik 808, 1070 Brussels (Belgium); Perez-Morga, David [Laboratory of Molecular Parasitology, Institut de Biologie et Médecine Moléculaires, Université Libre de Bruxelles, Gosselies (Belgium); De Vos, Rita [Pathology Department, U.Z. Leuven, Minderbroedersstraat 12, Leuven (Belgium); Snoeck, Robert, E-mail: Robert.Snoeck@Rega.kuleuven.be [Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven (Belgium); Marchant, Arnaud, E-mail: arnaud.marchant@ulb.ac.be [Institute for Medical Immunology, Université Libre de Bruxelles, Rue A. Bolland 8, B-6041 Charleroi (Belgium); ImmuneHealth, Rue A. Bolland 8, B-6041 Charleroi (Belgium)

    2014-07-15

    Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMV by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.

  4. Regional aerosol deposition in human upper airways. Progress report, March 1, 1992--February 28, 1993

    Energy Technology Data Exchange (ETDEWEB)

    Swift, D.L.

    1992-11-01

    Laboratory experimental studies were carried out to investigate the factors influencing the deposition of aerosols ranging in size from 1 nm to 10 {mu}m in the human nasal, oral, pharyngeal and laryngeal airways. These experimental studies were performed in replicate upper airway physical models and in human volunteer subjects. New replicate models of the oral passage of an infant, the oral passage of an adult at two openings and the combined nasal and oral airways of an adult were constructed during the period, adding to the existing models of adult, child and infant nasal and oral airways models. Deposition studies in the adult oral and adult nasal models were performed under simulated cyclic flow conditions with 1 nm particles to compare with previously measured constant flow studies. Similar studies with inertial particles (1--10 {mu}m diameter) were performed with the adult nasal model; in both instances, results with cyclic flow were similar to constant flow results using a simple average flow rate based on inspiratory volume and time of inspiration. Human subject studies were performed with particle sizes 5--20 nm for nasal inspiration; preliminary analysis shows good agreement with model studies at several representative flow rates. Nasal inspiratory inertial deposition of 1--4 {mu}m diameter particles was measured in several adults as a function of airway dimensions; dimensional changes of the valve area by decongestion did not produce concomitant deposition changes.

  5. Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells.

    Science.gov (United States)

    Jiang, Zhongjia; Song, Fuyang; Li, Yanan; Xue, Di; Deng, Guangcun; Li, Min; Liu, Xiaoming; Wang, Yujiong

    2017-01-01

    Mycoplasma ovipneumoniae ( M. ovipneumoniae ) is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS) of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI) model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR-) mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF- κ B), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1 β , TNF α , and IL8, and anti-inflammatory cytokines such as IL10 and TGF β of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae -induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae , which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

  6. Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Zhongjia Jiang

    2017-01-01

    Full Text Available Mycoplasma ovipneumoniae (M. ovipneumoniae is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR- mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB, activator protein-1 (AP-1, and interferon regulatory factor 3 (IRF3 as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1β, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFβ of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

  7. Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Bonnie H Y Yeung

    Full Text Available Stanniocalcin-1 (STC1, a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam formation, followed by cell migration. In this study, staurosporine (STS treatment induced human keratinocyte (HaCaT e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK, the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

  8. Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

    Science.gov (United States)

    Yeung, Bonnie H Y; Wong, Chris K C

    2011-01-01

    Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

  9. Overexpression of functional TrkA receptors after internalisation in human airway smooth muscle cells.

    Science.gov (United States)

    Freund-Michel, Véronique; Frossard, Nelly

    2008-10-01

    Trafficking of the TrkA receptor after stimulation by NGF is of emerging importance in structural cells in the context of airway inflammatory diseases. We have recently reported the expression of functional TrkA receptors in human airway smooth muscle cells (HASMC). We have here studied the TrkA trafficking mechanisms in these cells. TrkA disappearance from the cell membrane was induced within 5 min of NGF (3pM) stimulation. Co-immunoprecipitation of clathrin-TrkA was revealed, and TrkA internalisation inhibited either by clathrin inhibitors or by siRNA inducing downregulation of endogenous clathrin. TrkA internalised receptors were totally degraded in lysosomes, with no recycling phenomenon. Newly synthesized TrkA receptors were thereafter re-expressed at the cell membrane within 10 h. TrkA re-synthesis was inhibited by blockade of clathrin-dependent internalisation, but not of TrkA receptors lysosomal degradation. Finally, we observed that NGF multiple stimulations progressively increased TrkA expression in HASMC, which was associated with an increase in NGF/TrkA-dependent proliferation. In conclusion, we show here the occurrence of clathrin-dependent TrkA internalisation and lysosomal degradation in the airway smooth muscle, followed by upregulated re-synthesis of functional TrkA receptors and increased proliferative effect in the human airway smooth muscle. This may have pathophysiological consequences in airway inflammatory diseases.

  10. Anti-inflammatory effects of antibacterials on human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Hatz Rudolf

    2009-09-01

    Full Text Available Abstract Background Human Bronchial epithelial cells (hu-BEC have been claimed to play a significant role in the pathogenesis of chronic inflammatory airway diseases like COPD. In this context IL-8 and GM-CSF have been shown to be key cytokines. Some antibiotics which are routinely used to treat lower respiratory tract infections have been shown to exert additional immunomodulatory or anti-inflammatory effects. We investigated whether these effects can also be detected in hu-BEC. Methods Hu-BEC obtained from patients undergoing lung resections were transferred to air-liquid-interface (ALI culture. These cultures were incubated with cefuroxime (CXM, 10-62.5 mg/l, azithromycin (AZM, 0.1-1.5 mg/l, levofloxacin (LVX, 1-8 mg/l and moxifloxacin (MXF, 1-16 mg/l. The spontaneous and TNF-α (10 ng/ml induced expression and release of IL-8 and GM-CSF were measured using PCR and ELISA in the absence or presence of these antibiotics. Results The spontaneous IL-8 and GM-CSF release was significantly reduced with MXF (8 mg/l by 37 ± 20% and 45 ± 31%, respectively (both p Conclusion Using ALI cultures of hu-BEC we observed differential effects of antibiotics on spontaneous and TNF-α induced cytokine release. Our data suggest that MXF and AZM, beyond bactericidal effects, may attenuate the inflammatory process mediated by hu-BEC.

  11. High degree of overlap between responses to a virus and to the house dust mite allergen in airway epithelial cells

    NARCIS (Netherlands)

    Golebski, Korneliusz; Luiten, Silvia; van Egmond, Danielle; de Groot, Esther; Röschmann, Kristina Irene Lisolette; Fokkens, Wytske Johanna; van Drunen, Cornelis Maria

    2014-01-01

    Airway epithelium is widely considered to play an active role in immune responses through its ability to detect changes in the environment and to generate a microenvironment for immune competent cells. Therefore, besides its role as a physical barrier, epithelium affects the outcome of the immune

  12. Simulation of size-dependent aerosol deposition in a realistic model of the upper human airways

    NARCIS (Netherlands)

    Frederix, E.M.A.; Kuczaj, Arkadiusz K.; Nordlund, Markus; Belka, M.; Lizal, F.; Elcner, J.; Jicha, M.; Geurts, Bernardus J.

    An Eulerian internally mixed aerosol model is used for predictions of deposition inside a realistic cast of the human upper airways. The model, formulated in the multi-species and compressible framework, is solved using the sectional discretization of the droplet size distribution function to

  13. T cell subsets in human airways prior to and following endobronchial administration of endotoxin

    DEFF Research Database (Denmark)

    Ronit, Andreas; Plovsing, Ronni R; Gaardbo, Julie C

    2015-01-01

    BACKGROUND AND OBJECTIVES: Bronchial instillation of lipopolysaccharide (LPS) provides a reversible model of lung inflammation that may resemble early stages of acute respiratory distress syndrome (ARDS). We investigated the distributions of T-cell subsets in the human airways and sought to deter...

  14. TRANSPORT AND DEPOSITION OF NANO-SIZE PARTICLES IN THE UPPER HUMAN RESPIRATORY AIRWAYS

    Science.gov (United States)

    TRANSPORT AND DEPOSITION OF NANO-SIZE PARTICLES IN THE UPPER HUMAN RESPIRATORY AIRWAYS. Zhe Zhang*, Huawei Shi, Clement Kleinstreuer, Department of Mechanical and Aerospace Engineering, North Carolina State University, Raleigh, NC 27695-7910; Chong S. Kim, National Health and En...

  15. Common features of sexual dimorphism in the cranial airways of different human populations.

    Science.gov (United States)

    Bastir, Markus; Godoy, Paula; Rosas, Antonio

    2011-11-01

    Sexual dimorphism in the human craniofacial system is an important feature of intraspecific variation in recent and fossil humans. Although several studies have reported different morphological patterns of sexual dimorphism in different populations, this study searches for common morphological aspects related to functional anatomy of the respiratory apparatus. 3D geometric morphometrics were used to test the hypothesis that due to higher daily energy expenditure and associated greater respiratory air consumption as well as differences in body composition, males should have absolutely and relatively greater air passages in the bony cranial airways than females. We measured 25 3D landmarks in five populations (N = 212) of adult humans from different geographic regions. Male average cranial airways were larger in centroid sizes than female ones. Males tended to show relatively taller piriform apertures and, more consistently, relatively taller internal nasal cavities and choanae than females. Multivariate regressions and residual analysis further indicated that after standardizing to the same size, males still show relatively larger airway passages than females. Because the dimensions of the choanae are limiting factors for air transmission towards the noncranial part of the respiratory system, the identified sex-specific differences in cranial airways, possibly shared among human populations, may be linked with sex-specific differences in body size, composition, and energetics. These findings may be important to understanding trends in hominin facial evolution. Copyright © 2011 Wiley-Liss, Inc.

  16. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping, E-mail: wpxie@njmu.edu.cn; Wang, Hong, E-mail: hongwang@njmu.edu.cn

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation and migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that Ipt

  17. In vivo deposition of ultrafine aerosols in human nasal and oral airways

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsu-Chi; Swift, D.L. [John Hopkins Univ., Baltimore, MD (United States); Simpson, S.Q. [Univ. of New Mexico, Albuquerque, NM (United States)] [and others

    1995-12-01

    The extrathoracic airways, including the nasal passage, oral passage, pharynx, and larynx, are the first targets for inhaled particles and provide an important defense for the lung. Understanding the deposition efficiency of the nasal and oral passages is therefore crucial for assessing doses of inhaled particles to the extrathoracic airways and the lung. Significant inter-subject variability in nasal deposition has been shown in recent studies by Rasmussen, T.R. et al, using 2.6 {mu}m particles in 10 human subjects and in our preliminary studies using 0.004-0.15 {mu}m particles in four adult volunteers. No oral deposition was reported in either of these studies. Reasons for the intersubject variations have been frequently attributed to the geometry of the nasal passages. The aims of the present study were to measure in vivo the nasal airway dimensions and the deposition of ultrafine aerosols in both the nasal and oral passages, and to determine the relationship between nasal airway dimensions and aerosol deposition. A statistical procedure incorporated with the diffusion theory was used to model the dimensional features of the nasal airways which may be responsible for the biological variability in particle deposition. In summary, we have correlated deposition of particles in the size range of 0.004 to 0.15 {mu}m with the nasal dimensions of each subject.

  18. In vivo deposition of ultrafine aerosols in human nasal and oral airways

    International Nuclear Information System (INIS)

    Yeh, Hsu-Chi; Swift, D.L.; Simpson, S.Q.

    1995-01-01

    The extrathoracic airways, including the nasal passage, oral passage, pharynx, and larynx, are the first targets for inhaled particles and provide an important defense for the lung. Understanding the deposition efficiency of the nasal and oral passages is therefore crucial for assessing doses of inhaled particles to the extrathoracic airways and the lung. Significant inter-subject variability in nasal deposition has been shown in recent studies by Rasmussen, T.R. et al, using 2.6 μm particles in 10 human subjects and in our preliminary studies using 0.004-0.15 μm particles in four adult volunteers. No oral deposition was reported in either of these studies. Reasons for the intersubject variations have been frequently attributed to the geometry of the nasal passages. The aims of the present study were to measure in vivo the nasal airway dimensions and the deposition of ultrafine aerosols in both the nasal and oral passages, and to determine the relationship between nasal airway dimensions and aerosol deposition. A statistical procedure incorporated with the diffusion theory was used to model the dimensional features of the nasal airways which may be responsible for the biological variability in particle deposition. In summary, we have correlated deposition of particles in the size range of 0.004 to 0.15 μm with the nasal dimensions of each subject

  19. Simulation of deposition and activity distribution of radionuclides in human airways

    International Nuclear Information System (INIS)

    Farkas, A.; Balashazy, I.; Szoke, I.; Hofmann, W.; Golser, R.

    2002-01-01

    The aim of our research activities is the modelling of the biological processes related to the development of lung cancer at the large central-airways observed in the case of uranium miners caused by the inhalation of radionuclides (especially alpha-emitting radon decay products). Statistical data show that at the uranium miners the lung cancer has developed mainly in the 3-4.-5. airway generations and especially in the right upper lobe. Therefore, it is rather important to study the physical and biological effects in this section of the human airways to find relations between the radiation dose and the adverse health effects. These results may provide useful information about the validity or invalidity of the currently used LNT (Linear-No-Threshold) dose-effect hypothesis at low doses

  20. The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

    DEFF Research Database (Denmark)

    Petersen, Ole William; Nielsen, Helga Lind; Gudjonsson, Thorarinn

    2001-01-01

    The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neopl...

  1. Regulation of potassium transport in human lens epithelial cells.

    Science.gov (United States)

    Lauf, Peter K; Warwar, Ronald; Brown, Thomas L; Adragna, Norma C

    2006-01-01

    The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed 100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.

  2. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  3. Depleted uranium induces neoplastic transformation in human lung epithelial cells.

    Science.gov (United States)

    Xie, Hong; LaCerte, Carolyne; Thompson, W Douglas; Wise, John Pierce

    2010-02-15

    Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and noncombatants is frequent and widespread. Previous studies have shown that DU has both chemical and radiological toxicity and that the primary route of exposure of DU to humans is through inhalation and ingestion. However, there is limited research information on the potential carcinogenicity of DU in human bronchial cells. Accordingly, we determined the neoplastic transforming ability of particulate DU to human bronchial epithelial cells (BEP2D). We observed the loss of contact inhibition and anchorage independent growth in cells exposed to DU after 24 h. We also characterized these DU-induced transformed cell lines and found that 40% of the cell lines exhibit alterations in plating efficiency and no significant changes in the cytotoxic response to DU. Cytogenetic analyses showed that 53% of the DU-transformed cell lines possess a hypodiploid phenotype. These data indicate that human bronchial cells are transformed by DU and exhibit significant chromosome instability consistent with a neoplastic phenotype.

  4. Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

    International Nuclear Information System (INIS)

    Roberts, Joan E.; Wielgus, Albert R.; Boyes, William K.; Andley, Usha; Chignell, Colin F.

    2008-01-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C 60 (OH) 22-26 ] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C 60 (OH) 22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 μM. Exposure to either UVA or visible light in the presence of > 5 μM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 μM lutein, 1 mM N-acetyl cysteine, or 1 mM L-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA > visible light > dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein α-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C 60 (OH) 22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo

  5. Human lung mast cells modulate the functions of airway smooth muscle cells in asthma.

    Science.gov (United States)

    Alkhouri, H; Hollins, F; Moir, L M; Brightling, C E; Armour, C L; Hughes, J M

    2011-09-01

    Activated mast cell densities are increased on the airway smooth muscle in asthma where they may modulate muscle functions and thus contribute to airway inflammation, remodelling and airflow obstruction. To determine the effects of human lung mast cells on the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Freshly isolated human lung mast cells were stimulated with IgE/anti-IgE. Culture supernatants were collected after 2 and 24 h and the mast cells lysed. The supernatants/lysates were added to serum-deprived, subconfluent airway smooth muscle cells for up to 48 h. Released chemokines and extracellular matrix were measured by ELISA, proliferation was quantified by [(3) H]-thymidine incorporation and cell counting, and intracellular signalling by phospho-arrays. Mast cell 2-h supernatants reduced CCL11 and increased CXCL8 and fibronectin production from both asthmatic and nonasthmatic muscle cells. Leupeptin reversed these effects. Mast cell 24-h supernatants and lysates reduced CCL11 release from both muscle cell types but increased CXCL8 release by nonasthmatic cells. The 24-h supernatants also reduced asthmatic, but not nonasthmatic, muscle cell DNA synthesis and asthmatic cell numbers over 5 days through inhibiting extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI3)-kinase pathways. However, prostaglandins, thromboxanes, IL-4 and IL-13 were not involved in reducing the proliferation. Mast cell proteases and newly synthesized products differentially modulated the secretory and proliferative functions of airway smooth muscle cells from donors with and without asthma. Thus, mast cells may modulate their own recruitment and airway smooth muscle functions locally in asthma. © 2011 John Wiley & Sons A/S.

  6. Hold your horses: A comparison of human laryngomalacia with analogous equine airway pathology.

    Science.gov (United States)

    Lawrence, Rachael J; Butterell, Matthew J; Constable, James D; Daniel, Matija

    2018-02-01

    Laryngomalacia is the most common cause of stridor in infants. Dynamic airway collapse is also a well-recognised entity in horses and an important cause of surgical veterinary intervention. We compare the aetiology, clinical features and management of human laryngomalacia with equine dynamic airway collapse. A structured review of the PubMed, the Ovid Medline and the Cochrane Collaboration databases (Cochrane Central Register of Controlled Trials, Cochrane Database of Systemic Reviews). There are numerous equine conditions that cause dynamic airway collapse defined specifically by the anatomical structures involved. Axial Deviation of the Aryepiglottic Folds (ADAF) is the condition most clinically analogous to laryngomalacia in humans, and is likewise most prevalent in the immature equine airway. Both conditions are managed either conservatively, or if symptoms require it, with surgical intervention. The operative procedures performed for ADAF and laryngomalacia are technically comparable. Dynamic collapse of the equine larynx, especially ADAF, is clinically similar to human laryngomalacia, and both are treated in a similar fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

    International Nuclear Information System (INIS)

    Kan-o, Keiko; Matsumoto, Koichiro; Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi; Inoue, Hiromasa

    2013-01-01

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB

  8. Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells

    DEFF Research Database (Denmark)

    Poulsen, T.T.; Naizhen, X.; Linnoila, R.I.

    2008-01-01

    Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury...... neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed...... and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis Udgivelsesdato: 2008/9/26...

  9. CXCL9 Regulates TGF-β1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells.

    Science.gov (United States)

    O'Beirne, Sarah L; Walsh, Sinead M; Fabre, Aurélie; Reviriego, Carlota; Worrell, Julie C; Counihan, Ian P; Lumsden, Robert V; Cramton-Barnes, Jennifer; Belperio, John A; Donnelly, Seamas C; Boylan, Denise; Marchal-Sommé, Joëlle; Kane, Rosemary; Keane, Michael P

    2015-09-15

    Epithelial to mesenchymal cell transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial marker thyroid transcription factor-1 and the mesenchymal marker α-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-β1 and CXCL9, with Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-β1-induced EMT. A decrease in TGF-β1-induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-β1-induced EMT. Copyright © 2015 by The American Association of Immunologists, Inc.

  10. GM-CSF production from human airway smooth muscle cells is potentiated by human serum

    Directory of Open Access Journals (Sweden)

    Maria B. Sukkar

    2000-01-01

    Full Text Available Recent evidence suggests that airway smooth muscle cells (ASMC actively participate in the airway inflammatory process in asthma. Interleukin–1β (IL–1β and tumour necrosis factor–α (TNF–α induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1 allergic asthmatic serum (AAS modulates ASMC mediator release in response to IL–1β and TNF–α, and (2 IL–1β/TNF–α prime ASMC to release mediators in response to AAS. IL–5 and GMCSF were quantified by ELISA in culture supernatants of; (1 ASMC pre-incubated with either AAS, non-allergic non-asthmatic serum (NAS or MonomedTM (a serum substitute and subsequently stimulated with IL–1β and TNF–α and (2 ASMC stimulated with IL–1β/TNF–α and subsequently exposed to either AAS, NAS or MonomedTM. IL-1g and TNF–α induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or MonomedTM. IL–1β and TNF–α, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL–1β/TNF–α and serum exposure (AAS or NAS was significantly greater than that following IL–1β /TNF–α and MonomedTM exposure or IL–1β/TNF–α exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage.

  11. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

    Science.gov (United States)

    Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C

    2015-12-01

    Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

  12. Regional aerosol deposition in human upper airways: Progress report, June 1, 1988--February 28, 1989

    International Nuclear Information System (INIS)

    Swift, D.L.

    1989-01-01

    The objective of this research program is to elucidate important factors which influence overall and local deposition of aerosols in the human airways above the trachea, including nasal airways, oral passage, pharynx and larynx. The intent is to develop information which can be used for exposure models for radon from unattached radon progeny (/approximately/1nm) up to 10 μm. Special emphasis is upon flow rate and airway dimensions as influenced by age and respiratory condition, as no experimental data presently exist for age-related deposition. Because of ethical and practical considerations associated with measuring aerosol deposition in children and difficulties of measuring local deposition in vivo, our experimental approach is to construct faithful replicate models of the airways for several ages of humans in which detailed studies of deposition can be carried out with well-characterized aerosols. Initial studies of overall deposition of ultrafine aerosols in an adult model have been carried out using replicate nasal passage models provided from this laboratory. These studies demonstrate a significant deposition percent for particle sizes approaching that of unattached radon progeny (40--50%), decreasing as particle size approaches 0.1 μm. Studies of the effect of flow rate indicate that higher deposition percent is realized at lower flow rate over the range from 4--60 lmin -1

  13. Lipids in airway secretions

    International Nuclear Information System (INIS)

    Bhaskar, K.R.; DeFeudis O'Sullivan, D.; Opaskar-Hincman, H.; Reid, L.M.

    1987-01-01

    Lipids form a significant portion of airway mucus yet they have not received the same attention that epithelial glycoproteins have. We have analysed, by thin layer chromatography, lipids present in airway mucus under 'normal' and hypersecretory (pathological) conditions.The 'normals' included (1) bronchial lavage obtained from healthy human volunteers and from dogs and (2) secretions produced ''in vitro'' by human (bronchial) and canine (tracheal) explants. Hypersecretory mucus samples included (1) lavage from dogs made bronchitic by exposure to SO 2 , (2) bronchial aspirates from acute and chronic tracheostomy patients, (3) sputum from patients with cystic fibrosis and chronic bronchitis and (4) postmortem secretions from patients who died from sudden infant death syndrome (SIDS) or from status asthmaticus. Cholesterol was found to be the predominant lipid in 'normal' mucus with lesser amounts of phospholipids. No glycolipids were detected. In the hypersecretory mucus, in addition to neutral and phospholipids, glycolipids were present in appreciable amounts, often the predominant species, suggesting that these may be useful as markers of disease. Radioactive precursors 14 C acetate and 14 C palmitate were incorporated into lipids secreted ''in vitro'' by canine tracheal explants indicating that they are synthesised by the airway. (author)

  14. Oscillatory flow in the human airways from the mouth through several bronchial generations

    International Nuclear Information System (INIS)

    Banko, Andrew J.; Coletti, Filippo; Elkins, Christopher J.; Eaton, John K.

    2016-01-01

    Highlights: • Oscillatory flow in the human airways is studied experimentally. • The realistic anatomy is obtained from the CT scan of a healthy adult. • Integral parameters are calculated to quantify streamwise and lateral dispersion. • Flow in real human anatomy is qualitatively different from idealized models. - Abstract: The time-varying flow is studied experimentally in an anatomically accurate model of the human airways from the mouth through several generations of bronchial branching. The airway geometry is obtained from the CT scan of a healthy adult male of normal height and build. The three-component, three-dimensional mean velocity field is obtained throughout the entire model using phase-locked Magnetic Resonance Velocimetry. A pulsatile pump drives a sinusoidal waveform (inhalation and exhalation) with frequency and stroke-length such that the mean trachea Reynolds number at peak inspiration is 4200 and the Womersley number is 7. Integral parameters are defined to quantify the degree of velocity profile non-uniformity (related to axial dispersion) and secondary flow strength (lateral dispersion). It is found that the extrathoracic airways significantly modify the tracheal flow and that the flow at the first bifurcation is highly asymmetric. The effect of flow oscillation is to produce time dependent flow features which are asymmetric with respect to the acceleration and deceleration periods surrounding peak inhalation and exhalation. This is most pronounced in regions of separation and on the secondary flow structure, which are sensitive to local attributes of the real anatomy. This is reflected in the integral parameters, which behave non-monotonically between successive bronchial generations. In general, the measured oscillatory flow in a realistic anatomy confirms many trends derived from idealized models but also possesses qualitatively different large scale flow structures as compared to idealized representations of the upper airways.

  15. Role of aberrant WNT signalling in the airway epithelial response to cigarette smoke in chronic obstructive pulmonary disease

    NARCIS (Netherlands)

    Heijink, Hilde; de Bruin, Harold G.; van den Berge, Maarten; Bennink, Lisa J. C.; Brandenburg, Simone M.; Gosens, Reinoud; van Oosterhout, Antoon J.; Postma, Dirkje S.

    Background WNT signalling is activated during lung tissue damage and inflammation. We investigated whether lung epithelial expression of WNT ligands, receptors (frizzled; FZD) or target genes is dysregulated on cigarette smoking and/or in chronic obstructive pulmonary disease (COPD). Methods We

  16. Transcriptional profiling of putative human epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Koçer Salih S

    2008-07-01

    may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.

  17. Computational Thermodynamics Analysis of Vaporizing Fuel Droplets in the Human Upper Airways

    Science.gov (United States)

    Zhang, Zhe; Kleinstreuer, Clement

    The detailed knowledge of air flow structures as well as particle transport and deposition in the human lung for typical inhalation flow rates is an important precursor for dosimetry-and-health-effect studies of toxic particles as well as for targeted drug delivery of therapeutic aerosols. Focusing on highly toxic JP-8 fuel aerosols, 3-D airflow and fluid-particle thermodynamics in a human upper airway model starting from mouth to Generation G3 (G0 is the trachea) are simulated using a user-enhanced and experimentally validated finite-volume code. The temperature distributions and their effects on airflow structures, fuel vapor deposition and droplet motion/evaporation are discussed. The computational results show that the thermal effect on vapor deposition is minor, but it may greatly affect droplet deposition in human airways.

  18. Tungsten-induced carcinogenesis in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Laulicht, Freda; Brocato, Jason; Cartularo, Laura; Vaughan, Joshua; Wu, Feng; Kluz, Thomas; Sun, Hong [Department of Environmental Medicine, New York University Langone Medical Center, Tuxedo, NY 10987 (United States); Oksuz, Betul Akgol [Genome Technology Center, New York University Langone Medical Center, New York, NY 10016 (United States); Shen, Steven [Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, NY 10016 (United States); Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta [Department of Chemistry and Pharmacy, University of Sassari, Sassari (Italy); Costa, Max, E-mail: Max.Costa@nyumc.org [Department of Environmental Medicine, New York University Langone Medical Center, Tuxedo, NY 10987 (United States)

    2015-10-01

    Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans. - Highlights: • Tungsten (W) induces cell transformation and increases migration in vitro. • W increases xenograft growth in nude mice. • W altered the expression of cancer-related genes such as those involved in leukemia. • Some of the dysregulated leukemia genes include, CD74, CTGF, MST4, and HOXB5. • For the first time, data is presented that demonstrates tungsten's carcinogenic potential.

  19. A taxonomy of epithelial human cancer and their metastases

    Directory of Open Access Journals (Sweden)

    De Moor Bart

    2009-12-01

    Full Text Available Abstract Background Microarray technology has allowed to molecularly characterize many different cancer sites. This technology has the potential to individualize therapy and to discover new drug targets. However, due to technological differences and issues in standardized sample collection no study has evaluated the molecular profile of epithelial human cancer in a large number of samples and tissues. Additionally, it has not yet been extensively investigated whether metastases resemble their tissue of origin or tissue of destination. Methods We studied the expression profiles of a series of 1566 primary and 178 metastases by unsupervised hierarchical clustering. The clustering profile was subsequently investigated and correlated with clinico-pathological data. Statistical enrichment of clinico-pathological annotations of groups of samples was investigated using Fisher exact test. Gene set enrichment analysis (GSEA and DAVID functional enrichment analysis were used to investigate the molecular pathways. Kaplan-Meier survival analysis and log-rank tests were used to investigate prognostic significance of gene signatures. Results Large clusters corresponding to breast, gastrointestinal, ovarian and kidney primary tissues emerged from the data. Chromophobe renal cell carcinoma clustered together with follicular differentiated thyroid carcinoma, which supports recent morphological descriptions of thyroid follicular carcinoma-like tumors in the kidney and suggests that they represent a subtype of chromophobe carcinoma. We also found an expression signature identifying primary tumors of squamous cell histology in multiple tissues. Next, a subset of ovarian tumors enriched with endometrioid histology clustered together with endometrium tumors, confirming that they share their etiopathogenesis, which strongly differs from serous ovarian tumors. In addition, the clustering of colon and breast tumors correlated with clinico-pathological characteristics

  20. The expression of Egfl7 in human normal tissues and epithelial tumors.

    Science.gov (United States)

    Fan, Chun; Yang, Lian-Yue; Wu, Fan; Tao, Yi-Ming; Liu, Lin-Sen; Zhang, Jin-Fan; He, Ya-Ning; Tang, Li-Li; Chen, Guo-Dong; Guo, Lei

    2013-04-23

    To investigate the expression of Egfl7 in normal adult human tissues and human epithelial tumors.
 RT-PCR and Western blot were employed to detect Egfl7 expression in normal adult human tissues and 10 human epithelial tumors including hepatocellular carcinoma (HCC), lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. Immunohistochemistry and cytoimmunofluorescence were subsequently used to determine the localization of Egfl7 in human epithelial tumor tissues and cell lines. ELISA was also carried out to examine the serum Egfl7 levels in cancer patients. In addition, correlations between Egfl7 expression and clinicopathological features as well as prognosis of HCC and breast cancer were also analyzed on the basis of immunohistochemistry results.
 Egfl7 was differentially expressed in 19 adult human normal tissues and was overexpressed in all 10 human epithelial tumor tissues. The serum Egfl7 level was also significantly elevated in cancer patients. The increased Egfl7 expression in HCC correlated with vein invasion, absence of capsule formation, multiple tumor nodes and poor prognosis. Similarly, upregulation of Egfl7 in breast cancer correlated strongly with TNM stage, lymphatic metastasis, estrogen receptor positivity, Her2 positivity and poor prognosis. 
 Egfl7 is significantly upregulated in human epithelial tumor tissues, suggesting Egfl7 to be a potential biomarker for human epithelial tumors, especially HCC and breast cancer.

  1. Effect of Lead on Human Middle Ear Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shin Hye Kim

    2018-01-01

    Full Text Available Lead is a ubiquitous metal in the environment, but no studies have examined lead toxicity on the middle ear. Here, we investigated lead toxicity and its mechanism in human middle ear epithelial cells (HMEECs. Moreover, we investigated the protective effects of amniotic membrane extract (AME and chorionic membrane extract (CME against lead toxicity in HMEECs. Cell viability was analyzed using the cell counting kit, and reactive oxygen species (ROS activity was measured using a cellular ROS detection kit. After lead(II acetate trihydrate treatment, mRNA levels of various genes were assessed by semiquantitative real-time polymerase chain reaction. Following treatment with AME or CME after lead exposure, the changes in cell viability, ROS activity, and gene expression were analyzed. Exposure to >100 μg/mL of lead(II acetate trihydrate caused a significant decrease in cell viability and increased ROS production in HMEECs. Lead exposure significantly increased the mRNA expression of genes encoding inflammatory cytokines and mucins. Administration of AME or CME restored cell viability, reduced ROS activity, and ameliorated mRNA levels. Our findings suggest that environmental lead exposure is related to the development of otitis media, and AME and CME may have antioxidative and anti-inflammatory effects against lead toxicity.

  2. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

    Directory of Open Access Journals (Sweden)

    María Laura Chiribao

    2014-01-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response, a great number of transcription factors (including the majority of NFκB family members, and host metabolism (cholesterol, fatty acids, and phospholipids. These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

  3. Nanoceria have no genotoxic effect on human lens epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pierscionek, Barbara K; Yasseen, Akeel A [School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA (United Kingdom); Li, Yuebin; Schachar, Ronald A; Chen, Wei [Department of Physics, University of Texas at Arlington, Arlington, TX 76019 (United States); Colhoun, Liza M, E-mail: b.pierscionek@ulster.ac.uk, E-mail: weichen@uta.edu [Centre for Vision and Vascular Sciences, School of Medicine, Dentistry and Biomedical Sciences, Queen' s University Belfast, Grosvenor Road, Belfast, BT12 6BA (United Kingdom)

    2010-01-22

    There are no treatments for reversing or halting cataract, a disease of the structural proteins in the eye lens, that has associations with other age-related degenerative conditions such as Alzheimer's disease. The incidence of cataract and associated conditions is increasing as the average age of the population rises. Protein folding diseases are difficult to assess in vivo as proteins and their age-related changes are assessed after extraction. Nanotechnology can be used to investigate protein changes in the intact lens as well as for a potential means of drug delivery. Nanoparticles, such as cerium oxide (CeO{sub 2}) which have antioxidant properties, may even be used as a means of treating cataract directly. Prior to use in treatments, nanoparticle genotoxicity must be tested to assess the extent of any DNA or chromosomal damage. Sister chromatid exchanges were measured and DNA damage investigated using the alkaline COMET assay on cultured human lens epithelial cells, exposed to 5 and 10 {mu}g ml{sup -1} of CeO{sub 2} nanoparticles (nanoceria). Nanoceria at these dosages did not cause any DNA damage or significant increases in the number of sister chromatid exchanges. The absence of genotoxic effects on lens cells suggests that nanoceria, in the doses and exposures tested in this study, are not deleterious to the eye lens and have the potential for use in studying structural alterations, in developing non-surgical cataract treatments and in investigating other protein folding diseases.

  4. Effect of Formaldehyde on Human Middle Ear Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Shin Hye Kim

    2018-01-01

    Full Text Available Formaldehyde (FA is a familiar indoor air pollutant found in everything from cosmetics to clothing, but its impact on the middle ear is unknown. This study investigated whether FA causes cytotoxicity, inflammation, or induction of apoptosis in human middle ear epithelial cells (HMEECs. Cell viability was investigated using the trypan blue assay and a cell counting kit (CCK-8 in HMEECs treated with FA for 4 or 24 h. The expression of genes encoding the inflammatory cytokine tumor necrosis factor alpha (TNF-α and mucin (MUC5AC was analyzed using RT-PCR. Activation of the apoptosis pathway was determined by measuring mitochondrial membrane potential (MMP, cytochrome oxidase, caspase-9/Mch6/Apaf 3, and Caspase-Glo® 3/7 activities. The CCK-8 assay and trypan blue assay results showed a reduction in cell viability in FA-treated HMEECs. FA also increased the cellular expression of TNF-α and MUC5AC and reduced the activities of MMP and cytochrome oxidase. Caspase-9 activity increased in cells stimulated for 4 h, as well as caspase-3/7 activity in cells stimulated for 24 h. The decreased cell viability, the induction of inflammation and mucin gene expression, and the activation of the apoptosis pathway together indicate a link between environmental FA exposure and the development of otitis media.

  5. Feature Importance for Human Epithelial (HEp-2 Cell Image Classification

    Directory of Open Access Journals (Sweden)

    Vibha Gupta

    2018-02-01

    Full Text Available Indirect Immuno-Fluorescence (IIF microscopy imaging of human epithelial (HEp-2 cells is a popular method for diagnosing autoimmune diseases. Considering large data volumes, computer-aided diagnosis (CAD systems, based on image-based classification, can help in terms of time, effort, and reliability of diagnosis. Such approaches are based on extracting some representative features from the images. This work explores the selection of the most distinctive features for HEp-2 cell images using various feature selection (FS methods. Considering that there is no single universally optimal feature selection technique, we also propose hybridization of one class of FS methods (filter methods. Furthermore, the notion of variable importance for ranking features, provided by another type of approaches (embedded methods such as Random forest, Random uniform forest is exploited to select a good subset of features from a large set, such that addition of new features does not increase classification accuracy. In this work, we have also, with great consideration, designed class-specific features to capture morphological visual traits of the cell patterns. We perform various experiments and discussions to demonstrate the effectiveness of FS methods along with proposed and a standard feature set. We achieve state-of-the-art performance even with small number of features, obtained after the feature selection.

  6. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    International Nuclear Information System (INIS)

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R.

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV

  7. Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism

    NARCIS (Netherlands)

    Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker; van der Hoek, Lia

    2013-01-01

    The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in

  8. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    Directory of Open Access Journals (Sweden)

    Z. Liu

    2010-05-01

    Full Text Available Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines, allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39 and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV, may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

  9. Development of an in vitro cytotoxicity model for aerosol exposure using 3D reconstructed human airway tissue; application for assessment of e-cigarette aerosol.

    Science.gov (United States)

    Neilson, Louise; Mankus, Courtney; Thorne, David; Jackson, George; DeBay, Jason; Meredith, Clive

    2015-10-01

    Development of physiologically relevant test methods to analyse potential irritant effects to the respiratory tract caused by e-cigarette aerosols is required. This paper reports the method development and optimisation of an acute in vitro MTT cytotoxicity assay using human 3D reconstructed airway tissues and an aerosol exposure system. The EpiAirway™ tissue is a highly differentiated in vitro human airway culture derived from primary human tracheal/bronchial epithelial cells grown at the air-liquid interface, which can be exposed to aerosols generated by the VITROCELL® smoking robot. Method development was supported by understanding the compatibility of these tissues within the VITROCELL® system, in terms of airflow (L/min), vacuum rate (mL/min) and exposure time. Dosimetry tools (QCM) were used to measure deposited mass, to confirm the provision of e-cigarette aerosol to the tissues. EpiAirway™ tissues were exposed to cigarette smoke and aerosol generated from two commercial e-cigarettes for up to 6 h. Cigarette smoke reduced cell viability in a time dependent manner to 12% at 6 h. E-cigarette aerosol showed no such decrease in cell viability and displayed similar results to that of the untreated air controls. Applicability of the EpiAirway™ model and exposure system was demonstrated, showing little cytotoxicity from e-cigarette aerosol and different aerosol formulations when compared directly with reference cigarette smoke, over the same exposure time. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Direct numerical simulation of particle laden flow in a human airway bifurcation model

    International Nuclear Information System (INIS)

    Stylianou, Fotos S.; Sznitman, Josué; Kassinos, Stavros C.

    2016-01-01

    Highlights: • An anatomically realistic model of a human airway bifurcation is constructed. • Direct numerical simulations are used to study laminar and turbulent airflow. • Aerosol deposition in the bifurcation is studied with lagrangian particle tracking. • Carinal vortices forming during steady expiration are reported for the first time. • Stokes number determines deposition differences between inspiration and expiration. - Abstract: During the delivery of inhaled medicines, and depending on the size distribution of the particles in the formulation, airway bifurcations are areas of preferential deposition. Previous studies of laminar flow through airway bifurcations point to an interplay of inertial and centrifugal forces that leads to rich flow phenomena and controls particle deposition patterns. However, recent computational studies have shown that the airflow in the upper human airways is turbulent during much of the respiratory cycle. The question of how the presence of turbulence modifies these effects remains open. In this study, we perform for the first time Direct Numerical Simulations (DNS) of fully developed turbulent flow through a single human airway bifurcation model, emulating steady prolonged inspiration and expiration. We use the rich information obtained from the DNS in order to identify key structures in the flow field and scrutinize their role in determining deposition patterns in the bifurcation. We find that the vortical structures present in the bifurcation during expiration differ from those identified during inspiration. While Dean vortices are present in both cases, a set of three dimensional “carinal vortices” are identified only during expiration. A set of laminar simulations in the same geometries, but at lower Reynolds numbers, allow us to identify key differences in aerosol deposition patterns between laminar and turbulent respiration. We also report deposition fractions for representative Stokes numbers for both

  11. Airflow structures and nano-particle deposition in a human upper airway model

    Science.gov (United States)

    Zhang, Z.; Kleinstreuer, C.

    2004-07-01

    Considering a human upper airway model, or equivalently complex internal flow conduits, the transport and deposition of nano-particles in the 1-150 nm diameter range are simulated and analyzed for cyclic and steady flow conditions. Specifically, using a commercial finite-volume software with user-supplied programs as a solver, the Euler-Euler approach for the fluid-particle dynamics is employed with a low-Reynolds-number k- ω model for laminar-to-turbulent airflow and the mass transfer equation for dispersion of nano-particles or vapors. Presently, the upper respiratory system consists of two connected segments of a simplified human cast replica, i.e., the oral airways from the mouth to the trachea (Generation G0) and an upper tracheobronchial tree model of G0-G3. Experimentally validated computational fluid-particle dynamics results show the following: (i) transient effects in the oral airways appear most prominently during the decelerating phase of the inspiratory cycle; (ii) selecting matching flow rates, total deposition fractions of nano-size particles for cyclic inspiratory flow are not significantly different from those for steady flow; (iii) turbulent fluctuations which occur after the throat can persist downstream to at least Generation G3 at medium and high inspiratory flow rates (i.e., Qin⩾30 l/min) due to the enhancement of flow instabilities just upstream of the flow dividers; however, the effects of turbulent fluctuations on nano-particle deposition are quite minor in the human upper airways; (iv) deposition of nano-particles occurs to a relatively greater extent around the carinal ridges when compared to the straight tubular segments in the bronchial airways; (v) deposition distributions of nano-particles vary with airway segment, particle size, and inhalation flow rate, where the local deposition is more uniformly distributed for large-size particles (say, dp=100 nm) than for small-size particles (say, dp=1 nm); (vi) dilute 1 nm particle

  12. Assessment of the storz video Macintosh laryngoscope for use in difficult airways: A human simulator study.

    Science.gov (United States)

    Bair, Aaron E; Olmsted, Kalani; Brown, Calvin A; Barker, Tobias; Pallin, Daniel; Walls, Ron M

    2010-10-01

    Video laryngoscopy has been shown to improve glottic exposure when compared to direct laryngoscopy in operating room studies. However, its utility in the hands of emergency physicians (EPs) remains undefined. A simulated difficult airway was used to determine if intubation by EPs using a video Macintosh system resulted in an improved glottic view, was easier, was faster, or was more successful than conventional direct laryngoscopy. Emergency medicine (EM) residents and attending physicians at two academic institutions performed endotracheal intubation in one normal and two identical difficult airway scenarios. With the difficult scenarios, the participants used video laryngoscopy during the second case. Intubations were performed on a medium-fidelity human simulator. The difficult scenario was created by limiting cervical spine mobility and inducing trismus. The primary outcome was the proportion of direct versus video intubations with a grade I or II Cormack-Lehane glottic view. Ease of intubation (self-reported via 10-cm visual analog scale [VAS]), time to intubation, and success rate were also recorded. Descriptive statistics as well as medians with interquartile ranges (IQRs) are reported where appropriate. The Wilcoxon matched pairs signed-rank test was used for comparison testing of nonparametric data. Participants (n = 39) were residents (59%) and faculty. All had human intubation experience; 51% reported more than 100 prior intubations. On difficult laryngoscopy, a Cormack-Lehane grade I or II view was obtained in 20 (51%) direct laryngoscopies versus 38 (97%) of the video-assisted laryngoscopies (p < 0.01). The median VAS score for difficult airways was 50 mm (IQR = 28–73 mm) for direct versus 18 mm (IQR = 9–50 mm) for video (p < 0.01). The median time to intubation in difficult airways was 25 seconds (IQR = 16–44 seconds) for direct versus 20 seconds (IQR = 12–35 seconds) for video laryngoscopy (p < 0.01). All intubations were successful without

  13. Altered ion transport in normal human bronchial epithelial cells following exposure to chemically distinct metal welding fume particles

    Energy Technology Data Exchange (ETDEWEB)

    Fedan, Jeffrey S., E-mail: jsf2@cdc.gov; Thompson, Janet A.; Meighan, Terence G.; Zeidler-Erdely, Patti C.; Antonini, James M.

    2017-07-01

    Welding fume inhalation causes pulmonary toxicity, including susceptibility to infection. We hypothesized that airway epithelial ion transport is a target of fume toxicity, and investigated the effects of fume particulates from manual metal arc-stainless steel (MMA-SS) and gas metal arc-mild steel (GMA-MS) on ion transport in normal human bronchial epithelium (NHBE) cultured in air-interface. MMA-SS particles, more soluble than GMA-MS particles, contain Cr, Ni, Fe and Mn; GMA-MS particles contain Fe and Mn. MMA-SS or GMA-MS particles (0.0167–166.7 μg/cm{sup 2}) were applied apically to NHBEs. After 18 h transepithelial potential difference (V{sub t}), resistance (R{sub t}), and short circuit current (I{sub sc}) were measured. Particle effects on Na{sup +} and Cl¯ channels and the Na{sup +},K{sup +},2Cl¯-cotransporter were evaluated using amiloride (apical), 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, apical), and bumetanide (basolateral), respectively. MMA-SS (0.0167–16.7 μg/cm{sup 2}) increased basal V{sub t}. Only 16.7 μg/cm{sup 2} GMA-MS increased basal V{sub t} significantly. MMA-SS or GMA-MS exposure potentiated I{sub sc} responses (decreases) to amiloride and bumetanide, while not affecting those to NPPB, GMA-MS to a lesser degree than MMA-SS. Variable effects on R{sub t} were observed in response to amiloride, and bumetanide. Generally, MMA-SS was more potent in altering responses to amiloride and bumetanide than GMA-MS. Hyperpolarization occurred in the absence of LDH release, but decreases in V{sub t}, R{sub t}, and I{sub sc} at higher fume particulate doses accompanied LDH release, to a greater extent for MMA-SS. Thus, Na{sup +} transport and Na{sup +},K{sup +},2Cl¯-cotransport are affected by fume exposure; MMA-MS is more potent than GMA-MS. Enhanced Na{sup +} absorption and decreased airway surface liquid could compromise defenses against infection. - Highlights: • Welding fume particle toxicity was investigated in human bronchial

  14. Proinflammatory effects of cookstove emissions on human bronchial epithelial cells.

    Science.gov (United States)

    Hawley, B; Volckens, J

    2013-02-01

    Approximately half of the world's population uses biomass fuel for indoor cooking and heating. This form of combustion typically occurs in open fires or primitive stoves. Human exposure to emissions from indoor biomass combustion is a global health concern, causing an estimated 1.5 million premature deaths each year. Many 'improved' stoves have been developed to address this concern; however, studies that examine exposure-response with cleaner-burning, more efficient stoves are few. The objective of this research was to evaluate the effects of traditional and cleaner-burning stove emissions on an established model of the bronchial epithelium. We exposed well-differentiated, normal human bronchial epithelial cells to emissions from a single biomass combustion event using either a traditional three-stone fire or one of two energy-efficient stoves. Air-liquid interface cultures were exposed using a novel, aerosol-to-cell deposition system. Cellular expression of a panel of three pro-inflammatory markers was evaluated at 1 and 24 h following exposure. Cells exposed to emissions from the cleaner-burning stoves generated significantly fewer amounts of pro-inflammatory markers than cells exposed to emissions from a traditional three-stone fire. Particulate matter emissions from each cookstove were substantially different, with the three-stone fire producing the largest concentrations of particles (by both number and mass). This study supports emerging evidence that more efficient cookstoves have the potential to reduce respiratory inflammation in settings where solid fuel combustion is used to meet basic domestic needs. Emissions from more efficient, cleaner-burning cookstoves produced less inflammation in well-differentiated bronchial lung cells. The results support evidence that more efficient cookstoves can reduce the health burden associated with exposure to indoor pollution from the combustion of biomass. © 2012 John Wiley & Sons A/S.

  15. Ozone exposure increases respiratory epithelial permeability in humans

    International Nuclear Information System (INIS)

    Kehrl, H.R.; Vincent, L.M.; Kowalsky, R.J.; Horstman, D.H.; O'Neil, J.J.; McCartney, W.H.; Bromberg, P.A.

    1987-01-01

    Ozone is a respiratory irritant that has been shown to cause an increase in the permeability of the respiratory epithelium in animals. We used inhaled aerosolized /sup 99m/Tc-labeled diethylene triamine pentacetic acid (/sup 99m/Tc-DTPA) to investigate whether human respiratory epithelial permeability is similarly affected by exposure to ozone. In a randomized, crossover double-blinded study, 8 healthy, nonsmoking young men were exposed for 2 h to purified air and 0.4 ppm ozone while performing intermittent high intensity treadmill exercise (minute ventilation = 66.8 L/min). SRaw and FVC were measured before and at the end of exposures. Seventy-five minutes after the exposures, the pulmonary clearance of /sup 99m/Tc-DTPA was measured by sequential posterior lung imaging with a computer-assisted gamma camera. Ozone exposure caused respiratory symptoms in all 8 subjects and was associated with a 14 +/- 2.8% (mean +/- SEM) decrement in FVC (p less than 0.001) and a 71 +/- 22% increase in SRaw (p = 0.04). Compared with the air exposure day, 7 of the 8 subjects showed increased /sup 99m/Tc-DTPA clearance after the ozone exposure, with the mean value increasing from 0.59 +/- 0.08 to 1.75 +/- 0.43%/min (p = 0.03). These data show that ozone exposure sufficient to produce decrements in the pulmonary function of human subjects also causes an increase in /sup 99m/Tc-DTPA clearance

  16. Prevalence of human papillomavirus in epithelial ovarian cancer tissue. A meta-analysis of observational studies

    DEFF Research Database (Denmark)

    Svahn, Malene F; Faber, Mette Tuxen; Christensen, Jane

    2014-01-01

    The role of human papillomavirus (HPV) in the pathogenesis of ovarian cancer is controversial, and conflicting results have been published. We conducted a systematic review and meta-analysis to estimate the prevalence of HPV in epithelial ovarian cancer tissue....

  17. Activation of neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair.

    Science.gov (United States)

    Oslund, Karen L; Hyde, Dallas M; Putney, Leialoha F; Alfaro, Mario F; Walby, William F; Tyler, Nancy K; Schelegle, Edward S

    2008-09-01

    We investigated the importance of neurokinin (NK)-1 receptors in epithelial injury and repair and neutrophil function. Conscious Wistar rats were exposed to 1 ppm ozone or filtered air for 8 hours, followed by an 8-hour postexposure period. Before exposure, we administered either the NK-1 receptor antagonist, SR140333, or saline as a control. Ethidium homodimer was instilled into lungs as a marker of necrotic airway epithelial cells. After fixation, whole mounts of airway dissected lung lobes were immunostained for 5-bromo-2'-deoxyuridine, a marker of epithelial proliferation. Both ethidium homodimer and 5-bromo-2'-deoxyuridine-positive epithelial cells were quantified in specific airway generations. Rats treated with the NK-1 receptor antagonist had significantly reduced epithelial injury and epithelial proliferation compared with control rats. Sections of terminal bronchioles showed no significant difference in the number of neutrophils in airways between groups. In addition, staining ozone-exposed lung sections for active caspase 3 showed no apoptotic cells, but ethidium-positive cells colocalized with the orphan nuclear receptor, Nur77, a marker of nonapoptotic, programmed cell death mediated by the NK-1 receptor. An immortalized human airway epithelial cell line, human bronchial epithelial-1, showed no significant difference in the number of oxidant stress-positive cells during exposure to hydrogen peroxide and a range of SR140333 doses, demonstrating no antioxidant effect of the receptor antagonist. We conclude that activation of the NK-1 receptor during acute ozone inhalation contributes to epithelial injury and subsequent epithelial proliferation, a critical component of repair, but does not influence neutrophil emigration into airways.

  18. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    OpenAIRE

    Liu, Z.; Zhang, P.; Zhou, Y.; Qin, H.; Shen, T.

    2010-01-01

    Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithel...

  19. Human mast cell and airway smooth muscle cell interactions: implications for asthma.

    Science.gov (United States)

    Page, S; Ammit, A J; Black, J L; Armour, C L

    2001-12-01

    Asthma is characterized by inflammation, hyperresponsiveness, and remodeling of the airway. Human mast cells (HMCs) play a central role in all of these changes by releasing mediators that cause exaggerated bronchoconstriction, induce human airway smooth muscle (HASM) cell proliferation, and recruit and activate inflammatory cells. Moreover, the number of HMCs present on asthmatic HASM is increased compared with that on nonasthmatic HASM. HASM cells also have the potential to actively participate in the inflammatory process by synthesizing cytokines and chemokines and expressing surface molecules, which have the capacity to perpetuate the inflammatory mechanisms present in asthma. This review specifically examines how the mediators of HMCs have the capacity to modulate many functions of HASM; how the synthetic function of HASM, particularly through the release and expression of stem cell factor, has the potential to influence HMC number and activation in an extraordinarily potent and proinflammatory manner; and how these interactions between HMCs and HASM have potential consequences for airway structure and inflammation relevant to the disease process of asthma.

  20. Quantitative imaging of the human upper airway: instrument design and clinical studies

    Science.gov (United States)

    Leigh, M. S.; Armstrong, J. J.; Paduch, A.; Sampson, D. D.; Walsh, J. H.; Hillman, D. R.; Eastwood, P. R.

    2006-08-01

    Imaging of the human upper airway is widely used in medicine, in both clinical practice and research. Common imaging modalities include video endoscopy, X-ray CT, and MRI. However, no current modality is both quantitative and safe to use for extended periods of time. Such a capability would be particularly valuable for sleep research, which is inherently reliant on long observation sessions. We have developed an instrument capable of quantitative imaging of the human upper airway, based on endoscopic optical coherence tomography. There are no dose limits for optical techniques, and the minimally invasive imaging probe is safe for use in overnight studies. We report on the design of the instrument and its use in preliminary clinical studies, and we present results from a range of initial experiments. The experiments show that the instrument is capable of imaging during sleep, and that it can record dynamic changes in airway size and shape. This information is useful for research into sleep disorders, and potentially for clinical diagnosis and therapies.

  1. Human Bronchial Epithelial Cell Response to Heavy Particle Exposure

    Science.gov (United States)

    Story, Michael; Ding, Liang-Hao; Minna, John; Park, Seong-mi; Peyton, Michael; Larsen, Jill

    2012-07-01

    A battery of non-oncogenically immortalized human bronchial epithelial cells (HBECs) are being used to examine the molecular changes that lead to lung carcinogenesis after exposure to heavy particles found in the free space environment. The goal is to ultimately identify biomarkers of radioresponse that can be used for prediction of carcinogenic risk for fatal lung cancer. Our initial studies have focused on the cell line HBEC3 KT and the isogenic variant HBEC3 KTR53, which overexpresses the RASv12 mutant and where p53 has been knocked down by shRNA, and is considered to be a more oncogenically progressed variant. We have previously described the response of HBEC3 KT at the cellular and molecular level, however, the focus here is on the rate of cellular transformation after HZE radiation exposure and the molecular changes in transformed cells. When comparing the two cell lines we find that there is a maximum rate of cellular transformation at 0.25 Gy when cells are exposed to 1 GeV Fe particles, and, for the HBEC3 KTR53 there are multiple pathways upregulated that promote anchorage independent growth including the mTOR pathway, the TGF-1 pathway, RhoA signaling and the ERK/MAPK pathway as early as 2 weeks after radiation. This does not occur in the HBEC3 KT cell line. Transformed HBEC3 KT cells do not show any morphologic or phenotypic changes when grown as cell cultures. HBEC3 KTR53 cells on the other hand show substantial changes in morphology from a cobblestone epithelial appearance to a mesenchymal appearance with a lack of contact inhibition. This epithelial to mesenchymal change in morphology is accompanied by the expression of vimentin and a reduction in the expression of E-cadherin, which are hallmarks of epithelial to mesenchymal transition. Interestingly, for HBEC3 KT transformed cells there are no mutations in the p53 gene, 2 of 15 clones were found to be heterozygous for the RASV12 mutation, and 3 of 15 clones expressed high levels of BigH3, a TGFB

  2. Radiosensitizing effect of epothilone B on human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Baumgart, T.; Kriesen, S.; Hildebrandt, G.; Manda, K. [Univ. of Rostock (Germany). Dept. of Radiotherapy and Radiation Oncology; Klautke, G.; Fietkau, R. [Friedrich-Alexander-Univ. Erlangen-Nuernberg, Erlangen (Germany). Dept. of Radiation Oncology; Kuznetsov, S.A.; Weiss, D.G. [Univ. of Rostock (Germany). Inst. of Biological Sciences, Cell Biology, and Biosystems Technology

    2012-02-15

    A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a {gamma}H2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC{sub 50} values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The {gamma}H2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel. (orig.)

  3. Bone marrow contributes to epithelial cancers in mice and humans as developmental mimicry.

    Science.gov (United States)

    Cogle, Christopher R; Theise, Neil D; Fu, Dongtao; Ucar, Deniz; Lee, Sean; Guthrie, Steven M; Lonergan, Jean; Rybka, Witold; Krause, Diane S; Scott, Edward W

    2007-08-01

    Bone marrow cells have the capacity to contribute to distant organs. We show that marrow also contributes to epithelial neoplasias of the small bowel, colon, and lung, but not the skin. In particular, epithelial neoplasias found in patients after hematopoietic cell transplantations demonstrate that human marrow incorporates into neoplasias by adopting the phenotype of the surrounding neoplastic environment. To more rigorously evaluate marrow contribution to epithelial cancer, we employed mouse models of intestinal and lung neoplasias, which revealed specifically that the hematopoietic stem cell and its progeny incorporate within cancer. Furthermore, this marrow involvement in epithelial cancer does not appear to occur by induction of stable fusion. Whereas previous claims have been made that marrow can serve as a direct source of epithelial neoplasia, our results indicate a more cautionary note, that marrow contributes to cancer as a means of developmental mimicry. Disclosure of Potential Conflicts of Interest is found at the end of this article.

  4. Computational model of soft tissues in the human upper airway.

    Science.gov (United States)

    Pelteret, J-P V; Reddy, B D

    2012-01-01

    This paper presents a three-dimensional finite element model of the tongue and surrounding soft tissues with potential application to the study of sleep apnoea and of linguistics and speech therapy. The anatomical data was obtained from the Visible Human Project, and the underlying histological data was also extracted and incorporated into the model. Hyperelastic constitutive models were used to describe the material behaviour, and material incompressibility was accounted for. An active Hill three-element muscle model was used to represent the muscular tissue of the tongue. The neural stimulus for each muscle group was determined through the use of a genetic algorithm-based neural control model. The fundamental behaviour of the tongue under gravitational and breathing-induced loading is investigated. It is demonstrated that, when a time-dependent loading is applied to the tongue, the neural model is able to control the position of the tongue and produce a physiologically realistic response for the genioglossus.

  5. Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Pettigrew, Melinda M; Ahearn, Christian P; Gent, Janneane F; Kong, Yong; Gallo, Mary C; Munro, James B; D'Mello, Adonis; Sethi, Sanjay; Tettelin, Hervé; Murphy, Timothy F

    2018-04-03

    Nontypeable Haemophilus influenzae (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

  6. TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

    Science.gov (United States)

    Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

    2008-05-01

    Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

  7. Increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection

    Directory of Open Access Journals (Sweden)

    King Nicholas JC

    2006-05-01

    Full Text Available Abstract Background Exacerbations of asthma are associated with viral respiratory tract infections, of which rhinoviruses (RV are the predominant virus type. Airway smooth muscle is important in asthma pathogenesis, however little is known about the potential interaction of RV and human airway smooth muscle cells (HASM. We hypothesised that rhinovirus induction of inflammatory cytokine release from airway smooth muscle is augmented and differentially regulated in asthmatic compared to normal HASM cells. Methods HASM cells, isolated from either asthmatic or non-asthmatic subjects, were infected with rhinovirus. Cytokine production was assayed by ELISA, ICAM-1 cell surface expression was assessed by FACS, and the transcription regulation of IL-6 was measured by luciferase activity. Results RV-induced IL-6 release was significantly greater in HASM cells derived from asthmatic subjects compared to non-asthmatic subjects. This response was RV specific, as 5% serum- induced IL-6 release was not different in the two cell types. Whilst serum stimulated IL-8 production in cells from both subject groups, RV induced IL-8 production in only asthmatic derived HASM cells. The transcriptional induction of IL-6 was differentially regulated via C/EBP in the asthmatic and NF-κB + AP-1 in the non-asthmatic HASM cells. Conclusion This study demonstrates augmentation and differential transcriptional regulation of RV specific innate immune response in HASM cells derived from asthmatic and non-asthmatics, and may give valuable insight into the mechanisms of RV-induced asthma exacerbations.

  8. A Numerical Study of Water Loss Rate Distributions in MDCT-based Human Airway Models

    Science.gov (United States)

    Wu, Dan; Miyawaki, Shinjiro; Tawhai, Merryn H.; Hoffman, Eric A.; Lin, Ching-Long

    2015-01-01

    Both three-dimensional (3D) and one-dimensional (1D) computational fluid dynamics (CFD) methods are applied to study regional water loss in three multi-detector row computed-tomography (MDCT)-based human airway models at the minute ventilations of 6, 15 and 30 L/min. The overall water losses predicted by both 3D and 1D models in the entire respiratory tract agree with available experimental measurements. However, 3D and 1D models reveal different regional water loss rate distributions due to the 3D secondary flows formed at bifurcations. The secondary flows cause local skewed temperature and humidity distributions on inspiration acting to elevate the local water loss rate; and the secondary flow at the carina tends to distribute more cold air to the lower lobes. As a result, the 3D model predicts that the water loss rate first increases with increasing airway generation, and then decreases as the air approaches saturation, while the 1D model predicts a monotonic decrease of water loss rate with increasing airway generation. Moreover, the 3D (or 1D) model predicts relatively higher water loss rates in lower (or upper) lobes. The regional water loss rate can be related to the non-dimensional wall shear stress (τ*) by the non-dimensional mass transfer coefficient (h0*) as h0* = 1.15 τ*0.272, R = 0.842. PMID:25869455

  9. From single cilia to collective waves in human airway ciliated tissues

    Science.gov (United States)

    Cicuta, Pietro; Chioccioli, Maurizio; Feriani, Luigi; Pellicciotta, Nicola; Kotar, Jurij

    I will present experimental results on activity of motile cilia on various scales: from waveforms on individual cilia to the synchronised motion in cilia carpets of airway cells. Model synthetic experiments have given us an understanding of how cilia could couple with each other through forces transmitted by the fluid, and thus coordinate to beat into well organized waves (previous work is reviewed in Annu. Rev. Condens. Matter Phys. 7, 1-26 (2016)). Working with live imaging of airway human cells at the different scales, we can now test whether the biological system satisfies the ``simple'' behavior expected of the fluid flow coupling, or if other factors of mechanical forces transmission need to be accounted for. In general being able to link from the scale of molecular biological activity up to the phenomenology of collective dynamics requires to understand the relevant physical mechanism. This understanding then allows informed diagnostics (and perhaps therapeutic) approaches to a variety of diseases where mucociliary clearance in the airways is compromised. We have started exploring particularly cystic fibrosis, where the rheological properties of the mucus are affected and prevent efficient cilia synchronization. ERC Grant HydroSync.

  10. Comparative proteome analysis of human epithelial ovarian cancer

    Directory of Open Access Journals (Sweden)

    Gagné Jean-Philippe

    2007-09-01

    Full Text Available Abstract Background Epithelial ovarian cancer is a devastating disease associated with low survival prognosis mainly because of the lack of early detection markers and the asymptomatic nature of the cancer until late stage. Using two complementary proteomics approaches, a differential protein expression profile was carried out between low and highly transformed epithelial ovarian cancer cell lines which realistically mimic the phenotypic changes observed during evolution of a tumour metastasis. This investigation was aimed at a better understanding of the molecular mechanisms underlying differentiation, proliferation and neoplastic progression of ovarian cancer. Results The quantitative profiling of epithelial ovarian cancer model cell lines TOV-81D and TOV-112D generated using iTRAQ analysis and two-dimensional electrophoresis coupled to liquid chromatography tandem mass spectrometry revealed some proteins with altered expression levels. Several of these proteins have been the object of interest in cancer research but others were unrecognized as differentially expressed in a context of ovarian cancer. Among these, series of proteins involved in transcriptional activity, cellular metabolism, cell adhesion or motility and cytoskeleton organization were identified, suggesting their possible role in the emergence of oncogenic pathways leading to aggressive cellular behavior. Conclusion The differential protein expression profile generated by the two proteomics approaches combined to complementary characterizations studies will open the way to more exhaustive and systematic representation of the disease and will provide valuable information that may be helpful to uncover the molecular mechanisms related to epithelial ovarian cancer.

  11. The classical Starling resistor model often does not predict inspiratory airflow patterns in the human upper airway.

    Science.gov (United States)

    Owens, Robert L; Edwards, Bradley A; Sands, Scott A; Butler, James P; Eckert, Danny J; White, David P; Malhotra, Atul; Wellman, Andrew

    2014-04-15

    The upper airway is often modeled as a classical Starling resistor, featuring a constant inspiratory airflow, or plateau, over a range of downstream pressures. However, airflow tracings from clinical sleep studies often show an initial peak before the plateau. To conform to the Starling model, the initial peak must be of small magnitude or dismissed as a transient. We developed a method to simulate fast or slow inspirations through the human upper airway, to test the hypothesis that this initial peak is a transient. Eight subjects [4 obstructive sleep apnea (OSA), 4 controls] slept in an "iron lung" and wore a nasal mask connected to a continuous/bilevel positive airway pressure machine. Downstream pressure was measured using an epiglottic catheter. During non-rapid eye movement (NREM) sleep, subjects were hyperventilated to produce a central apnea, then extrathoracic pressure was decreased slowly (∼2-4 s) or abruptly (resistor model, the upper airway exhibits marked NED in some subjects.

  12. Local deposition patterns of inhaled radon progeny in human bronchial airways

    International Nuclear Information System (INIS)

    Heistracher, T.; Hofmann, W.; Balashazy, I.

    1996-01-01

    The local distribution of radon decay products deposited within bronchial bifurcations, particularly the formation of hot spots, may be more relevant for the determination of cellular doses in bronchial tissue than the commonly computed deposition efficiency, which is conceptually equivalent to the assumption of a uniform nuclide distribution. It is well known that the initial states of lung cancer in humans preferably occur in upper airways close to the cardinal location. In this study we use a recently developed geometric approach of a physiologically realistic bifurcation to demonstrate the site sensitivity of radon progeny deposition for two particle sizes, which are representative of the unattached and attached fraction of radon progeny

  13. Modelling the effect of non-uniform radon progeny activities on transformation frequencies in human bronchial airways

    International Nuclear Information System (INIS)

    Fakir, H.; Hofmann, W.; Aubineau-Laniece, I.

    2006-01-01

    The effects of radiological and morphological source heterogeneities in straight and Y-shaped bronchial airways on hit frequencies and Micro-dosimetric quantities in epithelial cells have been investigated previously. The goal of the present study is to relate these physical quantities to transformation frequencies in sensitive target cells and to radon-induced lung cancer risk. Based on an effect-specific track length model, computed linear energy transfer (LET) spectra were converted to corresponding transformation frequencies for different activity distributions and source - target configurations. Average transformation probabilities were considerably enhanced for radon progeny accumulations and target cells at the carinal ridge, relative to uniform activity distributions and target cells located along the curved and straight airway portions at the same exposure level. Although uncorrelated transformation probabilities produce a linear dose - effect relationship, correlated transformations first increase depending on the LET, but then decrease significantly when exceeding a defined number of hits or cumulative exposure level. (authors)

  14. Human factors in the development of complications of airway management: preliminary evaluation of an interview tool.

    Science.gov (United States)

    Flin, R; Fioratou, E; Frerk, C; Trotter, C; Cook, T M

    2013-08-01

    The 4th National Audit Project of the Royal College of Anaesthetists and the Difficult Airway Society (NAP4) analysed reports of serious events arising from airway management during anaesthesia, intensive care and the emergency department. We conducted supplementary telephone interviews with 12 anaesthetists who had reported to NAP4, aiming to identify causal factors using a method based on the Human Factors Investigation Tool (HFIT). We identified contributing human factors in all cases (median [range] 4.5 [1-10] per case). The most frequent related to: situation awareness (failures to anticipate, wrong decision) (nine cases); job factors (e.g. task difficulty; staffing, time pressure) (eight cases); and person factors (e.g. tiredness, hunger, stress) (six cases). Protective factors, such as teamwork and communication, were also revealed. The post-report HFIT interview method identified relevant human factors and this approach merits further testing as part of the investigation of anaesthetic incidents. Anaesthesia © 2013 The Association of Anaesthetists of Great Britain and Ireland.

  15. Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

    Science.gov (United States)

    Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

    2015-04-24

    The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. In vitro safety evaluation of human nasal epithelial cell monolayers exposed to carrageenan sinus wash.

    Science.gov (United States)

    Ramezanpour, Mahnaz; Murphy, Jae; Smith, Jason L P; Vreugde, Sarah; Psaltis, Alkis James

    2017-12-01

    Carrageenans have shown to reduce the viral load in nasal secretions and lower the incidence of secondary infections in children with common cold. Despite the widespread use of carrageenans in topical applications, the effect of carrageenans on the sinonasal epithelial barrier has not been elucidated. We investigate the effect of different carrageenans on the sinonasal epithelial barrier and inflammatory response in vitro. Iota and Kappa carrageenan delivered in saline irrigation solutions applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells from chronic rhinosinusitis patients and controls. Epithelial barrier structure was assessed by measuring the transepithelial electrical resistance (TEER) and immunolocalization of F actin. Ciliary beat frequency (CBF), toxicity, and inflammatory response was studied. Kappa or Iota carrageenan in the different solutions was not toxic, did not have detrimental effects on epithelial barrier structure and CBF. Rather, application of Kappa carrageenan significantly increased TEER and suppressed interleukin 6 (IL-6) secretion in ALI cultures from CRS patients. Kappa or Iota carrageenan solution was safe and did not negatively affect epithelial barrier function. Kappa carrageenan increased TEER and decreased IL-6 production in CRS patients, indicating positive effects on epithelial barrier function in vitro. © 2017 ARS-AAOA, LLC.

  17. Lipopolysaccharide hyperpolarizes guinea pig airway epithelium by increasing the activities of the epithelial Na(+) channel and the Na(+)-K(+) pump.

    Science.gov (United States)

    Dodrill, Michael W; Fedan, Jeffrey S

    2010-10-01

    Earlier, we found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference (V(t)) of tracheal epithelium in the isolated, perfused trachea (IPT) of the guinea pig 18 h after injection. As well, LPS increased the hyperpolarization component of the response to basolateral methacholine, and potentiated the epithelium-derived relaxing factor-mediated relaxation responses to hyperosmolar solutions applied to the apical membrane. We hypothesized that LPS stimulates the transepithelial movement of Na(+) via the epithelial sodium channel (ENaC)/Na(+)-K(+) pump axis, leading to hyperpolarization of V(t). LPS increased the V(t)-depolarizing response to amiloride (10 μM), i.e., offset the effect of LPS, indicating that Na(+) transport activity was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the Na(+)-K(+) pump with basolateral amphotericin B (7.5 μM). LPS had no effect on the hyperpolarization response to apical trypsin (100 U/ml) in the Ussing chamber, indicating that channel-activating proteases are not involved in the LPS-induced activation of ENaC. To assess Na(+)-K(+) pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater V(t) in the presence of amphotericin B in tracheas from LPS-treated animals compared with controls revealed that LPS increased Na(+)-K(+) pump activity. This finding was confirmed in the Ussing chamber by inhibiting the Na(+)-K(+) pump via extracellular K(+) removal, loading the epithelium with Na(+), and observing a greater hyperpolarization response to K(+) restoration. Together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the Na(+)-K(+) pump.

  18. Induction of human airway hyperresponsiveness by tumour necrosis factor-alpha.

    Science.gov (United States)

    Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

    1995-09-15

    Tumour necrosis factor-alpha (TNF alpha) is implicated in the pathogenesis of asthma; however, little is known of its direct effect on smooth muscle reactivity. We investigated the effect of TNF alpha on the responsiveness of human bronchial tissue to electrical field stimulation in vitro. Incubation of non-sensitized tissue with 1 nM, 3 nM and 10 nM TNF alpha significantly increased responsiveness to electrical field stimulation (113 +/- 8, 110 +/- 4 and 112 +/- 2% respectively) compared to control (99 +/- 2%) (P 0.05) nor were responses to exogenous acetylcholine (93 +/- 4% versus 73 +/- 7%, n = 3, P = 0.38). These results show that TNF alpha causes an increase in responsiveness of human bronchial tissue and that this occurs prejunctionally on the parasympathetic nerve pathway. This is the first report of a cytokine increasing human airway tissue responsiveness.

  19. The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

    International Nuclear Information System (INIS)

    William Petersen, Ole; Lind Nielsen, Helga; Gudjonsson, Thorarinn; Villadsen, René; Rønnov-Jessen, Lone; Bissell, Mina J

    2001-01-01

    The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression

  20. The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion

    Energy Technology Data Exchange (ETDEWEB)

    Petersen, Ole William; Nielsen, Helga Lind; Gudjonsson, Thorarinn; Villadsen, Ren& #233; ; Ronnov-Jessen, Lone; Bissell, Mina J.

    2001-05-12

    The human breast comprises three lineages: the luminal epithelial lineage, the myoepithelial lineage, and the mesenchymal lineage. It has been widely accepted that human breast neoplasia pertains only to the luminal epithelial lineage. In recent years, however, evidence has accumulated that neoplastic breast epithelial cells may be substantially more plastic in their differentiation repertoire than previously anticipated. Thus, along with an increasing availability of markers for the myoepithelial lineage, at least a partial differentiation towards this lineage is being revealed frequently. It has also become clear that conversions towards the mesenchymal lineage actually occur, referred to as epithelial to mesenchymal transitions. Indeed, some of the so-called myofibroblasts surrounding the tumor may indeed have an epithelial origin rather than a mesenchymal origin. Because myoepithelial cells, epithelial to mesenchymal transition-derived cells, genuine stromal cells and myofibroblasts share common markers, we now need to define a more ambitious set of markers to distinguish these cell types in the microenvironment of the tumors. This is necessary because the different microenvironments may confer different clinical outcomes. The aim of this commentary is to describe some of the inherent complexities in defining cellular phenotypes in the microenvironment of breast cancer and to expand wherever possible on the implications for tumor suppression and progression.

  1. Determining adaptive and adverse oxidative stress responses in human bronical epithelial cells exposed to zinc

    Science.gov (United States)

    Determining adaptive and adverse oxidative stress responses in human bronchial epithelial cells exposed to zincJenna M. Currier1,2, Wan-Yun Cheng1, Rory Conolly1, Brian N. Chorley1Zinc is a ubiquitous contaminant of ambient air that presents an oxidant challenge to the human lung...

  2. Oral focal epithelial hyperplasia: report of 3 cases with human papillomavirus DNA sequencing analysis.

    Science.gov (United States)

    Gültekin, S E; Tokman Yildirim, Benay; Sarisoy, S

    2011-01-01

    Focal epithelial hyperplasia (FEH), or Heck's disease, is a benign proliferative viral infection of the oral mucosa that is related to Human Papil-lomavirus (HPV), mainly subtypes 13 and 32. Although this condition is known to exist in numerous populations and ethnic groups, the reported cases among Caucasians are relatively rare. It presents as asymptomatic papules or nodules on the oral mucosa, gingiva, tongue, and lips. Histopathologically, it is characterized by parakeratosis, epithelial hyperplasia, focal acanthosis, fusion, and horizontal outgrowth of epithelial ridges and the cells named mitozoids. The purpose of this case report was to present 3 cases of focal epithelial hyperplasia in a pediatric age group. Histopathological and clinical features of cases are discussed and DNA sequencing analysis is reported in which HPV 13, HPV 32, and HPV 11 genomes are detected.

  3. Neurally mediated airway constriction in human and other species: a comparative study using precision-cut lung slices (PCLS.

    Directory of Open Access Journals (Sweden)

    Marco Schlepütz

    Full Text Available The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS respond to electric field stimulation (EFS and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM caused bronchoconstriction in human (4 from 7 and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC. However, this effect was notably smaller in human responder (30 ± 7.1% than in guinea pig (79 ± 5.1% PCLS. The transient receptor potential (TRP channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.

  4. Fluid absorption related to ion transport in human airway epithelial spheroids

    DEFF Research Database (Denmark)

    Pedersen, P S; Holstein-Rathlou, N H; Larsen, P L

    1999-01-01

    , and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 microl x cm(-2) x h(-1)), 2) amiloride inhibited fluid absorption to a lower residual level...... in non-CF than in CF spheroids, 3) Cl(-)-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5......) osmotic water permeabilities were equal in non-CF and CF spheroids ( approximately 27 x 10(-7) cm x s(-1) x atm(-1))....

  5. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    Science.gov (United States)

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  6. Nanodiamond particles induce IL-8 expression through a transcript stabilization mechanism in human airway epithelial cells

    Science.gov (United States)

    Nanodiamond particles (NDP) prepared by detonational processes have a number of industrial and analytical applications. Previous in vitro studies have reported NDP to be biologically inert with negligible cytotoxicity, implying that they are potentially suitable for biomedical ap...

  7. MECHANISMS OF NANODIAMOND PARTICLE INDUCED IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS

    Science.gov (United States)

    Nanodiamond particles (NDP) prepared by detonation under confined conditions have a number of industrial and analytical applications. Previous in vitro studies have reported NDP to be biologically inert with negligible cytotoxicity, implying that they are potentially suitable for...

  8. HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

    Science.gov (United States)

    Mishina, Kei; Shinkai, Masaharu; Shimokawaji, Tadasuke; Nagashima, Akimichi; Hashimoto, Yusuke; Inoue, Yoriko; Inayama, Yoshiaki; Rubin, Bruce K; Ishigatsubo, Yoshiaki; Kaneko, Takeshi

    2015-12-01

    Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 μM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

  9. Inhibition of NFkappaB activation and IL-8 expression in human bronchial epithelial cells by acrolein.

    Science.gov (United States)

    Valacchi, Giuseppe; Pagnin, Elisa; Phung, Anh; Nardini, Mirella; Schock, Bettina C; Cross, Carroll E; van der Vliet, Albert

    2005-01-01

    Lipid oxidation and environmental pollutants are major sources of alpha,beta-unsaturated aldehydes such as acrolein and 4-hydroxynonenal. Acrolein (2-propenal), a major product of organic combustion such as tobacco smoke, represents the most reactive alpha,beta-unsaturated aldehyde, with high reactivity toward nucleophilic targets such as sulfhydryl groups. To investigate how acrolein affects respiratory tract cell activation, we exposed either primary (NHBE) or immortalized human bronchial epithelial cells (HBE1) to 0-25 microM acrolein, and determined effects on basal and tumor necrosis factor-alpha (TNFalpha)-induced production of the chemokine interleukin (IL)-8. Cell exposure to acrolein dose-dependently suppressed IL-8 mRNA levels in HBE1 cells (26, 40, and 79% at 5, 10, and 25 microM acrolein concentrations, respectively) and resulted in corresponding decreases in IL-8 production. Studies of nuclear factor-kappaB (NFkappaB) activation, an essential event in IL-8 production, showed decreased TNFalpha-induced NFkappaB activation by acrolein, illustrated by inhibition of nuclear translocation of NFkappaB and reduced IkappaBalpha degradation. Immunochemical analysis of IkappaB kinase (IKK), a redox-sensitive regulator of NFkappaB activation, indicated direct modification of the IKK beta-subunit by acrolein, suggesting that acrolein may act directly on IKK. In summary, our results demonstrate that acrolein can suppress inflammatory processes in the airways by inhibiting epithelial IL-8 production through direct or indirect inhibitory effects on NFkappaB activation.

  10. Measurement of IL-13–Induced iNOS-Derived Gas Phase Nitric Oxide in Human Bronchial Epithelial Cells

    Science.gov (United States)

    Suresh, Vinod; Mih, Justin D.; George, Steven C.

    2007-01-01

    Exhaled nitric oxide (NO) is altered in numerous diseases including asthma, and is thought broadly to be a noninvasive marker of inflammation. However, the precise source of exhaled NO has yet to be identified, and the interpretation is further hampered by significant inter-subject variation. Using fully differentiated normal human bronchial epithelial (NHBE) cells, we sought to determine (1) the rate of NO release (flux, pl·s−1.cm−2) into the gas; (2) the effect of IL-13, a prominent mediator of allergic inflammation, on NO release; and (3) inter-subject/donor variability in NO release. NHBE cells from three different donors were cultured at an air–liquid interface and stimulated with different concentrations of IL-13 (0, 1, and 10 ng/ml) for 48 h. Gas phase NO concentrations in the headspace over the cells were measured using a chemiluminescence analyzer. The basal NO flux from the three donors (0.05 ± 0.03) is similar in magnitude to that estimated from exhaled NO concentrations, and was significantly increased by IL-13 in a donor-specific fashion. The increase in NO release was strongly correlated with inducible nitric oxide synthase (iNOS) gene and protein expression. There was a trend toward enhanced production of nitrate relative to nitrite as an end product of NO metabolism in IL-13–stimulated cells. NO release from airway epithelial cells can be directly measured. The rate of release in response to IL-13 is strongly dependent on the individual donor, but is primarily due to the expression of iNOS. PMID:17347445

  11. Measurement of IL-13-induced iNOS-derived gas phase nitric oxide in human bronchial epithelial cells.

    Science.gov (United States)

    Suresh, Vinod; Mih, Justin D; George, Steven C

    2007-07-01

    Exhaled nitric oxide (NO) is altered in numerous diseases including asthma, and is thought broadly to be a noninvasive marker of inflammation. However, the precise source of exhaled NO has yet to be identified, and the interpretation is further hampered by significant inter-subject variation. Using fully differentiated normal human bronchial epithelial (NHBE) cells, we sought to determine (1) the rate of NO release (flux, pl.s(-1.)cm(-2)) into the gas; (2) the effect of IL-13, a prominent mediator of allergic inflammation, on NO release; and (3) inter-subject/donor variability in NO release. NHBE cells from three different donors were cultured at an air-liquid interface and stimulated with different concentrations of IL-13 (0, 1, and 10 ng/ml) for 48 h. Gas phase NO concentrations in the headspace over the cells were measured using a chemiluminescence analyzer. The basal NO flux from the three donors (0.05 +/- 0.03) is similar in magnitude to that estimated from exhaled NO concentrations, and was significantly increased by IL-13 in a donor-specific fashion. The increase in NO release was strongly correlated with inducible nitric oxide synthase (iNOS) gene and protein expression. There was a trend toward enhanced production of nitrate relative to nitrite as an end product of NO metabolism in IL-13-stimulated cells. NO release from airway epithelial cells can be directly measured. The rate of release in response to IL-13 is strongly dependent on the individual donor, but is primarily due to the expression of iNOS.

  12. Chlamydophila spp. infection in horses with recurrent airway obstruction: similarities to human chronic obstructive disease

    Directory of Open Access Journals (Sweden)

    Hotzel Helmut

    2008-01-01

    Full Text Available Abstract Background Recurrent airway obstruction (RAO in horses is a naturally occurring dust-induced disease mainly characterized by bronchiolitis which shows histological and pathophysiological similarities to human chronic obstructive pulmonary disease (COPD. In human COPD previous investigations indicated an association with Chlamydophila psittaci infection. The present study was designed (1 to clarify a possible role of this infectious agent in RAO and (2 to investigate the suitability of this equine disorder as a model for human COPD. Methods Clinico-pathological parameters of a total of 45 horses (25 horses with clinical signs of RAO and 20 clinically healthy controls were compared to histological findings in lung tissue samples and infection by Chlamydiaceae using light microscopy, immunohistochemistry, and PCR. Results Horses with clinical signs of RAO vs. controls revealed more inflammatory changes in histology (p = 0.01, and a higher detection rate of Chlamydia psittaci antigens in all cells (p OmpA sequencing identified Chlamydophila psittaci (n = 9 and Chlamydophila abortus (n = 13 in both groups with no significant differences. Within the group of clinically healthy horses subgroups with no changes (n = 15 and slight inflammation of the small airways (n = 5 were identified. Also in the group of animals with RAO subgroups with slight (n = 16 and severe (n = 9 bronchiolitis could be formed. These four subgroups can be separated in parts by the number of cells positive for Chlamydia psittaci antigens. Conclusion Chlamydophila psittaci or abortus were present in the lung of both clinically healthy horses and those with RAO. Immunohistochemistry revealed acute chlamydial infections with inflammation in RAO horses, whereas in clinically healthy animals mostly persistent chlamydial infection and no inflammatory reactions were seen. Stable dust as the known fundamental abiotic factor in RAO is comparable to smoking in human disease. These

  13. Test of the Starling resistor model in the human upper airway during sleep.

    Science.gov (United States)

    Wellman, Andrew; Genta, Pedro R; Owens, Robert L; Edwards, Bradley A; Sands, Scott A; Loring, Stephen H; White, David P; Jackson, Andrew C; Pedersen, Ole F; Butler, James P

    2014-12-15

    The human pharyngeal airway during sleep is conventionally modeled as a Starling resistor. However, inspiratory flow often decreases with increasing effort (negative effort dependence, NED) rather than remaining fixed as predicted by the Starling resistor model. In this study, we tested a major prediction of the Starling resistor model--that the resistance of the airway upstream from the site of collapse remains fixed during flow limitation. During flow limitation in 24 patients with sleep apnea, resistance at several points along the pharyngeal airway was measured using a pressure catheter with multiple sensors. Resistance between the nose and the site of collapse (the upstream segment) was measured before and after the onset of flow limitation to determine whether the upstream dimensions remained fixed (as predicted by the Starling resistor model) or narrowed (a violation of the Starling resistor model). The upstream resistance from early to mid inspiration increased considerably during flow limitation (by 35 ± 41 cmH2O · liter(-1) · s(-1), P < 0.001). However, there was a wide range of variability between patients, and the increase in upstream resistance was strongly correlated with the amount of NED (r = 0.75, P < 0.001). Therefore, patients with little NED exhibited little upstream narrowing (consistent with the Starling model), and patients with large NED exhibited large upstream narrowing (inconsistent with the Starling model). These findings support the idea that there is not a single model of pharyngeal collapse, but rather that different mechanisms may dominate in different patients. These differences could potentially be exploited for treatment selection. Copyright © 2014 the American Physiological Society.

  14. SRC-mediated EGF Receptor Activation Regulates Ozone-induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

    Science.gov (United States)

    BACKGROUND: Human exposure to ozone (03) results in pulmonary function decrements and airway inflammation. The mechanisms underlying these adverse effects remain unclear. Epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of lung inflammation. ...

  15. Translocation of SiO2-NPs across in vitro human bronchial epithelial monolayer

    International Nuclear Information System (INIS)

    George, I; Vranic, S; Boland, S; Borot, M C; Marano, F; Baeza-Squiban, A

    2013-01-01

    Safe development and application of nanotechnologies in many fields require better knowledge about their potential adverse effects on human health. Evidence of abilities of nanoparticles (NPs) to cross epithelial barriers and reach secondary organs via the bloodstream led us to investigate the translocation of SiO 2 NPs of 50 nm (50 nm-SiO 2 -NPs) across human bronchial epithelial cells that are primary targets after exposure to inhaled NPs. We quantified the translocation of fluorescently labelled SiO 2 NPs at non-cytotoxic concentrations (5 and 10 μg/cm 2 ) across Calu-3 epithelial monolayer. After 14 days in culture Calu-3 cells seeded onto 3 μm-polycarbonate Transwell membranes formed an efficient bronchial barrier assessed by measurement of the transepithelial electric resistance and quantification of the permeability of the monolayer. After 24 hours of exposure, we observed a significant translocation of NPs that was more important when the initial NP concentration decreased. Confocal microscopy observations revealed NP uptake by cells and an important NP retention inside the porous membrane. In conclusion, 50 nm-SiO 2 -NPs can cross the human bronchial epithelial barrier without affecting the integrity of the epithelial cell monolayer.

  16. Differential growth factor induction and modulation of human gastric epithelial regeneration

    International Nuclear Information System (INIS)

    Tetreault, Marie-Pier; Chailler, Pierre; Rivard, Nathalie; Menard, Daniel

    2005-01-01

    While several autocrine/paracrine growth factors (GFs) can all stimulate epithelial regeneration in experimentally wounded primary gastric cultures, clinical relevance for their non-redundant cooperative actions in human gastric ulcer healing is suggested by the sequential pattern of GF gene induction in vivo. Using new HGE cell lines able to form a coherent monolayer with tight junctions as well as using primary human gastric epithelial cultures, we show that EGF, TGFα, HGF and IGFs accelerate epithelial restitution upon wounding, independently of the TGFβ pathway (as opposed to intestinal cells). However, they differently modulate cell behavior: TGFα exerts strong effects (even more than EGF) on cytoplasmic spreading and non-oriented protruding activity of bordering cells whereas HGF preferentially coordinates single lamella formation, cell elongation and migration into the wound. IGF-I and IGF-II rather induce the alignment of bordering cells and maintain a compact monolayer front. The number of mitotic cells maximally increases with EGF, followed by TGFα and IGF-I,-II. The current study demonstrates that GFs differentially regulate the regeneration of human gastric epithelial cells through specific modulation of cell shape adaptation, migration and proliferation, further stressing that a coordination of GF activities would be necessary for the normal progression of post-wounding epithelial repair

  17. Effects of gasoline and ethanol-gasoline exhaust exposure on human bronchial epithelial and natural killer cells in vitro.

    Science.gov (United States)

    Roth, Michèle; Usemann, Jakob; Bisig, Christoph; Comte, Pierre; Czerwinski, Jan; Mayer, Andreas C R; Beier, Konstantin; Rothen-Rutishauser, Barbara; Latzin, Philipp; Müller, Loretta

    2017-12-01

    Air pollution exposure, including passenger car emissions, may cause substantial respiratory health effects and cancer death. In western countries, the majority of passenger cars are driven by gasoline fuel. Recently, new motor technologies and ethanol fuels have been introduced to the market, but potential health effects have not been thoroughly investigated. We developed and verified a coculture model composed of bronchial epithelial cells (ECs) and natural killer cells (NKs) mimicking the human airways to compare toxic effects between pure gasoline (E0) and ethanol-gasoline-blend (E85, 85% ethanol, 15% gasoline) exhaust emitted from a flexfuel gasoline car. We drove a steady state cycle, exposed ECs for 6h and added NKs. We assessed exhaust effects in ECs alone and in cocultures by RT-PCR, flow cytometry, and oxidative stress assay. We found no toxic effects after exposure to E0 or E85 compared to air controls. Comparison between E0 and E85 exposure showed a weak association for less oxidative DNA damage after E85 exposure compared to E0. Our results indicate that short-term exposure to gasoline exhaust may have no major toxic effects in ECs and NKs and that ethanol as part of fuel for gasoline cars may be favorable. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures.

    Science.gov (United States)

    Schlage, Walter K; Iskandar, Anita R; Kostadinova, Radina; Xiang, Yang; Sewer, Alain; Majeed, Shoaib; Kuehn, Diana; Frentzel, Stefan; Talikka, Marja; Geertz, Marcel; Mathis, Carole; Ivanov, Nikolai; Hoeng, Julia; Peitsch, Manuel C

    2014-10-01

    Smoking has been associated with diseases of the lung, pulmonary airways and oral cavity. Cytologic, genomic and transcriptomic changes in oral mucosa correlate with oral pre-neoplasia, cancer and inflammation (e.g. periodontitis). Alteration of smoking-related gene expression changes in oral epithelial cells is similar to that in bronchial and nasal epithelial cells. Using a systems toxicology approach, we have previously assessed the impact of cigarette smoke (CS) seen as perturbations of biological processes in human nasal and bronchial organotypic epithelial culture models. Here, we report our further assessment using in vitro human oral organotypic epithelium models. We exposed the buccal and gingival organotypic epithelial tissue cultures to CS at the air-liquid interface. CS exposure was associated with increased secretion of inflammatory mediators, induction of cytochrome P450s activity and overall weak toxicity in both tissues. Using microarray technology, gene-set analysis and a novel computational modeling approach leveraging causal biological network models, we identified CS impact on xenobiotic metabolism-related pathways accompanied by a more subtle alteration in inflammatory processes. Gene-set analysis further indicated that the CS-induced pathways in the in vitro buccal tissue models resembled those in the in vivo buccal biopsies of smokers from a published dataset. These findings support the translatability of systems responses from in vitro to in vivo and demonstrate the applicability of oral organotypical tissue models for an impact assessment of CS on various tissues exposed during smoking, as well as for impact assessment of reduced-risk products.

  19. Response of cultured normal human mammary epithelial cells to X rays

    International Nuclear Information System (INIS)

    Yang, T.C.; Stampfer, M.R.; Smith, H.S.

    1983-01-01

    The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D 0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique

  20. Human epithelial cells increase their rigidity with ageing in vitro: direct measurements

    International Nuclear Information System (INIS)

    Berdyyeva, Tamara K; Woodworth, Craig D; Sokolov, Igor

    2005-01-01

    The decrease in elasticity of epithelial tissues with ageing contributes to many human diseases. This change was previously attributed to increased crosslinking of extracellular matrix proteins. Here we show that individual human epithelial cells also become significantly more rigid during ageing in vitro. Using atomic force microscopy (AFM), we found that the Young's modulus of viable cells was consistently increased two- to four-fold in older versus younger cells. Direct visualization of the cytoskeleton using a novel method involving the AFM suggested that increased rigidity of ageing cells was due to a higher density of cytoskeletal fibres. Our results identify a unique mechanism that might contribute to the age-related loss of elasticity in epithelial tissues

  1. Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: Implications for human health.

    Science.gov (United States)

    Xiang, Ping; Liu, Rong-Yan; Li, Chao; Gao, Peng; Cui, Xin-Yi; Ma, Lena Q

    2017-11-01

    Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is one of the most detected organophosphorus flame retardants (OPFRs) in the environment, especially in indoor dust. Continuous daily exposure to TDCPP-containing dust may adversely impact human cornea. However, its detrimental effects on human corneal epithelium are largely unknown. In this study, we investigated the cell apoptosis in normal human corneal epithelial cells (HCECs) after TDCPP exposure and elucidated the underlying molecular mechanisms. Our data indicated a dose-dependent decrease of cell viability after TDCPP exposure with LC 50 at 202 μg/mL. A concentration-dependent apoptotic sign was observed in HCECs after exposing to ≥2 μg/mL TDCPP. Endoplasmic reticulum stress induction was evidenced by up-regulation of its biomarker genes (ATF-4, CHOP, BiP, and XBP1). Furthermore, alternation of Bcl-2/Bax expression, mitochondrial membrane potential loss, cellular ATP content decrease, and caspase-3 and -9 activity increase were observed after exposing to 2 or 20 μg/mL TDCPP. Taken together, the data implicated the involvement of endoplasmic reticulum stress in TDCPP-induced HCEC apoptosis, probably mediated by mitochondrial apoptotic pathway. Our findings showed TDCPP exposure induced toxicity to human cornea. Due to TDCPP's presence at high levels in indoor dust, further study is warranted to evaluate its health risk on human corneas. Published by Elsevier Ltd.

  2. Chronic Exposure to Particulate Nickel Induces Neoplastic Transformation in Human Lung Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Amie L. Holmes

    2013-11-01

    Full Text Available Nickel is a well-known human lung carcinogen with the particulate form being the most potent; however, the carcinogenic mechanism remains largely unknown. Few studies have investigated the genotoxicity and carcinogenicity of nickel in its target cell, human bronchial epithelial cells. Thus, the goal of this study was to investigate the effects of particulate nickel in human lung epithelial cells. We found that nickel subsulfide induced concentration- and time-dependent increases in both cytotoxicity and genotoxicity in human lung epithelial cells (BEP2D. Chronic exposure to nickel subsulfide readily induced cellular transformation, inducing 2.55, 2.9 and 2.35 foci per dish after exposure to 1, 2.5 and 5 μg/cm2 nickel subsulfide, respectively. Sixty-one, 100 and 70 percent of the foci isolated from 1, 2.5, and 5 μg/cm2 nickel subsulfide treatments formed colonies in soft agar and the degree of soft agar colony growth increased in a concentration-dependent manner. Thus, chronic exposure to particulate nickel induces genotoxicity and cellular transformation in human lung epithelial cells.

  3. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs.

  4. Chromosomal changes in cultured human epithelial cells transformed by low- and high-LET radiation

    International Nuclear Information System (INIS)

    Yang, Tracy Chui-hsu; Craise, L.M; Prioleau, J.C.; Stampfer, M.R.; Rhim, J.S.

    1990-11-01

    For a better assessment of radiation risk in space, an understanding of the responses of human cells, especially the epithelial cells, to low- and high-LET radiation is essential. In our laboratory, we have successfully developed techniques to study the neoplastic transformation of two human epithelial cell systems by ionizing radiation. These cell systems are human mammary epithelial cells (H184B5) and human epidermal keratinocytes (HEK). Both cell lines are immortal, anchorage dependent for growth, and nontumorigenic in athymic nude nice. Neoplastic transformation was achieved by irradiation cells successively. Our results showed that radiogenic cell transformation is a multistep process and that a single exposure of ionizing radiation can cause only one step of transformation. It requires, therefore, multihits to make human epithelial cells fully tumorigenic. Using a simple karyotyping method, we did chromosome analysis with cells cloned at various stages of transformation. We found no consistent large terminal deletion of chromosomes in radiation-induced transformants. Some changes of total number of chromosomes, however, were observed in the transformed cells. These transformants provide an unique opportunity for further genetic studies at a molecular level. 15 refs., 9 figs., 2 tabs

  5. Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia

    Directory of Open Access Journals (Sweden)

    Nagayasu Egawa

    2015-07-01

    Full Text Available Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted.

  6. Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia

    Science.gov (United States)

    Egawa, Nagayasu; Egawa, Kiyofumi; Griffin, Heather; Doorbar, John

    2015-01-01

    Papillomaviruses have evolved over many millions of years to propagate themselves at specific epithelial niches in a range of different host species. This has led to the great diversity of papillomaviruses that now exist, and to the appearance of distinct strategies for epithelial persistence. Many papillomaviruses minimise the risk of immune clearance by causing chronic asymptomatic infections, accompanied by long-term virion-production with only limited viral gene expression. Such lesions are typical of those caused by Beta HPV types in the general population, with viral activity being suppressed by host immunity. A second strategy requires the evolution of sophisticated immune evasion mechanisms, and allows some HPV types to cause prominent and persistent papillomas, even in immune competent individuals. Some Alphapapillomavirus types have evolved this strategy, including those that cause genital warts in young adults or common warts in children. These strategies reflect broad differences in virus protein function as well as differences in patterns of viral gene expression, with genotype-specific associations underlying the recent introduction of DNA testing, and also the introduction of vaccines to protect against cervical cancer. Interestingly, it appears that cellular environment and the site of infection affect viral pathogenicity by modulating viral gene expression. With the high-risk HPV gene products, changes in E6 and E7 expression are thought to account for the development of neoplasias at the endocervix, the anal and cervical transformation zones, and the tonsilar crypts and other oropharyngeal sites. A detailed analysis of site-specific patterns of gene expression and gene function is now prompted. PMID:26193301

  7. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    Science.gov (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  8. Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo

    International Nuclear Information System (INIS)

    Larios-Rodriguez, E; Rangel-Ayon, C; Herrera-Urbina, R; Castillo, S J; Zavala, G

    2011-01-01

    Healthy epithelial cells, in vivo, have the ability to synthesize gold nanoparticles when aqueous tetrachloroauric acid is made to react with human skin. Neither a reducing agent nor a protecting chemical is needed for this bio-synthesis method. The first indication of gold nanoparticle formation is the staining of the skin, which turns deep purple. Stereoscopic optical micrographs of human skin tissue in contact with aqueous tetrachloroauric acid clearly show the staining of the epithelial cells. The UV-Vis spectrum of these epithelial cells shows an absorption band with a maximum at 553 nm. This absorption peak is within the wavelength region where the surface plasmon resonance (SPR) band of aqueous colloidal gold exhibits a maximum. Transmission electron micrographs show that gold nanoparticles synthesized by epithelial cells have sizes between 1 and 100 nm. The electron diffraction pattern of these nanoparticles reveals a crystalline structure whose interplanar distances correspond to fcc metallic gold. Transmission electron micrographs of ultra-thin (70 nm thick) slices of epithelial cells clearly and undoubtedly demonstrate that gold nanoparticles are inside the cell. According to high resolution transmission electron micrographs of intracellular single gold nanoparticles, they have the shape of a polyhedron.

  9. Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Larios-Rodriguez, E; Rangel-Ayon, C; Herrera-Urbina, R [Departamento de Ingenieria Quimica y Metalurgia, Universidad de Sonora, Rosales y Luis Encinas S/N, Hermosillo, Sonora, C.P. 83000 (Mexico); Castillo, S J [Departamento de Investigacion en Fisica, Universidad de Sonora, Rosales y Luis Encinas S/N, Hermosillo, Sonora, C.P. 83000 (Mexico); Zavala, G, E-mail: elarios@polimeros.uson.mx [Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos (Mexico)

    2011-09-02

    Healthy epithelial cells, in vivo, have the ability to synthesize gold nanoparticles when aqueous tetrachloroauric acid is made to react with human skin. Neither a reducing agent nor a protecting chemical is needed for this bio-synthesis method. The first indication of gold nanoparticle formation is the staining of the skin, which turns deep purple. Stereoscopic optical micrographs of human skin tissue in contact with aqueous tetrachloroauric acid clearly show the staining of the epithelial cells. The UV-Vis spectrum of these epithelial cells shows an absorption band with a maximum at 553 nm. This absorption peak is within the wavelength region where the surface plasmon resonance (SPR) band of aqueous colloidal gold exhibits a maximum. Transmission electron micrographs show that gold nanoparticles synthesized by epithelial cells have sizes between 1 and 100 nm. The electron diffraction pattern of these nanoparticles reveals a crystalline structure whose interplanar distances correspond to fcc metallic gold. Transmission electron micrographs of ultra-thin (70 nm thick) slices of epithelial cells clearly and undoubtedly demonstrate that gold nanoparticles are inside the cell. According to high resolution transmission electron micrographs of intracellular single gold nanoparticles, they have the shape of a polyhedron.

  10. Structure of neuro-endocrine and neuro-epithelial interactions in human foetal pancreas.

    Science.gov (United States)

    Krivova, Yuliya; Proshchina, Alexandra; Barabanov, Valeriy; Leonova, Olga; Saveliev, Sergey

    2016-12-01

    In the pancreas of many mammals including humans, endocrine islet cells can be integrated with the nervous system components into neuro-insular complexes. The mechanism of the formation of such complexes is not clearly understood. The present study evaluated the interactions between the nervous system components, epithelial cells and endocrine cells in the human pancreas. Foetal pancreas, gestational age 19-23 weeks (13 cases) and 30-34 weeks (7 cases), were studied using double immunohistochemical labeling with neural markers (S100 protein and beta III tubulin), epithelial marker (cytokeratin 19 (CK19)) and antibodies to insulin and glucagon. We first analyse the structure of neuro-insular complexes using confocal microscopy and provide immunohistochemical evidences of the presence of endocrine cells within the ganglia or inside the nerve bundles. We showed that the nervous system components contact with the epithelial cells located in ducts or in clusters outside the ductal epithelium and form complexes with separate epithelial cells. We observed CK19-positive cells inside the ganglia and nerve bundles which were located separately or were integrated with the islets. Therefore, we conclude that neuro-insular complexes may forms as a result of integration between epithelial cells and nervous system components at the initial stages of islets formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Repair activity of oxidatively damaged DNA and telomere length in human lung epithelial cells after exposure to multi-walled carbon nanotubes

    DEFF Research Database (Denmark)

    Borghini, Andrea; Roursgaard, Martin; Andreassi, Maria Grazia

    2017-01-01

    One type of carbon nanotubes (CNTs) (MWCNT-7, from Mitsui) has been classified as probably carcinogenic to humans, however insufficient data does not warrant the same classification for other types of CNTs. Experimental data indicate that CNT exposure can result in oxidative stress and DNA damage...... the cells toward replicative senescence, assessed by attrition of telomeres. To investigate this, H2O2 and KBrO3 were used to induce DNA damage in the cells and the effect of pre-exposure to MWCNT tested for a change in repair activity inside the cells or in the extract of treated cells. The effect of MWCNT...... in cultured cells, whereas these materials appear to induce low or no mutagenicity. Therefore, the present study aimed to investigate whether in vitro exposure of cultured airway epithelial cells (A549) to multi-walled CNTs (MWCNTs) could increase the DNA repair activity of oxidatively damaged DNA and drive...

  12. Effect of Perinatal secondhand tobacco smoke exposure on in vivo and intrinsic airway structure/function in non-human primates

    International Nuclear Information System (INIS)

    Joad, Jesse P.; Kott, Kayleen S.; Bric, John M.; Peake, Janice L.; Pinkerton, Kent E.

    2009-01-01

    Infants exposed to second hand smoke (SHS) experience more problems with wheezing. This study was designed to determine if perinatal SHS exposure increases intrinsic and/or in vivo airway responsiveness to methacholine and whether potential structural/cellular alterations in the airway might explain the change in responsiveness. Pregnant rhesus monkeys were exposed to filtered air (FA) or SHS (1 mg/m 3 total suspended particulates) for 6 h/day, 5 days/week starting at 50 days gestational age. The mother/infant pairs continued the SHS exposures postnatally. At 3 months of age each infant: 1) had in vivo lung function measurements in response to inhaled methacholine, or 2) the right accessory lobe filled with agarose, precision-cut to 600 μm slices, and bathed in increasing concentrations of methacholine. The lumenal area of the central airway was determined using videomicrometry followed by fixation and histology with morphometry. In vivo tests showed that perinatal SHS increases baseline respiratory rate and decreases responsiveness to methacholine. Perinatal SHS did not alter intrinsic airway responsiveness in the bronchi. However in respiratory bronchioles, SHS exposure increased airway responsiveness at lower methacholine concentrations but decreased it at higher concentrations. Perinatal SHS did not change eosinophil profiles, epithelial volume, smooth muscle volume, or mucin volume. However it did increase the number of alveolar attachments in bronchi and respiratory bronchioles. In general, as mucin increased, airway responsiveness decreased. We conclude that perinatal SHS exposure alters in vivo and intrinsic airway responsiveness, and alveolar attachments

  13. Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

    Science.gov (United States)

    Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

    2012-01-01

    Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

  14. Overexpression of human sperm protein 17 increases migration and decreases the chemosensitivity of human epithelial ovarian cancer cells

    International Nuclear Information System (INIS)

    Li, Fang-qiu; Han, Yan-ling; Liu, Qun; Wu, Bo; Huang, Wen-bin; Zeng, Su-yun

    2009-01-01

    Most deaths from ovarian cancer are due to metastases that are resistant to conventional therapies. But the factors that regulate the metastatic process and chemoresistance of ovarian cancer are poorly understood. In the current study, we investigated the aberrant expression of human sperm protein 17 (HSp17) in human epithelial ovarian cancer cells and tried to analyze its influences on the cell behaviors like migration and chemoresistance. Immunohistochemistry and immunocytochemistry were used to identify HSp17 in paraffin embedded ovarian malignant tumor specimens and peritoneal metastatic malignant cells. Then we examined the effect of HSp17 overexpression on the proliferation, migration, and chemoresistance of ovarian cancer cells to carboplatin and cisplatin in a human ovarian carcinoma cell line, HO8910. We found that HSp17 was aberrantly expressed in 43% (30/70) of the patients with primary epithelial ovarian carcinomas, and in all of the metastatic cancer cells of ascites from 8 patients. The Sp17 expression was also detected in the metastatic lesions the same as in ovarian lesions. None of the 7 non-epithelial tumors primarily developed in the ovaries was immunopositive for HSp17. Overexpression of HSp17 increased the migration but decreased the chemosensitivity of ovarian carcinoma cells to carboplatin and cisplatin. HSp17 is aberrantly expressed in a significant proportion of epithelial ovarian carcinomas. Our results strongly suggest that HSp17 plays a role in metastatic disease and resistance of epithelial ovarian carcinoma to chemotherapy

  15. The protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Xue - Fang Chen

    2013-06-01

    Full Text Available AIM: To investigate the protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis. METHODS:Subcultured human lens epithelial cell line, ultraviolet induced cell apoptosis, 20μmol/L resveratrol pretreated cell, the indicators change was observed: rate of apoptosis was detected by flow cytometry and apoptosis-related factors of caspses-3 and caspase-9 were detected by colorimetric detection, ultrastructure changes were observed under transmission electron microscope. RESULTS: Flow cytometry instrument testing found that resveratrol can suppress the apoptosis induced by ultraviolet irradiation, caspses-3 and caspase-9 content in positive control group were significantly higher than that of the negative control group at the same time period, the difference was statistically significant(P<0.05; caspses-3 and caspase-9 content in experimental group were lower than that in the positive control group at the same time, the difference was statistically significant(P<0.05. In addition, the damage of human lens epithelial cells was alleviated with the incubation time of resveratrol elongated. CONCLUSION:Resveratrol may inhibit ultraviolet-induced apoptosis of human lens epithelial cells, it has preventive function against radioactive cataract, and it can provide reliable evidence for pursuing effective medicine to prevent and treat cataract.

  16. Research of transport and deposition of aerosol in human airway replica

    Science.gov (United States)

    Lizal, Frantisek; Jedelsky, Jan; Elcner, Jakub; Durdina, Lukas; Halasova, Tereza; Mravec, Filip; Jicha, Miroslav

    2012-04-01

    Growing concern about knowledge of aerosol transport in human lungs is caused by great potential of use of inhaled pharmaceuticals. Second substantial motive for the research is an effort to minimize adverse effects of particular matter emitted by traffic and industry on human health. We created model geometry of human lungs to 7th generation of branching. This model geometry was used for fabrication of two physical models. The first one is made from thin walled transparent silicone and it allows a measurement of velocity and size of aerosol particles by Phase Doppler Anemometry (PDA). The second one is fabricated by stereolithographic method and it is designed for aerosol deposition measurements. We provided a series of measurements of aerosol transport in the transparent model and we ascertained remarkable phenomena linked with lung flow. The results are presented in brief. To gather how this phenomena affects aerosol deposition in human lungs we used the second model and we developed a technique for deposition fraction and deposition efficiency assessment. The results confirmed that non-symmetric and complicated shape of human airways essentially affects transport and deposition of aerosol. The research will now focus on deeper insight in aerosol deposition.

  17. Research of transport and deposition of aerosol in human airway replica

    Directory of Open Access Journals (Sweden)

    Mravec Filip

    2012-04-01

    Full Text Available Growing concern about knowledge of aerosol transport in human lungs is caused by great potential of use of inhaled pharmaceuticals. Second substantial motive for the research is an effort to minimize adverse effects of particular matter emitted by traffic and industry on human health. We created model geometry of human lungs to 7th generation of branching. This model geometry was used for fabrication of two physical models. The first one is made from thin walled transparent silicone and it allows a measurement of velocity and size of aerosol particles by Phase Doppler Anemometry (PDA. The second one is fabricated by stereolithographic method and it is designed for aerosol deposition measurements. We provided a series of measurements of aerosol transport in the transparent model and we ascertained remarkable phenomena linked with lung flow. The results are presented in brief. To gather how this phenomena affects aerosol deposition in human lungs we used the second model and we developed a technique for deposition fraction and deposition efficiency assessment. The results confirmed that non-symmetric and complicated shape of human airways essentially affects transport and deposition of aerosol. The research will now focus on deeper insight in aerosol deposition.

  18. Macrophages are related to goblet cell hyperplasia and induce MUC5B but not MUC5AC in human bronchus epithelial cells.

    Science.gov (United States)

    Silva, Manuel A; Bercik, Premysl

    2012-06-01

    Airway goblet cell hyperplasia (GCH)--detectable by mucin staining--and abnormal macrophage infiltrate are pathological features present in many chronic respiratory disorders. However, it is unknown if both factors are associated. Using in-vivo and in-vitro models, we investigated whether macrophages are related with GCH and changes in mucin immunophenotypes. Lung sections from Sprague-Dawley rats treated for 48 h with one intra-tracheal dose of PBS or LPS (n=4-6 per group) were immunophenotyped for rat-goblet cells, immune, and proliferation markers. Human monocyte-derived macrophages (MDM) were pre-treated with or without LPS, immunophenotyped, and their supernatant, as well as cytokines at levels equivalent to supernatant were used to challenge primary culture of normal human bronchus epithelial cells (HBEC) in air-liquid interface, followed by MUC5B and MUC5AC mucin immunostaining. An association between increased bronchiolar goblet cells and terminal-bronchiolar proliferative epithelial cells confirmed the presence of GCH in our LPS rat model, which was related with augmented bronchiolar CD68 macrophage infiltration. The in-vitro experiments have shown that MUC5AC phenotype was inhibited when HBEC were challenged with supernatant from MDM pre-treated with or without LPS. In contrast, TNF-α and interleukin-1β at levels equivalent to supernatant from LPS-treated MDM increased MUC5AC. MUC5B was induced by LPS, supernatant from LPS-treated MDM, a mix of cytokines including TNF-α and TNF-α alone at levels present in supernatant from LPS-treated MDM. We demonstrated that macrophages are related with bronchiolar GCH, and that they induced MUC5B and inhibited MUC5AC in HBEC, suggesting a role for them in the pathogenesis of airway MUC5B-related GCH.

  19. Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

    Science.gov (United States)

    Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

    2007-11-23

    Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

  20. Lipopolysaccharide attenuates induction of pro-allergic cytokines, thymic stromal lymphopoietin and interleukin 33, in respiratory epithelial cells stimulated with polyI:C and human parechovirus

    Directory of Open Access Journals (Sweden)

    Tsang-Hsiung Lin

    2016-10-01

    Full Text Available Epidemiological studies based on the hygiene hypothesis declare that the level of childhood exposure to environmental microbial products is inversely related to the incidence of allergic diseases in later life. Multiple types of immune cell-mediated immune regulation networks support the hygiene hypothesis. Epithelial cells are the first line of response to microbial products in the environment and bridge the innate and adaptive immune systems; however, their role in the hygiene hypothesis is unknown. To demonstrate the hygiene hypothesis in airway epithelial cells, we examined the effect of lipopolysaccharide (LPS; Toll-like receptor 4 ligand on the expression of the proallergic cytokines thymic stromal lymphopoietin (TSLP and interleukin 33 (IL33 in H292 cells (pulmonary mucoepidermoid carcinoma cells. Stimulation with the TLR ligand polyI:C and human parechovirus type 1 (HPeV1 but not LPS induced TSLP and IL33 through interferon (IFN regulatory factor 3 (IRF3 and NFB activity, which was further validated by using inhibitors (dexamethasone and Bay 11-7082 and shRNA-mediated gene knockdown. Importantly, polyI:C and HPeV1-stimulated TSLP and IL33 induction was reduced by LPS treatment by attenuating TANK-binding kinase 1, IRF3 and NFB activation. Interestingly, the basal mRNA levels of TLR signaling proteins were downregulated with long-term LPS treatment of H292 cells, which suggests that such long-term exposure modulates the expression of innate immunity signaling molecules in airway epithelial cells to mitigate the allergic response. In contrast to the effects of LPS treatment, the alarmin HMBG1 (high mobility group protein B1 acts in synergy with polyI:C to promote TSLP and IL33 expression. Our data support part of the hygiene hypothesis in airway epithelia cells in vitro. In addition to therapeutic targeting of TSLP and IL33, local application of non-pathogenic LPS may be a rational strategy to prevent allergies.

  1. Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

    Science.gov (United States)

    Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

    2011-02-01

    Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

    Science.gov (United States)

    Ambhore, Nilesh Sudhakar; Katragadda, Rathnavali; Raju Kalidhindi, Rama Satyanarayana; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S; Sathish, Venkatachalem

    2018-04-20

    Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways

  3. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  4. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  5. Diesel exhaust particles increase IL-1β-induced human β-defensin expression via NF-κB-mediated pathway in human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Lee Chun

    2006-05-01

    Full Text Available Abstract Background Human β-defensin (hBD-2, antimicrobial peptide primarily induced in epithelial cells, is a key factor in the innate immune response of the respiratory tract. Several studies showed increased defensin levels in both inflammatory lung diseases, such as cystic fibrosis, diffuse panbronchiolitis, idiopathic pulmonary fibrosis and acute respiratory distress syndrome, and infectious diseases. Recently, epidemiologic studies have demonstrated acute and serious adverse effects of particulate air pollution on respiratory health, especially in people with pre-existing inflammatory lung disease. To elucidate the effect of diesel exhaust particles (DEP on pulmonary innate immune response, we investigated the hBD-2 and interleukin-8 (IL-8 expression to DEP exposure in interleukin-1 beta (IL-1β-stimulated A549 cells. Results IL-1β markedly up-regulated the hBD-2 promoter activity, and the subsequent DEP exposure increased dose-dependently the expression of hBD-2 and inflammatory cytokine IL-8 at the transcriptional level. In addition, DEP further induced the NF-κB activation in IL-1β-stimulated A549 cells more rapidly than in unstimulated control cells, which was showed by nuclear translocation of p65 NF-κB and degradation of IκB-α. The experiment using two NF-κB inhibitors, PDTC and MG132, confirmed that this increase of hBD-2 expression following DEP exposure was regulated through NF-κB-mediated pathway. Conclusion These results demonstrated that DEP exposure increases the expression of antimicrobial peptide and inflammatory cytokine at the transcriptional level in IL-1β-primed A549 epithelial cells and suggested that the increase is mediated at least partially through NF-κB activation. Therefore, DEP exposure may contribute to enhance the airway-responsiveness especially on the patients suffering from chronic respiratory disease.

  6. Platelets are a possible regulator of human endometrial re-epithelialization during menstruation.

    Science.gov (United States)

    Suginami, Koh; Sato, Yukiyasu; Horie, Akihito; Matsumoto, Hisanori; Kyo, Satoru; Araki, Yoshihiko; Konishi, Ikuo; Fujiwara, Hiroshi

    2017-01-01

    The human endometrium periodically breaks down and regenerates. As platelets have been reported to contribute to the tissue remodeling process, we examined the possible involvement of platelets in endometrial regeneration. The distribution of extravasating platelets throughout the menstrual cycle was immunohistochemically examined using human endometrial tissues. EM-E6/E7/hTERT cells, a human endometrial epithelial cell-derived immortalized cell line, were co-cultured with platelets, and the effects of platelets on the epithelialization response of EM-E6/E7/hTERT cells were investigated by attachment and permeability assays, immunohistochemical staining, and Western blot analysis. Immunohistochemical study showed numerous extravasated platelets in the subluminar stroma during the menstrual phase. The platelets promoted the cell-to-matrigel attachment of EM-E6/E7/hTERT cells concomitantly with the phosphorylation of focal adhesion kinase. They also promoted cell-to-cell contact among EM-E6/E7/hTERT cells in parallel with E-cadherin expression. These results indicate the possible involvement of platelets in the endometrial epithelial re-epithelialization process. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

    Science.gov (United States)

    2007-06-01

    human mammary epithelial cell types by human papilloma virus 16 e6 or e7. Proc Nat Acad Sci USA 1995; 92:3687-91. 54. Shay JW, Pereira-Smith OM, Wright...Liu X-L, Chu Q, Gao Q, Band V. Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 e6 or e7. Proc Nat Acad

  8. Divergent pro-inflammatory profile of human dendritic cells in response to commensal and pathogenic bacteria associated with the airway microbiota

    NARCIS (Netherlands)

    Larsen, J.M.; Steen-Jensen, D.B.; Laursen, J.M.; Sondergaard, J.N.; Musavian, H.S.; Butt, T.M.; Brix, S.

    2012-01-01

    Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties

  9. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Romain Ferru-Clément

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR is a chloride channel that is expressed on the apical plasma membrane (PM of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o- expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  10. Mechanism research of miR-181 regulating human lens epithelial cell apoptosis

    Directory of Open Access Journals (Sweden)

    Yu Qin

    2015-05-01

    Full Text Available AIM: To investigate the expression of miR-181 in the lens tissue of cataract and the regulating mechanism of miR-181 on apoptosis of human lens epithelial cell.METHODS:Real time q-PCR was used to measure the expression of miR-181 in the anterior lens capsules of age-related cataract and human lens epithelial cell apoptosis model. miR-181 mimic and inhibitor were transfected using Lipofectamine 2 000 to regulate the expression of miR-181, and then Real time q-PCR was used to verify transfection efficiency. Flow cytometry was used to detect the change of cell apoptosis rate. RESULTS: Compared with control group, the expression of miR-181 was significantly higher in both the anterior lens capsules of age-related cataract and human lens epithelial cell apoptosis model; the relative expression of miR-181 in lens epithelial cells transfected with miR-181 mimic was increased, whereas decreased in cells transfected with miR-181 inhibitor; the apoptosis rate of cells transfected with miR-181 mimic was increased, while reduced in miR-181 inhibitor group. Each result was statistically significant(PCONCLUSION: High expression of miR-181 is detected in anterior lens capsule of age-related cataract. miR-181 might play a certain role in the pathogenesis of cataract via promoting human lens epithelial cell apoptosis. miR-181 probably becomes a new approach for the nonoperative treatment of cataract, but the concrete mechanism still needs to be further studied.

  11. Characteristics of turbulent particle transport in human airways under steady and cyclic flows

    International Nuclear Information System (INIS)

    Jedelsky, Jan; Lizal, Frantisek; Jicha, Miroslav

    2012-01-01

    Highlights: ► PDA data allow to estimate PSD of particle velocity fluctuations in realistic model. ► PSD of micron-sized particles is independent of their size up to 700 Hz. ► Such particles follow air flow and turb. diffusion contributes to their deposition. ► Cyclic flow PSDs contain more TKE at high freq. than equivalent steady-flow PSDs. ► Exp. breathing phase differs from insp. phase at high frequency part of the spectra. - Abstract: Motion of monodispersed aerosol particles suspended in air flow has been studied on realistic transparent model of human airways using Phase Doppler Particle Analyser (P/DPA). Time-resolved velocity data for particles in size range 1–8 μm were processed using Fuzzy Slotting Technique to estimate the power spectral density (PSD) of velocity fluctuations. The optimum processing setup for our data was found and recommendations for future experiments to improve PSD quality were suggested. Typical PSD plots at mainstream positions of the trachea and the upper bronchi are documented and differences among (1) steady-flow regimes and equivalent cyclic breathing regimes, (2) inspiration and expiration breathing phase and (3) behaviour of particles of different sizes are described in several positions of the airway model. Systematically higher level of velocity fluctuations in the upper part of the frequency range (30–500 Hz) was found for cyclic flows in comparison with corresponding steady flows. Expiratory flows in both the steady and cyclic cases produce more high-frequency fluctuations compared to inspiratory flows. Negligible differences were found for flow of particles in the inspected size range 1–8 μm at frequencies below 500 Hz. This finding was explained by Stokes number analysis. Implied match of the air and particle flows thereby indicates turbulent diffusion as important deposition mechanism and confirms the capability to use the P/DPA data as the air flow velocity estimate.

  12. Human Lung Mast Cell Products Regulate Airway Smooth Muscle CXCL10 Levels.

    Science.gov (United States)

    Alkhouri, H; Cha, V; Tong, K; Moir, L M; Armour, C L; Hughes, J M

    2014-01-01

    In asthma, the airway smooth muscle (ASM) produces CXCL10 which may attract CXCR3(+) mast/T cells to it. Our aim was to investigate the effects of mast cell products on ASM cell CXCL10 production. ASM cells from people with and without asthma were stimulated with IL-1 β , TNF- α , and/or IFN γ and treated with histamine (1-100  μ M) ± chlorpheniramine (H1R antagonist; 1  μ M) or ranitidine (H2R antagonist; 50  μ M) or tryptase (1 nM) ± leupeptin (serine protease inhibitor; 50  μ M), heat-inactivated tryptase, or vehicle for 4 h or 24 h. Human lung mast cells (MC) were isolated and activated with IgE/anti-IgE and supernatants were collected after 2 h or 24 h. The supernatants were added to ASM cells for 48 h and ASM cell CXCL10 production detected using ELISA (protein) and real-time PCR (mRNA). Histamine reduced IL-1 β /TNF- α -induced CXCL10 protein, but not mRNA, levels independent of H1 and H2 receptor activation, whereas tryptase and MC 2 h supernatants reduced all cytokine-induced CXCL10. Tryptase also reduced CXCL10 levels in a cell-free system. Leupeptin inhibited the effects of tryptase and MC 2 h supernatants. MC 24 h supernatants contained TNF- α and amplified IFN γ -induced ASM cell CXCL10 production. This is the first evidence that MC can regulate ASM cell CXCL10 production and its degradation. Thus MC may regulate airway myositis in asthma.

  13. MicroRNA Mediated Chemokine Responses in Human Airway Smooth Muscle Cells.

    Directory of Open Access Journals (Sweden)

    Mythili Dileepan

    Full Text Available Airway smooth muscle (ASM cells play a critical role in the pathophysiology of asthma due to their hypercontractility and their ability to proliferate and secrete inflammatory mediators. microRNAs (miRNAs are gene regulators that control many signaling pathways and thus serve as potential therapeutic alternatives for many diseases. We have previously shown that miR-708 and miR-140-3p regulate the MAPK and PI3K signaling pathways in human ASM (HASM cells following TNF-α exposure. In this study, we investigated the regulatory effect of these miRNAs on other asthma-related genes. Microarray analysis using the Illumina platform was performed with total RNA extracted from miR-708 (or control miR-transfected HASM cells. Inhibition of candidate inflammation-associated gene expression was further validated by qPCR and ELISA. The most significant biologic functions for the differentially expressed gene set included decreased inflammatory response, cytokine expression and signaling. qPCR revealed inhibition of expression of CCL11, CXCL10, CCL2 and CXCL8, while the release of CCL11 was inhibited in miR-708-transfected cells. Transfection of cells with miR-140-3p resulted in inhibition of expression of CCL11, CXCL12, CXCL10, CCL5 and CXCL8 and of TNF-α-induced CXCL12 release. In addition, expression of RARRES2, CD44 and ADAM33, genes known to contribute to the pathophysiology of asthma, were found to be inhibited in miR-708-transfected cells. These results demonstrate that miR-708 and miR-140-3p exert distinct effects on inflammation-associated gene expression and biological function of ASM cells. Targeting these miRNA networks may provide a novel therapeutic mechanism to down-regulate airway inflammation and ASM proliferation in asthma.

  14. Endogenous laminin is required for human airway smooth muscle cell maturation

    Directory of Open Access Journals (Sweden)

    Tran Thai

    2006-09-01

    Full Text Available Abstract Background Airway smooth muscle (ASM contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells. Methods Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured. Results Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype. Conclusion While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the

  15. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    Science.gov (United States)

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  16. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform

    Directory of Open Access Journals (Sweden)

    Kenji Kozuka

    2017-12-01

    Full Text Available Summary: We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound. : Siegel and colleagues describe their development of a human and mouse intestinal epithelial cell monolayer platform that maintains the cellular, molecular, and functional characteristics of tissue for each intestinal segment. They demonstrate the platform's application to drug discovery by screening a library of over 2,000 compounds to identify an inhibitor of potassium ion transport in the mouse distal colon. Keywords: intestinal epithelium, organoids, monolayer, colon, small intestine, phenotype screening assays, enteroid, colonoid

  17. The effect of inhaled menthol on upper airway resistance in humans: A randomized controlled crossover study

    Directory of Open Access Journals (Sweden)

    Effie J Pereira

    2013-01-01

    Full Text Available BACKGROUND: Menthol (l-menthol is a naturally-occurring cold receptor agonist commonly used to provide symptomatic relief for upper airway congestion. Menthol can also reduce the sensation of dyspnea. It is unclear whether the physiological action of menthol in dyspnea reduction is through its cold receptor agonist effect or whether associated mechanical changes occur in the upper airway.

  18. Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

    Science.gov (United States)

    Ayaki, Masahiko; Yaguchi, Shigeo; Iwasawa, Atsuo; Koide, Ryohei

    2008-08-01

    The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

  19. Altered ion transport in normal human bronchial epithelial cells following exposure to chemically distinct metal welding fume particles.

    Science.gov (United States)

    Fedan, Jeffrey S; Thompson, Janet A; Meighan, Terence G; Zeidler-Erdely, Patti C; Antonini, James M

    2017-07-01

    Welding fume inhalation causes pulmonary toxicity, including susceptibility to infection. We hypothesized that airway epithelial ion transport is a target of fume toxicity, and investigated the effects of fume particulates from manual metal arc-stainless steel (MMA-SS) and gas metal arc-mild steel (GMA-MS) on ion transport in normal human bronchial epithelium (NHBE) cultured in air-interface. MMA-SS particles, more soluble than GMA-MS particles, contain Cr, Ni, Fe and Mn; GMA-MS particles contain Fe and Mn. MMA-SS or GMA-MS particles (0.0167-166.7μg/cm 2 ) were applied apically to NHBEs. After 18h transepithelial potential difference (V t ), resistance (R t ), and short circuit current (I sc ) were measured. Particle effects on Na + and Cl¯ channels and the Na + ,K + ,2Cl¯-cotransporter were evaluated using amiloride (apical), 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, apical), and bumetanide (basolateral), respectively. MMA-SS (0.0167-16.7μg/cm 2 ) increased basal V t . Only 16.7μg/cm 2 GMA-MS increased basal V t significantly. MMA-SS or GMA-MS exposure potentiated I sc responses (decreases) to amiloride and bumetanide, while not affecting those to NPPB, GMA-MS to a lesser degree than MMA-SS. Variable effects on R t were observed in response to amiloride, and bumetanide. Generally, MMA-SS was more potent in altering responses to amiloride and bumetanide than GMA-MS. Hyperpolarization occurred in the absence of LDH release, but decreases in V t , R t , and I sc at higher fume particulate doses accompanied LDH release, to a greater extent for MMA-SS. Thus, Na + transport and Na + ,K + ,2Cl¯-cotransport are affected by fume exposure; MMA-MS is more potent than GMA-MS. Enhanced Na + absorption and decreased airway surface liquid could compromise defenses against infection. Published by Elsevier Inc.

  20. Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

    Science.gov (United States)

    Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

    2013-01-01

    The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

  1. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    Science.gov (United States)

    2012-04-01

    control expression of many of these miRNA genes. Many of the epigenetically regulated miRNAs identified are deregulated in breast cancer-derived...review board and Health Insurance Portability and Accountability Act regulations. At the time of surgery, a 1 to 3 cm section of the tumor was immediately...transformation process for- ward; the early deregulation of the HOX gene family clusters, which are decisively linked to human carcinogenesis, are one clear

  2. Aldose reductase inhibition prevents allergic airway remodeling through PI3K/AKT/GSK3β pathway in mice.

    Directory of Open Access Journals (Sweden)

    Umesh C S Yadav

    Full Text Available Long-term and unresolved airway inflammation and airway remodeling, characteristic features of chronic asthma, if not treated could lead to permanent structural changes in the airways. Aldose reductase (AR, an aldo-sugar and lipid aldehyde metabolizing enzyme, mediates allergen-induced airway inflammation in mice, but its role in the airway remodeling is not known. In the present study, we have examined the role of AR on airway remodeling using ovalbumin (OVA-induced chronic asthma mouse model and cultured human primary airway epithelial cells (SAECs and mouse lung fibroblasts (mLFs.Airway remodeling in chronic asthma model was established in mice sensitized and challenged twice a week with OVA for 6 weeks. AR inhibitor, fidarestat, was administered orally in drinking water after first challenge. Inflammatory cells infiltration in the lungs and goblet cell metaplasia, airway thickening, collagen deposition and airway hyper-responsiveness (AHR in response to increasing doses of methacholine were assessed. The TGFβ1-induced epithelial-mesenchymal transition (EMT in SAECs and changes in mLFs were examined to investigate AR-mediated molecular mechanism(s of airway remodeling.In the OVA-exposed mice for 6 wks inflammatory cells infiltration, levels of inflammatory cytokines and chemokines, goblet cell metaplasia, collagen deposition and AHR were significantly decreased by treatment with AR inhibitor, fidarestat. Further, inhibition of AR prevented TGFβ1-induced altered expression of E-cadherin, Vimentin, Occludin, and MMP-2 in SAECs, and alpha-smooth muscle actin and fibronectin in mLFs. Further, in SAECs, AR inhibition prevented TGFβ1- induced activation of PI3K/AKT/GSK3β pathway but not the phosphorylation of Smad2/3.Our results demonstrate that allergen-induced airway remodeling is mediated by AR and its inhibition blocks the progression of remodeling via inhibiting TGFβ1-induced Smad-independent and PI3K/AKT/GSK3β-dependent pathway.

  3. IL-13 regulates human nasal epithelial cell differentiation via H3K4me3 modification

    Directory of Open Access Journals (Sweden)

    Yu L

    2018-01-01

    Full Text Available Lei Yu,1 Na Li,1 Jisheng Zhang,2 Yan Jiang1 1Department of Otorhinolaryngology, 2Key Laboratory of Otolaryngology-Head and Neck Surgery, Affiliated Hospital of Qingdao University, Qingdao, China Introduction: Epigenetic regulation has been shown to play an important role in the development of inflammatory diseases, including chronic rhinosinusitis and nasal polyps. The latter are characterized by epithelial mis-differentiation and infiltration of inflammatory cytokines. H3K4me3 has been shown to be involved in regulating lineage commitment. However, the underlying mechanisms, especially in human nasal epithelial cells (HNEpC, remain underexplored. The objective of this study was to investigate the role of H3K4me3 in HNEpC differentiation treated with the Th2 cytokine IL-13. Patients and methods: The expression levels of mRNA and proteins were investigated using reverse transcription-polymerase chain reaction (RT-PCR assays and Western blot in nasal polyp tissues and human nasal epithelial cells respectively. We measured these levels of H3K4me3, MLL1 and targeted genes compared with control subjects.Results: We demonstrate that expression of H3K4me3 and its methyltransferase MLL1 was significantly upregulated in IL-13-treated HNEpC. This elevation was also observed in nasal polyps. Expression of cilia-related transcription factors FOXJ1 and DNAI2 decreased, while goblet cell-derived genes CLCA1 and MUC5a increased upon IL-13 treatment. Mechanistically, knockdown of MLL1 restored expression of these four genes induced by IL-13. Conclusion: These findings suggest that H3K4me3 is a critical regulator in control of nasal epithelial cell differentiation. MLL1 may be a potential therapeutic target for nasal inflammatory diseases. Keywords: IL-13, H3K4me3 modification, nasal epithelial cell, differentiation 

  4. Epigenetic influences of low-dose bisphenol A in primary human breast epithelial cells

    International Nuclear Information System (INIS)

    Weng, Yu-I; Hsu, Pei-Yin; Liyanarachchi, Sandya; Liu, Joseph; Deatherage, Daniel E.; Huang Yiwen; Zuo Tao; Rodriguez, Benjamin; Lin, Ching-Hung; Cheng, Ann-Lii; Huang, Tim H.-M.

    2010-01-01

    Substantial evidence indicates that exposure to bisphenol A (BPA) during early development may increase breast cancer risk later in life. The changes may persist into puberty and adulthood, suggesting an epigenetic process being imposed in differentiated breast epithelial cells. The molecular mechanisms by which early memory of BPA exposure is imprinted in breast progenitor cells and then passed onto their epithelial progeny are not well understood. The aim of this study was to examine epigenetic changes in breast epithelial cells treated with low-dose BPA. We also investigated the effect of BPA on the ERα signaling pathway and global gene expression profiles. Compared to control cells, nuclear internalization of ERα was observed in epithelial cells preexposed to BPA. We identified 170 genes with similar expression changes in response to BPA. Functional analysis confirms that gene suppression was mediated in part through an ERα-dependent pathway. As a result of exposure to BPA or other estrogen-like chemicals, the expression of lysosomal-associated membrane protein 3 (LAMP3) became epigenetically silenced in breast epithelial cells. Furthermore, increased DNA methylation in the LAMP3 CpG island was this repressive mark preferentially occurred in ERα-positive breast tumors. These results suggest that the in vitro system developed in our laboratory is a valuable tool for exposure studies of BPA and other xenoestrogens in human cells. Individual and geographical differences may contribute to altered patterns of gene expression and DNA methylation in susceptible loci. Combination of our exposure model with epigenetic analysis and other biochemical assays can give insight into the heritable effect of low-dose BPA in human cells.

  5. N-Myc Drives Neuroendocrine Prostate Cancer Initiated from Human Prostate Epithelial Cells

    Science.gov (United States)

    Lee, John K.; Phillips, John W.; Smith, Bryan A.; Park, Jung Wook; Stoyanova, Tanya; McCaffrey, Erin F.; Baertsch, Robert; Sokolov, Artem; Meyerowitz, Justin G.; Mathis, Colleen; Cheng, Donghui; Stuart, Joshua M.; Shokat, Kevan M.; Gustafson, W. Clay; Huang, Jiaoti; Witte, Owen N.

    2016-01-01

    SUMMARY MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention. PMID:27050099

  6. Transplantation of human amniotic epithelial cells repairs brachial plexus injury:pathological and biomechanical analyses

    Institute of Scientific and Technical Information of China (English)

    Qi Yang; Min Luo; Peng Li; Hai Jin

    2014-01-01

    A brachial plexus injury model was established in rabbits by stretching the C6 nerve root. Imme-diately after the stretching, a suspension of human amniotic epithelial cells was injected into the injured brachial plexus. The results of tensile mechanical testing of the brachial plexus showed that the tensile elastic limit strain, elastic limit stress, maximum stress, and maximum strain of the injured brachial plexuses were signiifcantly increased at 24 weeks after the injection. The treat-ment clearly improved the pathological morphology of the injured brachial plexus nerve, as seen by hematoxylin eosin staining, and the functions of the rabbit forepaw were restored. These data indicate that the injection of human amniotic epithelial cells contributed to the repair of brachial plexus injury, and that this technique may transform into current clinical treatment strategies.

  7. Comparison of radiosensitivities of human autologous normal and neoplastic thyroid epithelial cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Kopecky, K.J.; Hiraoka, T.; Ezaki, H.; Clifton, K.H.

    1986-01-01

    Studies were conducted to examine differences between the radiosensitivities of normal and neoplastic epithelial cells of the human thyroid. Freshly excised thyroid tissues from the tumours of eight patients with papillary carcinoma (PC) and five with follicular adenoma (FA) were cultured in vitro separately from normal thyroid tissue obtained from the surgical margins of the same patients. Plating efficiency of unirradiated control tissue was lower, on average for tumour tissue compared with normal tissue. Radiosensitivity, measured by the 37% inactivation dose D 0 , was greater for carcinoma tissue than for normal tissue in seven out of eight PC cases. Adenomatous tissue was less radiosensitive than normal tissue in four out of five FA cases. This is the first report comparing the radiosensitivity of autologous normal and abnormal epithelial tissue from the human thyroid. (author)

  8. Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

    Science.gov (United States)

    Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

    2005-09-01

    Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

  9. Small-Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Alexandra Mikhailova

    2014-02-01

    Full Text Available Human induced pluripotent stem cells (hiPSCs offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.

  10. Neural differentiation of choroid plexus epithelial cells: role of human traumatic cerebrospinal fluid

    Directory of Open Access Journals (Sweden)

    Elham Hashemi

    2017-01-01

    Full Text Available As the key producer of cerebrospinal fluid (CSF, the choroid plexus (CP provides a unique protective system in the central nervous system. CSF components are not invariable and they can change based on the pathological conditions of the central nervous system. The purpose of the present study was to assess the effects of non-traumatic and traumatic CSF on the differentiation of multipotent stem-like cells of CP into the neural and/or glial cells. CP epithelial cells were isolated from adult male rats and treated with human non-traumatic and traumatic CSF. Alterations in mRNA expression of Nestin and microtubule-associated protein (MAP2, as the specific markers of neurogenesis, and astrocyte marker glial fibrillary acidic protein (GFAP in cultured CP epithelial cells were evaluated using quantitative real-time PCR. The data revealed that treatment with CSF (non-traumatic and traumatic led to increase in mRNA expression levels of MAP2 and GFAP. Moreover, the expression of Nestin decreased in CP epithelial cells treated with non-traumatic CSF, while treatment with traumatic CSF significantly increased its mRNA level compared to the cells cultured only in DMEM/F12 as control. It seems that CP epithelial cells contain multipotent stem-like cells which are inducible under pathological conditions including exposure to traumatic CSF because of its compositions.

  11. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  12. Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

    Science.gov (United States)

    Kim, Tae-Hyun; Park, Young-Woo; Ahn, Jae-Sang; Ahn, Jeong-Taek; Kim, Se-Eun; Jeong, Man-Bok; Seo, Min-Su; Kang, Kyung-Sun

    2013-01-01

    This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits. PMID:23388445

  13. Polarized secretion of interleukin (IL-6 and IL-8 by human airway epithelia 16HBE14o- cells in response to cationic polypeptide challenge.

    Directory of Open Access Journals (Sweden)

    Alison Wai-ming Chow

    Full Text Available BACKGROUND: The airway epithelium participates in asthmatic inflammation in many ways. Target cells of the epithelium can respond to a variety of inflammatory mediators and cytokines. Damage to the surface epithelium occurs following the secretion of eosinophil-derived, highly toxic cationic proteins. Moreover, the surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells. To mimic the damage seen during asthmatic inflammation, the bronchial epithelium can be challenged with highly charged cationic polypeptides such as poly-L-arginine. METHODOLOGY/PRINCIPAL FINDINGS: In this study, human bronchial epithelial cells, 16HBE14o- cells, were "chemically injured" by exposing them to poly-l-arginine as a surrogate of the eosinophil cationic protein. Cytokine antibody array data showed that seven inflammatory mediators were elevated out of the 40 tested, including marked elevation in interleukin (IL-6 and IL-8 secretion. IL-6 and IL-8 mRNA expression levels were elevated as measured with real-time PCR. Cell culture supernatants from apical and basolateral compartments were collected, and the IL-6 and IL-8 production was quantified with ELISA. IL-6 and IL-8 secretion by 16HBE14o- epithelia into the apical compartment was significantly higher than that from the basolateral compartment. Using specific inhibitors, the production of IL-6 and IL-8 was found to be dependent on p38 MAPK, ERK1/2 MAPK, and NF-kappaB pathways. CONCLUSIONS/SIGNIFICANCE: The results clearly demonstrate that damage to the bronchial epithelia by poly-L-arginine stimulates polarized IL-6 and IL-8 secretion. This apically directed secretion of cytokines may play an important role in orchestrating epithelial cell responses to inflammation.

  14. Effect of resveratrol and zinc on intracellular zinc status in normal human prostate epithelial cells

    Science.gov (United States)

    To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated with 6 levels of resveratrol (0, 0.5, 1, 2.5, 5 and 10 microM) and 4 levels of zinc [0, 4, 16, and 32 microM for zinc-deficient (ZD), zinc-normal (ZN), zinc-adequate (ZA), an...

  15. Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

    OpenAIRE

    Foltz, LP; Clegg, DO

    2017-01-01

    We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embry...

  16. IL-17 suppresses immune effector functions in human papillomavirus-associated epithelial hyperplasia.

    Science.gov (United States)

    Gosmann, Christina; Mattarollo, Stephen R; Bridge, Jennifer A; Frazer, Ian H; Blumenthal, Antje

    2014-09-01

    Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL-23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skin-grafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against the graft. IL-17 was produced by CD3(+) T cells, predominantly CD4(+) T cells in human, and CD4(+) and γδ T cells in mouse hyperplastic lesions. IL-23 and IL-1β, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

  17. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    International Nuclear Information System (INIS)

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J.

    1995-01-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to α-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMVΒ vector; and (2) the antibiotic hygromycin-resistant transfected cells

  18. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J. [and others

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

  19. Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

    Science.gov (United States)

    Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

    2017-08-01

    An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. BENIGN EPITHELIAL NEOPLASIA ASSOCIATED WITH BETA-HUMAN PAPILLOMA VIRUS

    Directory of Open Access Journals (Sweden)

    V. A. Molochkov

    2014-01-01

    Full Text Available Aim: To study an association between acrochordon and human papilloma virus (HPV using quantitative analysis of viral desoxyribonucleic acid (DNA; to detect different phenotypes of beta-HPV. Materials and methods: We examined 52  patients (22 immuno-suppressed patients and 30 immunocompetent subjects in the Dermatovenereology and Dermato-Oncology Department and Chronic Dialysis and Kidney Transplantation Department of the Moscow Regional Research and Clinical Institute (MONIKI. Control group included 49 healthy donors. Burr biopsy samples (micro-samples of acrochordon and intact skin (apper arm were collected in sterile conditions. After sample procession and DNA isolation using DNK-sorb-C kit (Central Research Institute for Epidemiology – CRIE, polymerase chain reaction for HPV was performed with real-time fluorescent hybridization detection. For DNA amplification and detection we used RotorGene 3000 analyzer (Corbett Research, Australia. In the beta-HPV assay, recombinant plasmids were used as positive controls and control human beta-globin gene fragments (CRIE. 4 oligo-nucleotide systems (group-specific primers and probes were used for the detection of beta-HPV DNA. Results: Preliminary data indicated that acrochordons of open and covered skin regions were common in renal transplant recipients. Beta-HPV DNA was more frequent in acrochordons and intact skin (64% and 54% of renal transplant recipients compared to healthy donors (47%. 57% of renal transplant recipients demonstrated mixed infection in acrochordons. Conclusion: HPV DNA was frequently detected in acrochordons and intact skin of renal transplant recipients. In immunocompetent patients prevalence of HPV DNA in acrochordons was significantly higher compared to intact skin.

  1. Superoxide production and expression of NAD(P)H oxidases by transformed and primary human colonic epithelial cells

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Pedersen, G

    2003-01-01

    Superoxide (O(2)(-)) generation through the activity of reduced nicotinamide dinucleotide (NADH) or reduced nicotinamide dinucleotide phosphate (NADPH) oxidases has been demonstrated in a variety of cell types, but not in human colonic epithelial cells....

  2. Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

    Science.gov (United States)

    Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

    2013-01-01

    Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

  3. [Expression of thioredoxin-2 in human lens epithelial cells with oxidative damage and its significance].

    Science.gov (United States)

    Che, Xuanyi; Zhao, Qingxia; Li, Di

    2018-03-28

    To explore whether thioredoin-2 (Trx-2) is involved in the development of cataract and to study the effect of Trx-2 on hydrogen peroxide (H2O2)-induced injury in human lens epithelial cells.
 Methods: A total of 10 volunteers (removing the lens due totraumatism) and 30 patients received phacoemulsification (age more than 60 years) were selected. The expression of Trx-2 protein in lens epithelial cells from cataract patients and volunteers were detected by the immunohistochemical streptavidin-peroxidase (SP) method. SRA01/04 cells were cultured and were divided into six groups according to different treatment: a control group, H2O2-treated groups at 20, 50 or 
100 μmol/L, a negative control group (transfected with pCMV6 plasmid plus 100 μmol/L H2O2), and a Trx-2 overexpression group (transfected with pCMV6-Trx-2 plasmid plus 100 μmol/L H2O2). Methyl thiazolyltetrazolium (MTT) assay and flow cytometry was performed to measure the cell viability and apoptosis for SRA01/04 cells, respectively. The activities of superoxide dismutase (SOD) and catalase (CAT), the content of glutathione (GSH) and malondialdehyde (MDA) in human lens epithelial cells were measured via chemical chromatometry. Western blot was used to measure the protein levels of Trx-2, B-cell lymphoma 2 protein (Bcl-2), Bcl-2 associated X protein (Bax) and caspase-3.
 Results: Compared with the volunteers, the expression of Trx-2 was significantly decreased in lens epithelial cells in patients with cataract (PTrx-2 protein in the 20, 50 or 100 μmol/L H2O2 groups was decreased (all PTrx-2 and Bcl-2 expression and up-regulated Bax and caspase-3 expression (all PTrx-2 overexpression group (PTrx-2 and Bcl-2 expression and down-regulated Bax and caspase-3 expression (PTrx-2 might be involved in the apoptosis of lens epithelial cells in patients with cataract. The overexpression of Trx-2 obviously attenuated H2O2-induced injury of human lens epithelial cells, which might be associated with the

  4. Differential effects of airway anesthesia on ozone-induced pulmonary responses in human subjects.

    Science.gov (United States)

    Schelegle, E S; Eldridge, M W; Cross, C E; Walby, W F; Adams, W C

    2001-04-01

    We examined the effect of tetracaine aerosol inhalation, a local anesthetic, on lung volume decrements, rapid shallow breathing, and subjective symptoms of breathing discomfort induced by the acute inhalation of 0.30 ppm ozone for 65 min in 22 ozone-sensitive healthy human subjects. After 50 min of ozone inhalation FEV(1) was reduced 24%, breathing frequency was increased 40%, tidal volume was decreased 31%, and total subjective symptom score was increased (71.2, compared with 3.8 for filtered air exposure). Inhalation of tetracaine aerosol resulted in marked reductions in ozone-induced subjective symptoms of throat tickle and/or irritation (92.1%), cough (78.5%), shortness of breath (72.5%), and pain on deep inspiration (69.4%). In contrast, inhalation of tetracaine aerosol (mass median aerodynamic diameter of 3.52 microm with a geometric standard deviation of 1.92) resulted in only minor and inconsistent rectification of FEV(1) decrements (5.0%) and breathing frequency (-3.8%) that was not significantly different from that produced by saline aerosol alone (FEV(1), 5.1% and breathing frequency, -2.7%). Our data are consistent with afferent endings located within the large conducting airways of the tracheobronchial tree being primarily responsible for ozone-induced subjective symptoms and provides strong evidence that ozone-induced inhibition of maximal inspiratory effort is not dependent on conscious sensations of inspiratory discomfort.

  5. IMAGE-BASED IN VIVO QUANTITATIVE ASSESSMENT OF HUMAN AIRWAY OPENING AND CONTRACTILITY BY FIBER OPTICAL NASOPHARYNGOSCOPY IN HEALTHY AND ASTHMATIC SUBJECTS

    Directory of Open Access Journals (Sweden)

    LINHONG DENG

    2013-04-01

    Full Text Available Assessment of human airway lumen opening is important in diagnosing and understanding the mechanisms of airway dysfunctions such as the excessive airway narrowing in asthma and chronic obstructive pulmonary disease (COPD. Although there are indirect methods to evaluate the airway calibre, direct in vivo measurement of the airway calibre has not been commonly available. With recent advent of the flexible fiber optical nasopharyngoscope with video recording it has become possible to directly visualize the passages of upper and lower airways. However, quantitative analysis of the recorded video images has been technically challenging. Here, we describe an automatic image processing and analysis method that allows for batch analysis of the images recorded during the endoscopic procedure, thus facilitates image-based quantification of the airway opening. Video images of the airway lumen of volunteer subject were acquired using a fiber optical nasopharyngoscope, and subsequently processed using Gaussian smoothing filter, threshold segmentation, differentiation, and Canny image edge detection, respectively. Thus the area of the open airway lumen was identified and computed using a predetermined converter of the image scale to true dimension of the imaged object. With this method we measured the opening/narrowing of the glottis during tidal breathing with or without making "Hee" sound or cough. We also used this method to measure the opening/narrowing of the primary bronchus of either healthy or asthmatic subjects in response to histamine and/or albuterol treatment, which also provided an indicator of the airway contractility. Our results demonstrate that the image-based method accurately quantified the area change waveform of either the glottis or the bronchus as observed by using the optical nasopharygoscope. Importantly, the opening/narrowing of the airway lumen generally correlated with the airflow and resistance of the airways, and could

  6. Transcriptional response of bronchial epithelial cells to Pseudomonas aeruginosa: identification of early mediators of host defense.

    NARCIS (Netherlands)

    Vos, J.B.; Sterkenburg, M.A. van; Rabe, K.F.; Schalkwijk, J.; Hiemstra, P.S.; Datson, N.A.

    2005-01-01

    The airway epithelium responds to microbial exposure by altering expression of a variety of genes to increase innate host defense. We aimed to delineate the early transcriptional response in human primary bronchial epithelial cells exposed for 6 h to a mixture of IL-1beta and TNF-alpha or

  7. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

    Science.gov (United States)

    Sato, Toshiro; Stange, Daniel E; Ferrante, Marc; Vries, Robert G J; Van Es, Johan H; Van den Brink, Stieneke; Van Houdt, Winan J; Pronk, Apollo; Van Gorp, Joost; Siersema, Peter D; Clevers, Hans

    2011-11-01

    We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  8. Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

    LENUS (Irish Health Repository)

    Greene, C M

    2010-05-01

    alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

  9. IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

    Science.gov (United States)

    Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

    2017-08-01

    Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

  10. Deep RNA-Seq analysis reveals unexpected features of human prostate basal epithelial cells

    Directory of Open Access Journals (Sweden)

    Dingxiao Zhang

    2016-03-01

    Full Text Available Prostate cancer is the second leading cause of cancer-related deaths among American men [1]. The prostate gland mainly contains basal and luminal cells, which are constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here, for the first time, we describe a whole-genome transcriptome analysis of human benign prostatic basal and luminal populations by using deep RNA sequencing (GSE67070 [2]. Combined with comprehensive molecular and biological characterizations, we show that the differential gene expression profiles account for their distinct functional phenotypes. Strikingly, in contrast to luminal cells, basal cells preferentially express gene categories associated with stem cells, neural and neuronal development, and RNA processing. Of clinical relevance, the treatment failed castration-resistant and anaplastic prostate cancers molecularly resemble a basal-like phenotype. We also identified genes associated with patient clinical outcome. Therefore, we provide a gene expression resource for understanding human prostate epithelial lineages, and link the cell-type specific gene signatures to subtypes of prostate cancer development. Keywords: Prostate epithelial cells, Basal cells, Luminal cells, RNA-seq

  11. The parietal epithelial cell is crucially involved in human idiopathic focal segmental glomerulosclerosis.

    Science.gov (United States)

    Dijkman, Henry; Smeets, Bart; van der Laak, Jeroen; Steenbergen, Eric; Wetzels, Jack

    2005-10-01

    Focal segmental glomerulosclerosis (FSGS) is one of the most common patterns of glomerular injury encountered in human renal biopsies. Epithelial hyperplasia, which can be prominent in FSGS, has been attributed to dedifferentiation and proliferation of podocytes. Based on observations in a mouse model of FSGS, we pointed to the role of parietal epithelial cells (PECs). In the present study we investigated the relative role of PECs and podocytes in human idiopathic FSGS. We performed a detailed study of lesions from a patient with recurrent idiopathic FSGS by serial sectioning, marker analysis and three-dimensional reconstruction of glomeruli. We have studied the expression of markers for podocytes, PECs, mesangial cells, endothelium, and myofibroblasts. We also looked at proliferation and composition of the deposited extracellular matrix (ECM). We found that proliferating epithelial cells in FSGS lesions are negative for podocyte and macrophage markers, but stain for PEC markers. The composition of the matrix deposited by these cells is identical to Bowman's capsule. Our study demonstrates that PECs are crucially involved in the pathogenesis of FSGS lesions.

  12. Airway branching morphogenesis in three dimensional culture

    Directory of Open Access Journals (Sweden)

    Gudjonsson Thorarinn

    2010-11-01

    Full Text Available Abstract Background Lungs develop from the fetal digestive tract where epithelium invades the vascular rich stroma in a process called branching morphogenesis. In organogenesis, endothelial cells have been shown to be important for morphogenesis and the maintenance of organ structure. The aim of this study was to recapitulate human lung morphogenesis in vitro by establishing a three dimensional (3D co-culture model where lung epithelial cells were cultured in endothelial-rich stroma. Methods We used a human bronchial epithelial cell line (VA10 recently developed in our laboratory. This cell line cell line maintains a predominant basal cell phenotype, expressing p63 and other basal markers such as cytokeratin-5 and -14. Here, we cultured VA10 with human umbilical vein endothelial cells (HUVECs, to mimic the close interaction between these cell types during lung development. Morphogenesis and differentiation was monitored by phase contrast microscopy, immunostainings and confocal imaging. Results We found that in co-culture with endothelial cells, the VA10 cells generated bronchioalveolar like structures, suggesting that lung epithelial branching is facilitated by the presence of endothelial cells. The VA10 derived epithelial structures display various complex patterns of branching and show partial alveolar type-II differentiation with pro-Surfactant-C expression. The epithelial origin of the branching VA10 colonies was confirmed by immunostaining. These bronchioalveolar-like structures were polarized with respect to integrin expression at the cell-matrix interface. The endothelial-induced branching was mediated by soluble factors. Furthermore, fibroblast growth factor receptor-2 (FGFR-2 and sprouty-2 were expressed at the growing tips of the branching structures and the branching was inhibited by the FGFR-small molecule inhibitor SU5402. Discussion In this study we show that a human lung epithelial cell line can be induced by endothelial cells to

  13. Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

    Science.gov (United States)

    Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

    2015-07-15

    Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

  14. Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.

    Science.gov (United States)

    Mebratu, Yohannes A; Schwalm, Kurt; Smith, Kevin R; Schuyler, Mark; Tesfaigzi, Yohannes

    2011-06-01

    Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. We screened for dysregulated expression of the Bcl-2 family members. We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.

  15. PPARγ ligand ciglitazone inhibits TNFα-induced ICAM-1 in human airway smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Chien-Da Huang

    2014-08-01

    Full Text Available Background: Modification of human airway smooth muscle (ASM function by proinflammatory cytokines has been regarded as a potential mechanism underlying bronchial hyperresponsiveness in asthma. Human ASM cells express intercellular adhesion molecule (ICAM-1 in response to cytokines. Synthetic ligands for peroxisome proliferator-activated receptor (PPARγ reportedly possess anti-inflammatory and immunomodulatory properties. In this study, we examined whether ciglitazone, a synthetic PPARγ ligand, can modulate the basal and tumor necrosis factor (TNFα-induced ICAM1 gene expression in human ASM cells. Methods: Human ASM cells were treated with TNFα. ICAM-1 expression was assessed by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. PPARγ activity was inhibited by target-specific small interfering (si RNA targeting PPARγ and GW9662, a PPARγ antagonist. Activity of nuclear factor (NF-κB was assessed by using immunoblot analysis, immune-confocal images, and electrophoretic mobility shift assay (EMSA. Results: By flow cytometry, ciglitazone alone had no effect on ICAM-1 expression in ASM cells, but inhibited ICAM-1 expression in response to TNFα (10 ng/ml in a dose-dependent manner (1-10 μM. It also inhibited TNFα-induced ICAM1 gene expression by RT-PCR analysis. Knockdown of PPARγ gene by target-specific siRNA targeting PPARγ enhanced ICAM-1 expression and the inhibitory effect of ciglitazone on TNFα-induced ICAM-1 expression was reversed by PPARγ siRNA and GW9662. SN-50 (10 μg/ml, an inhibitor for nuclear translocation of NF-κB, inhibited TNFα-induced ICAM-1 expression. Ciglitazone did not prevent TNFα-induced degradation of the cytosolic inhibitor of NF-κB (IκB, but inhibited the nuclear translocation of p65 induced by TNFα and suppressed the NF-κB/DNA binding activity. Conclusion: These findings suggest that ciglitazone inhibits TNFα-induced ICAM1 gene expression in human ASM cells through

  16. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses.

    Science.gov (United States)

    Ozbun, Michelle A; Patterson, Nicole A

    2014-08-01

    Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Copyright © 2014 John Wiley

  17. Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

    Science.gov (United States)

    Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

    2014-01-01

    Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

  18. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    Science.gov (United States)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  19. MAPK Activation Is Essential for Waddlia chondrophila Induced CXCL8 Expression in Human Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Skye Storrie

    Full Text Available Waddlia chondrophila (W. chondrophila is an emerging agent of respiratory and reproductive disease in humans and cattle. The organism is a member of the order Chlamydiales, and shares many similarities at the genome level and in growth studies with other well-characterised zoonotic chlamydial agents, such as Chlamydia abortus (C. abortus. The current study investigated the growth characteristics and innate immune responses of human and ruminant epithelial cells in response to infection with W. chondrophila.Human epithelial cells (HEp2 were infected with W. chondrophila for 24h. CXCL8 release was significantly elevated in each of the cell lines by active-infection with live W. chondrophila, but not by exposure to UV-killed organisms. Inhibition of either p38 or p42/44 MAPK significantly inhibited the stimulation of CXCL8 release in each of the cell lines. To determine the pattern recognition receptor through which CXCL8 release was stimulated, wild-type HEK293 cells which express no TLR2, TLR4, NOD2 and only negligible NOD1 were infected with live organisms. A significant increase in CXCL8 was observed.W. chondrophila actively infects and replicates within both human and ruminant epithelial cells stimulating CXCL8 release. Release of CXCL8 is significantly inhibited by inhibition of either p38 or p42/44 MAPK indicating a role for this pathway in the innate immune response to W. chondrophila infection. W. chondrophila stimulation of CXCL8 secretion in HEK293 cells indicates that TLR2, TLR4, NOD2 and NOD1 receptors are not essential to the innate immune response to infection.

  20. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  1. Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

    International Nuclear Information System (INIS)

    Matsuzaki, Shinichi; Ishizuka, Tamotsu; Yamada, Hidenori; Kamide, Yosuke; Hisada, Takeshi; Ichimonji, Isao; Aoki, Haruka; Yatomi, Masakiyo; Komachi, Mayumi; Tsurumaki, Hiroaki; Ono, Akihiro; Koga, Yasuhiko; Dobashi, Kunio; Mogi, Chihiro; Sato, Koichi; Tomura, Hideaki; Mori, Masatomo; Okajima, Fumikazu

    2011-01-01

    Highlights: → The involvement of extracellular acidification in airway remodeling was investigated. → Extracellular acidification alone induced CTGF production in human ASMCs. → Extracellular acidification enhanced TGF-β-induced CTGF production in human ASMCs. → Proton-sensing receptor OGR1 was involved in acidic pH-stimulated CTGF production. → OGR1 may play an important role in airway remodeling in asthma. -- Abstract: Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP 3 ) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G q/11 protein and inositol-1,4,5-trisphosphate-induced Ca 2+ mobilization in human ASMCs.

  2. Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine

    Directory of Open Access Journals (Sweden)

    Mohammad Rasoul Khazaei

    2016-09-01

    Full Text Available Objective(s: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9 were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml. Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0, 10, 25, 50 and 100 micromole/liter (µM concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO–Ethidium Bromide (EB double staining and Terminal deoxynucleotidyl transferase (TdT dUTP Nick-End Labeling (TUNEL assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P

  3. Three-dimensional epithelial tissues generated from human embryonic stem cells.

    Science.gov (United States)

    Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

    2009-11-01

    The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

  4. Down-regulation of a calmodulin-related gene during transformation of human mammary epithelial cells

    International Nuclear Information System (INIS)

    Yaswen, P.; Smoll, A.; Stampfer, M.R.; Peehl, D.M.; Trask, D.K.; Sager, R.

    1990-01-01

    A human cDNA library obtained from cultured normal mammary epithelial cells (HMECs) was searched by subtractive hybridization for genes whose decrease in expression might be relevant to epithelial transformation. One clone identified by this procedure corresponded to a 1.4 kilobase mRNA, designated NB-1, whose expression was decreased >50-fold in HMECs tumorigenically transformed in vitro after exposure to benzo[α]pyrene and Kirsten sarcoma virus. Sequence analysis of NB-1 cDNA revealed an open reading frame with a high degree of homology to calmodulin. NB-1 expression could be demonstrated by polymerase chain reaction amplification in normal breast, prostate, cervix, and epidermal tissues. The presence of NB-1 transcripts was variable in primary breast carcinoma tissues and undetectable in tumor-derived cell lines of breast, prostate, or other origins. NB-1 mRNA expression could be down-regulated in cultured HMECs by exposure to reconstituted extracellular matrix material, while exposure to transforming growth factor type β increased its relative abundance. The protein encoded by NB-1 may have Ca 2 plus binding properties and perform functions similar to those of authentic calmodulin. Its possible roles in differentiation and/or suppression of tumorigenicity in epithelial tissues remain to be examined

  5. In vitro effects of three blood derivatives on human corneal epithelial cells.

    Science.gov (United States)

    Freire, Vanesa; Andollo, Noelia; Etxebarria, Jaime; Durán, Juan A; Morales, María-Celia

    2012-08-15

    We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

  6. Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

    International Nuclear Information System (INIS)

    Gutierrez-Barrera, Angelica M.; Menter, David G.; Abbruzzese, James L.; Reddy, Shrikanth A.G.

    2007-01-01

    Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7 organized into spheroids with what appeared to be apical and basal membranes and a luminal space. Immunostaining experiments indicated that protein kinase Akt was phosphorylated (Ser473) and CTMP, a negative Akt regulator, was expressed in both HPDE-E6E7 and cancer cells. However, a nuclear pool of CTMP was detectable in HPDE-E6E7 cells that showed a dynamic concentrated expression pattern, a feature that further distinguished HPDE-E637 cells from cancer cells. Collectively, these data suggest that 3D cultures of HPDE-E6E7 cells are useful for investigating signaling and morphological abnormalities in pancreatic cancer cells

  7. Recovery from radiation-induced damage in primary cultures of human epithelial thyroid cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Enno, Masumi; Takeichi, Nobuo.

    1985-01-01

    Human thyroid epithelial tissues from 23 individuals were obtained from surgical tissue, and cultured in vitro. Dose response survival curves showed thyroid cells, when compared to mammary epithelial and skin fibroblast cells of human origin, to be only slightly more radiosensitive to X-rays. Cell survival curves from the cell strains showed wide variability in radiation sensitivity. Of the 23 cell strains tested, 21 strains displayed significant shoulders (nonzero quasi-threshold (D q ) values and extrapolation number (n) values greater than 1) at low dose exposures. The ability of human cells to recover from radiation damage was further studied by dose fractionation. Two cell strains were given a total X-ray dose of 304 cGy in two equal fractions separated by varying time intervals. Maximal cell survival was observed when the time interval exceeded two hours. When the two cell strains were exposed to 152 cGy of X-rays followed four hours later by second graded doses, cell survival was enhanced as compared to survival after single dose exposures. However, no benefit of dose splitting was observed when cells were exposed to low second doses. These results support previous studies showing that human cells are capable of repair but require relatively large doses to elicit a repair response. (author)

  8. Recovery from radiation-induced damage in primary cultures of human epithelial thyroid cells

    International Nuclear Information System (INIS)

    Miller, R.C.; Hiraoka, Toshio; Enno, Masumi; Takeichi, Nobuo.

    1985-09-01

    Human thyroid epithelial tissue from 23 individuals was obtained from surgical tissue, and cultured in vitro. Dose-response survival curves showed thyroid cells, when compared to mammary epithelial and skin fibroblast cells of human origin, to be only slightly more radiosensitive to X rays. Cell survival curves from the cell strains showed wide variability in radiation sensitivity. Of the 23 cell strains tested, 21 strains displayed significant shoulders (nonzero quasi-threshold (Dsub(q)) values and extrapolation number (n) values greater than 1)* at low dose exposures. The ability of human cells to recover from radiation damage was further studied by dose fractionation. Two cell strains were given a total X-ray dose of 304 cGy in two equal fractions separated by varying time intervals. Maximal cell survival was observed when the time interval exceeded two hours. When the two cell strains were exposed to 152 cGy of X rays followed four hours later by second graded doses, cell survival was enhanced as compared to survival after single dose exposures. However, no benefit of dose splitting was observed when cells were exposed to low second doses. These results support previous studies showing that human cells are capable of repair but require relatively large doses to elicit a repair response. (author)

  9. Markers of Airway Remodeling in Bronchopulmonary Diseases

    Directory of Open Access Journals (Sweden)

    O.Ye. Chernyshova

    2014-10-01

    Full Text Available The article presents information about markers of airway remodeling in bronchopulmonary diseases. There is described the influence of matrix metalloproteinases, tissue inhibitor of matrix metalloproteinase, transforming growth factor, collagen autoantibodies III type, endothelin-1 on the processes of morphological airway reconstruction as smooth muscle hypertrophy, enhanced neovascularization, epithelial cell hyperplasia, collagen deposition, compaction of the basal membrane, observed in bronchial asthma.

  10. Differential responses of human dendritic cells to metabolites from the oral/airway microbiome.

    Science.gov (United States)

    Whiteson, K; Agrawal, S; Agrawal, A

    2017-06-01

    Small molecule metabolites that are produced or altered by host-associated microbial communities are emerging as significant immune response modifiers. However, there is a key gap in our knowledge of how oral microbial metabolites affect the immune response. Here, we examined the effects of metabolites from five bacterial strains found commonly in the oral/airway microbial communities of humans. The five strains, each isolated from cystic fibrosis patient sputum, were Pseudomonas aeruginosa FLR01 non-mucoid (P1) and FLR02 mucoid (P2) forms, Streptococcus pneumoniae (Sp), S. salivarius (Ss) and Rothia mucilaginosa (Rm). The effect of bacterial metabolites on dendritic cell (DC) activation, T cell priming and cytokine secretion was determined by exposing DCs to bacterial supernatants and individual metabolites of interest. Supernatants from P1 and P2 induced high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-12 and IL-6 from DCs and primed T cells to secrete interferon (IFN)-γ, IL-22 compared to supernatants from Sp, Ss and Rm. Investigations into the composition of supernatants using gas chromatography-mass spectroscopy (GC-MS) revealed signature metabolites for each of the strains. Supernatants from P1 and P2 contained high levels of putrescine and glucose, while Sp and Ss contained high levels of 2,3-butanediol. The individual metabolites replicated the results of whole supernatants, although the magnitudes of their effects were reduced significantly. Altogether, our data demonstrate for the first time that the signature metabolites produced by different bacteria have different effects on DC functions. The identification of signature metabolites and their effects on the host immune system can provide mechanistic insights into diseases and may also be developed as biomarkers. © 2017 British Society for Immunology.

  11. CCL20 and Beta-Defensin 2 Production by Human Lung Epithelial Cells and Macrophages in Response to Brucella abortus Infection

    Science.gov (United States)

    Fernández, Andrea G.; Bonetto, Josefina; Giambartolomei, Guillermo H.; Fossati, Carlos A.; Baldi, Pablo C.

    2015-01-01

    Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site. PMID:26448160

  12. Sulfation of chlorotyrosine and nitrotyrosine by human lung endothelial and epithelial cells: Role of the human SULT1A3

    International Nuclear Information System (INIS)

    Yasuda, Shin; Yasuda, Tomoko; Liu, Ming-Yih; Shetty, Sreerama; Idell, Steven; Boggaram, Vijayakumar; Suiko, Masahito; Sakakibara, Yoichi; Fu Jian; Liu, Ming-Cheh

    2011-01-01

    During inflammation, potent reactive oxidants formed may cause chlorination and nitration of both free and protein-bound tyrosine. In addition to serving as biomarkers of inflammation-mediated oxidative stress, elevated levels of chlorotyrosine and nitrotyrosine have been linked to the pathogenesis of lung and vascular disorders. The current study was designed to investigate whether the lung cells are equipped with mechanisms for counteracting these tyrosine derivatives. By metabolic labeling, chlorotyrosine O-[ 35 S]sulfate and nitrotyrosine O-[ 35 S]sulfate were found to be generated and released into the labeling media of human lung endothelial and epithelial cells labeled with [ 35 S]sulfate in the presence of added chlorotyrosine and nitrotyrosine. Enzymatic assays using the eleven known human cytosolic sulfotransferases (SULTs) revealed SULT1A3 as the enzyme responsible for catalyzing the sulfation of chlorotyrosine and nitrotyrosine. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the expression of SULT1A3 in the lung endothelial and epithelial cells used in this study. Kinetic constants of the sulfation of chlorotyrosine and nitrotyrosine by SULT1A3 were determined. Collectively, these results suggest that sulfation by SULT1A3 in lung endothelial and epithelial cells may play a role in the inactivation and/or disposal of excess chlorotyrosine and nitrotyrosine generated during inflammation.

  13. Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity.

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Quaranta

    Full Text Available The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.

  14. Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization.

    Science.gov (United States)

    Tang, L; Wu, J J; Ma, Q; Cui, T; Andreopoulos, F M; Gil, J; Valdes, J; Davis, S C; Li, J

    2010-07-01

    Human lactoferrin (hLF), a member of the transferrin family, is known for its antimicrobial and anti-inflammatory effects. Recent studies on various nonskin cell lines indicate that hLF may have a stimulatory effect on cell proliferation. To study the potential role of hLF in wound re-epithelialization. The effects of hLF on cell growth, migration, attachment and survival were assessed, with a rice-derived recombinant hLF (holo-rhLF), using proliferation analysis, scratch migration assay, calcein-AM/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) method, respectively. The mechanisms of hLF on cell proliferation and migration were explored using specific pathway inhibitors. The involvement of lactoferrin receptor low-density lipoprotein receptor-related protein 1 (LRP1) was examined with RNA interference technique. An in vivo swine second-degree burn wound model was also used to assess wound re-epithelialization. Studies revealed that holo-rhLF significantly stimulated keratinocyte proliferation which could be blocked by mitogen-activated protein kinase (MAPK) kinase 1 inhibitor. Holo-rhLF also showed strong promoting effects on keratinocyte migration, which could be blocked by either inhibition of the MAPK, Src and Rho/ROCK pathways, or downregulation of the LRP1 receptor. With cells under starving or 12-O-tetradecanoylphorbol-13-acetate exposure, the addition of holo-rhLF was found greatly to increase cell viability and inhibit cell apoptosis. Additionally, holo-rhLF significantly increased the rate of wound re-epithelialization in swine second-degree burn wounds. Our studies demonstrate the direct effects of holo-rhLF on wound re-epithelialization including the enhancement of keratinocyte proliferation and migration as well as the protection of cells from apoptosis. The data strongly indicate its potential therapeutic applications in wound healing.

  15. Transport and Deposition of Welding Fume Agglomerates in a Realistic Human Nasal Airway.

    Science.gov (United States)

    Tian, Lin; Inthavong, Kiao; Lidén, Göran; Shang, Yidan; Tu, Jiyuan

    2016-07-01

    Welding fume is a complex mixture containing ultra-fine particles in the nanometer range. Rather than being in the form of a singular sphere, due to the high particle concentration, welding fume particles agglomerate into long straight chains, branches, or other forms of compact shapes. Understanding the transport and deposition of these nano-agglomerates in human respiratory systems is of great interest as welding fumes are a known health hazard. The neurotoxin manganese (Mn) is a common element in welding fumes. Particulate Mn, either as soluble salts or oxides, that has deposited on the olfactory mucosa in human nasal airway is transported along the olfactory nerve to the olfactory bulb within the brain. If this Mn is further transported to the basal ganglia of the brain, it could accumulate at the part of the brain that is the focal point of its neurotoxicity. Accounting for various dynamic shape factors due to particle agglomeration, the current computational study is focused on the exposure route, the deposition pattern, and the deposition efficiency of the inhaled welding fume particles in a realistic human nasal cavity. Particular attention is given to the deposition pattern and deposition efficiency of inhaled welding fume agglomerates in the nasal olfactory region. For particles in the nanoscale, molecular diffusion is the dominant transport mechanism. Therefore, Brownian diffusion, hydrodynamic drag, Saffman lift force, and gravitational force are included in the model study. The deposition efficiencies for single spherical particles, two kinds of agglomerates of primary particles, two-dimensional planar and straight chains, are investigated for a range of primary particle sizes and a range of number of primary particles per agglomerate. A small fraction of the inhaled welding fume agglomerates is deposited on the olfactory mucosa, approximately in the range 0.1-1%, and depends on particle size and morphology. The strong size dependence of the deposition

  16. Epithelial hyperplasia in human polycystic kidney diseases. Its role in pathogenesis and risk of neoplasia.

    OpenAIRE

    Bernstein, J.; Evan, A. P.; Gardner, K. D.

    1987-01-01

    The importance of tubular epithelial hyperplasia in polycystic kidney diseases has become apparent during the last decade. Micropapillary hyperplasia occurs in autosomal dominant polycystic kidney disease, in localized cystic disease, and in acquired cystic disease. Neoplastic or severely dysplastic epithelial hyperplasia occurs in von Hippel-Lindau disease. A histopathologically distinctive epithelial hyperplasia occurs in tuberous sclerosis. In each of these conditions, epithelial hyperplas...

  17. Numerical modelling of local deposition patients, activity distributions and cellular hit probabilities of inhaled radon progenies in human airways

    International Nuclear Information System (INIS)

    Farkas, A.; Balashazy, I.; Szoeke, I.

    2003-01-01

    The general objective of our research is modelling the biophysical processes of the effects of inhaled radon progenies. This effort is related to the rejection or support of the linear no threshold (LNT) dose-effect hypothesis, which seems to be one of the most challenging tasks of current radiation protection. Our approximation and results may also serve as a useful tool for lung cancer models. In this study, deposition patterns, activity distributions and alpha-hit probabilities of inhaled radon progenies in the large airways of the human tracheobronchial tree are computed. The airflow fields and related particle deposition patterns strongly depend on the shape of airway geometry and breathing pattern. Computed deposition patterns of attached an unattached radon progenies are strongly inhomogeneous creating hot spots at the carinal regions and downstream of the inner sides of the daughter airways. The results suggest that in the vicinity of the carinal regions the multiple hit probabilities are quite high even at low average doses and increase exponentially in the low-dose range. Thus, even the so-called low doses may present high doses for large clusters of cells. The cell transformation probabilities are much higher in these regions and this phenomenon cannot be modeled with average burdens. (authors)

  18. Construction of predictive promoter models on the example of antibacterial response of human epithelial cells

    Directory of Open Access Journals (Sweden)

    Wingender Edgar

    2005-01-01

    Full Text Available Abstract Background Binding of a bacteria to a eukaryotic cell triggers a complex network of interactions in and between both cells. P. aeruginosa is a pathogen that causes acute and chronic lung infections by interacting with the pulmonary epithelial cells. We use this example for examining the ways of triggering the response of the eukaryotic cell(s, leading us to a better understanding of the details of the inflammatory process in general. Results Considering a set of genes co-expressed during the antibacterial response of human lung epithelial cells, we constructed a promoter model for the search of additional target genes potentially involved in the same cell response. The model construction is based on the consideration of pair-wise combinations of transcription factor binding sites (TFBS. It has been shown that the antibacterial response of human epithelial cells is triggered by at least two distinct pathways. We therefore supposed that there are two subsets of promoters activated by each of them. Optimally, they should be "complementary" in the sense of appearing in complementary subsets of the (+-training set. We developed the concept of complementary pairs, i.e., two mutually exclusive pairs of TFBS, each of which should be found in one of the two complementary subsets. Conclusions We suggest a simple, but exhaustive method for searching for TFBS pairs which characterize the whole (+-training set, as well as for complementary pairs. Applying this method, we came up with a promoter model of antibacterial response genes that consists of one TFBS pair which should be found in the whole training set and four complementary pairs. We applied this model to screening of 13,000 upstream regions of human genes and identified 430 new target genes which are potentially involved in antibacterial defense mechanisms.

  19. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    International Nuclear Information System (INIS)

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia; Deng, Yubin; Zeng, Mian

    2016-01-01

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  20. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Deng, Yubin, E-mail: dengyub@mail.sysu.edu.cn [Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Zeng, Mian, E-mail: zengmian2004@163.com [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China)

    2016-05-20

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  1. Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

    Science.gov (United States)

    Durante, M.; Grossi, G. F.; Gialanella, G.; Pugliese, M.; Nappo, M.; Yang, T. C.

    1995-01-01

    We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of alpha-particles emitted from radon daughters, we concentrated our studies on the efficiency of alpha-particles. Confluent cultures of M/10 cells were exposed to accelerated alpha-particles [beam energy incident at the cell monolayer = 3.85 MeV, incident linear energy transfer (LET) in cell = 109 keV/microns] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for alpha-particles (D0 = 0.73 +/- 0.04 Gy), while a shoulder was observed for x-rays (alpha/beta = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET alpha-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for alpha-particles and linearquadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges.(ABSTRACT TRUNCATED AT 250 WORDS).

  2. Characterization of primary human mammary epithelial cells isolated and propagated by conditional reprogrammed cell culture.

    Science.gov (United States)

    Jin, Liting; Qu, Ying; Gomez, Liliana J; Chung, Stacey; Han, Bingchen; Gao, Bowen; Yue, Yong; Gong, Yiping; Liu, Xuefeng; Amersi, Farin; Dang, Catherine; Giuliano, Armando E; Cui, Xiaojiang

    2018-02-20

    Conditional reprogramming methods allow for the inexhaustible in vitro proliferation of primary epithelial cells from human tissue specimens. This methodology has the potential to enhance the utility of primary cell culture as a model for mammary gland research. However, few studies have systematically characterized this method in generating in vitro normal human mammary epithelial cell models. We show that cells derived from fresh normal breast tissues can be propagated and exhibit heterogeneous morphologic features. The cultures are composed of CK18, desmoglein 3, and CK19-positive luminal cells and vimentin, p63, and CK14-positive myoepithelial cells, suggesting the maintenance of in vivo heterogeneity. In addition, the cultures contain subpopulations with different CD49f and EpCAM expression profiles. When grown in 3D conditions, cells self-organize into distinct structures that express either luminal or basal cell markers. Among these structures, CK8-positive cells enclosing a lumen are capable of differentiation into milk-producing cells in the presence of lactogenic stimulus. Furthermore, our short-term cultures retain the expression of ERα, as well as its ability to respond to estrogen stimulation. We have investigated conditionally reprogrammed normal epithelial cells in terms of cell type heterogeneity, cellular marker expression, and structural arrangement in two-dimensional (2D) and three-dimensional (3D) systems. The conditional reprogramming methodology allows generation of a heterogeneous culture from normal human mammary tissue in vitro . We believe that this cell culture model will provide a valuable tool to study mammary cell function and malignant transformation.

  3. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  4. Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

    OpenAIRE

    Li, Qiong; Kumar, Ashok; Gui, Jian-Fang; Yu, Fu-Shin X.

    2007-01-01

    Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-κB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-κB activation in HUCL cells after lipoprotein lipa...

  5. Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

    Science.gov (United States)

    O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

    2018-04-15

    JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

  6. The V protein of canine distemper virus is required for virus replication in human epithelial cells.

    Directory of Open Access Journals (Sweden)

    Noriyuki Otsuki

    Full Text Available Canine distemper virus (CDV becomes able to use human receptors through a single amino acid substitution in the H protein. In addition, CDV strains possessing an intact C protein replicate well in human epithelial H358 cells. The present study showed that CDV strain 007Lm, which was isolated from lymph node tissue of a dog with distemper, failed to replicate in H358 cells, although it possessed an intact C protein. Sequence analyses suggested that a cysteine-to-tyrosine substitution at position 267 of the V protein caused this growth defect. Analyses using H358 cells constitutively expressing the CDV V protein showed that the V protein with a cysteine, but not that with a tyrosine, at this position effectively blocked the interferon-stimulated signal transduction pathway, and supported virus replication of 007Lm in H358 cells. Thus, the V protein as well as the C protein appears to be functional and essential for CDV replication in human epithelial cells.

  7. Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

    2016-03-10

    Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

  8. Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures.

    Science.gov (United States)

    Essaidi-Laziosi, Manel; Brito, Francisco; Benaoudia, Sacha; Royston, Léna; Cagno, Valeria; Fernandes-Rocha, Mélanie; Piuz, Isabelle; Zdobnov, Evgeny; Huang, Song; Constant, Samuel; Boldi, Marc-Olivier; Kaiser, Laurent; Tapparel, Caroline

    2018-06-01

    The leading cause of acute illnesses, respiratory viruses, typically cause self-limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized. We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia. Tissues were infected with RV-A55, RV-A49, RV-B48, RV-C8, and RV-C15; respiratory EV-D68; influenza virus H3N2; RSV-B; and human coronavirus (HCoV)-OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing. RVs, RSV-B, and HCoV-OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV-D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV-B48 and HCoV-OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV-OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population. Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  9. Tacrolimus Modulates TGF-β Signaling to Induce Epithelial-Mesenchymal Transition in Human Renal Proximal Tubule Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jason Bennett

    2016-04-01

    Full Text Available Epithelial-mesenchymal transition (EMT, a process which describes the trans-differentiation of epithelial cells into motile mesenchymal cells, is pivotal in stem cell behavior, development and wound healing, as well as contributing to disease processes including fibrosis and cancer progression. Maintenance immunosuppression with calcineurin inhibitors (CNIs has become routine management for renal transplant patient, but unfortunately the nephrotoxicity of these drugs has been well documented. HK-2 cells were exposed to Tacrolimus (FK506 and EMT markers were assessed by RT PCR and western blot. FK506 effects on TGF-β mRNA were assessed by RT PCR and TGF-β secretion was measured by ELISA. The impact of increased TGF-β secretion on Smad signaling pathways was investigated. The impact of inhibition of TGF-β signaling on EMT processes was assessed by scratch-wound assay. The results presented in this study suggest that FK506 initiates EMT processes in the HK-2 cell line, with altered expression of epithelial and myofibroblast markers evident. Additionally, the study demonstrates that FK506 activation of the TGF-β/ SMAD pathways is an essential step in the EMT process. Overall the results demonstrate that EMT is heavily involved in renal fibrosis associated with CNI nephrotoxicity.

  10. Lentiviral Vector Gene Transfer to Porcine Airways

    Directory of Open Access Journals (Sweden)

    Patrick L Sinn

    2012-01-01

    Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

  11. Human Skin Is the Largest Epithelial Surface for Interaction with Microbes.

    Science.gov (United States)

    Gallo, Richard L

    2017-06-01

    Human skin contains an abundant and diverse population of microbial organisms. Many of these microbes inhabit follicular structures of the skin. Furthermore, numerous studies have shown that the interaction of some members of the skin microbiome with host cells will result in changes in cell function. However, estimates of the potential for the microbiome to influence human health through skin have ignored the inner follicular surface, and therefore vastly underestimated the potential of the skin microbiome to have a systemic effect on the human body. By calculating the surface area of follicular and the interfollicular epithelial surface it is shown that skin provides a vast interface for interactions with the microbiome. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  12. Detection of genomic instability in normal human bronchial epithelial cells exposed to 238Pu

    International Nuclear Information System (INIS)

    Kennedy, C.H.; Fukushima, N.H.; Neft, R.E.; Lechner, J.F.

    1994-01-01

    Alpha particle-emitting radon daughters constitute a risk for development of lung cancer in humans. The development of this disease involves multiple genetic alterations. These changes and the time course they follow are not yet defined despite numerous in vitro endeavors to transform human lung cells with various physical or chemical agents. However, genomic instability, characterized both by structural and numerical chromosomal aberrations and by elevated rates of point mutations, is a common feature of tumor cells. Further, both types of genomic instability have been reported in the noncancerous progeny of normal murine hemopoietic cells exposed in vitro to α-particles. The purpose of this investigation was to determine if genomic instability is also a prominent feature of normal human bronchial epithelial cells exposed to α-particle irradiation from the decay of inhaled radon daughters

  13. Design, characterization and use of replicate human upper airways for radon dosimetry studies

    International Nuclear Information System (INIS)

    Swift, D.L.; Cheng, Y.S.; Su, Y.F.; Yeh, H.C.

    1992-01-01

    The size distribution of inhaled radon progeny aerosols is a significant factor in dosimetry. The role of the airways above the trachea is an important determinant of the respiratory distribution of both attached and unattached progeny aerosols. In order to provide information on the effect of particle size and breathing conditions on the overall and local deposition, we have developed a method to produce a replicate airway model from an in vivo magnetic resonance imaging coronal scan. The model consists of a sandwich of methacrylate elements, each element having the thickness of the scan interval. The transition between successive scan outlines traced on the front and back surfaces of each element is handsculpted in the plastic. The hollow model of the nasal passages thus produced has been characterized both morphologically and fluid-mechanically and has a flow resistance typical of a normal adult. The model has several distinct advantages for studies of radon progeny aerosol deposition. After exposure to a radioaerosol (or to an aerosol of an otherwise measurable substance) the individual elements can be separated to determine local deposition. The dimensions of specific upper-airway regions can be changed by replacing a small number of elements. The model has been incorporated in an exposure system for determining overall nandregional deposition of aerosols whose median diameter is approximately 1.7 nm. Measurements at several flow rates are presented to demonstrate use of the model in radon dosimetry. The model should also be useful for determining the airway deposition of other environmental aerosols

  14. Surfactant Proteins A, B, C and D in the Human Nasal Airway

    DEFF Research Database (Denmark)

    Gaunsbaek, Maria Q; Kjeldsen, Anette D; Svane-Knudsen, Viggo

    2014-01-01

    Aims: To investigate the presence of surfactant protein (SP) A, B, C and D in nasal airways and to determine whether the proteins exert their main functions in nasal secretions or in the deeper layers of the nasal mucosa. Methods: Volunteers were recruited from the Department of ENT Head and Neck...