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Sample records for respiratory epithelial cell

  1. Respiratory epithelial cells orchestrate pulmonary innate immunity

    PubMed Central

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and ‘instruct’ the professional immune system to protect the lungs from infection and injury. PMID:25521682

  2. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  3. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

    PubMed

    Lamberti, Yanina; Gorgojo, Juan; Massillo, Cintia; Rodriguez, Maria E

    2013-12-01

    Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

  4. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    PubMed

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  5. Growth of respiratory syncytial virus in mink lung epithelial cells.

    PubMed

    Yeolekar, L R; Damle, R G; Basu, A; Rao, B L

    2002-12-01

    Mink lung epithelial cells (Mv-1-Lu) were tested for their ability to support the growth and serial passage of respiratory syncytial virus (RSV) in vitro. Indian isolates of RSV induced distinctive cytopathic effect with typical rounding of cells followed by detachment with more than 50 per cent cells showing bright fluorescence using anti-RSV monoclonal antibodies in immunofluorescence test. Serial passage of RSV was possible in Mv-1-Lu cells without loss of sensitivity of the cells for virus growth. Titration of cell associated virus and virus released in the supernatant indicated that 60 per cent of the virus was released in the supernatant, and 40 per cent remained cell associated. Transmission electron microscopic studies of negatively stained RSV particles and ultra-thin sections of RSV infected Mv-1-Lu cells showed roughly spherical particles with club shaped projections, budding from the cytoplasmic membrane. These results indicate that Mv-1-Lu cell line is suitable for the growth and propagation of RSV.

  6. Biological effects of desert dust in respiratory epithelial cells and a murine model.

    EPA Science Inventory

    Abstract As a result of the challenge of recent dust storms to public health, we tested the postulate that desert dust collected in the southwestern United States could impact a biological effect in respiratory epithelial cells and an animal model. Two samples of surface sedime...

  7. DIESEL EXHAUST ENHANCES TOLL-LIKE RECEPTOR 3 EXPRESSION AND SIGNALING IN RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    Our previous studies have shown that prior exposure of respiratory epithelial cells to an aqueous-trapped solution of DE (DEas) enhances the susceptibility to Influenza infections. Here we examined the effect of DEas on the toll-like receptor 3 (TLR3) pathway, which is responsib...

  8. Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface.

    PubMed

    Lam, Hilaire C; Choi, Augustine M K; Ryter, Stefan W

    2011-01-01

    Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems. PMID:21372793

  9. Modelling the effects of microgravity on the permeability of air interface respiratory epithelial cell layers

    NASA Astrophysics Data System (ADS)

    dos Santos, Marlise A.; Bosquillon, Cynthia; Russomano, Thais; Sundaresan, Alamelu; Falcão, Felipe; Marriott, Christopher; Forbes, Ben

    2010-09-01

    Although it has been suggested that microgravity might affect drug absorption in vivo, drug permeability across epithelial barriers has not yet been investigated in vitro during modelled microgravity. Therefore, a cell culture/diffusion chamber was designed specifically to accommodate epithelial cell layers in a 3D-clinostat and allow epithelial permeability to be measured under microgravity conditions in vitro with minimum alteration to established cell culture techniques. Human respiratory epithelial Calu-3 cell layers were used to model the airway epithelium. Cells grown at an air interface in the diffusion chamber from day 1 or day 5 after seeding on 24-well polyester Transwell cell culture inserts developed a similar transepithelial electrical resistance (TER) to cells cultured in conventional cell culture plates. Confluent Calu-3 layers exposed to modelled microgravity in the 3D-clinostat for up to 48 h maintained their high TER. The permeability of the paracellular marker 14C-mannitol was unaffected after a 24 h rotation of the cell layers in the 3D-clinostat, but was increased 2-fold after 48 h of modelled microgravity. It was demonstrated that the culture/diffusion chamber developed is suitable for culturing epithelial cell layers and, when subjected to rotation in the 3D-clinostat, will be a valuable in vitro system in which to study the influence of microgravity on epithelial permeability and drug transport.

  10. Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in rhesus monkeys exposed to ozone.

    PubMed Central

    Castleman, W. L.; Dungworth, D. L.; Schwartz, L. W.; Tyler, W. S.

    1980-01-01

    The pathogenesis of acute respiratory bronchiolitis was examined in rhesus monkeys exposed to 0.8 ppm ozone fpr 4--50 hours. Epithelial injury and renewal was qualitatively and quantitatively characterized by correlated techniques of scanning and transmission electron microscopy as well as by light-microscopic autoradiography following labeling with tritiated thymidine. Extensive degeneration and necrosis of Type 1 epithelial cells occurred on the respiratory bronchiolar wall during the initial 4--12 hours of exposure. Increased numbers of labeled epithelial cells were present in this region after 18 hours of exposure, and the highest labeling index (18% was measured after 50 hours of exposure. Most (67--80%) of the labeled cells and all the mitotic epithelial cells (22) observed ultrastructurally were cuboidal bronchiolar epithelial cells. Of the labeled epithelial cells, 20--33% were Type 2 epithelial cells. After 50 hours of exposure the respiratory bronchiolar epithelium was hyperplastic. The predominant inflammatory cell in respiratory bronchiolar exudate was the alveolar macrophage. Monkeys that were exposed for 50 hours and allowed to recover in unozonized air for 7 days had incomplete resolution of respiratory bronchiolar epithelial hyperplasia. The results indicate that Type 1 epithelial cells lining respiratory bronchioles are the cell type most sensitive to injury and that both cuboidal bronchiolar epithelial cells and Type 2 epithelial cells function as stem cells in epithelial renewal. Images Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 23 Figure 24 Figure 25 Figure 9 Figure 10 Figure 26 Figure 27 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 PMID:6767409

  11. DIESEL EXHAUST ENHANCES INFLUENZA VIRUS INFECTIONS IN RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    Several factors, such as age and nutritional status can affect the susceptibility to influenza infections. Moreover, exposure to air pollutants, such as diesel exhaust (DE), has been shown to affect respiratory virus infections in rodent models. Influenza virus primarily infects ...

  12. An ERp57-mediated disulphide exchange promotes the interaction between Burkholderia cenocepacia and epithelial respiratory cells

    PubMed Central

    Pacello, Francesca; D’Orazio, Melania; Battistoni, Andrea

    2016-01-01

    Previous studies have demonstrated that extracellular glutathione reduces the ability of the Cystic Fibrosis pathogen Burkholderia cenocepacia to infect primary or immortalized epithelial respiratory cells. We report here that the adhesion and invasion ability of B. cenocepacia is limited also by thiol-oxidizing and disulphide-reducing agents and by protein disulfide isomerase (PDI) inhibitors. PDI inhibitors also reduce the proinflammatory response elicited by cells in response to Burkholderia. These findings indicate that a membrane-associated PDI catalyzes thiol/disulphide exchange reactions which favor bacterial infection. The combined use of selective PDI inhibitors, RNA silencing and specific antibodies identified ERp57 as a major PDI involved in the interaction between B. cenocepacia and epithelial cells. This study contributes to the elucidation of the Burkholderia pathogenic mechanisms by showing that this microorganism exploits a membrane-associated host protein to infect epithelial cells and identifies ERp57 as a putative pharmacological target for the treatment of Burkholderia lung infections. PMID:26879174

  13. Evaluation of a rat tracheal epithelial cell culture assay system to identify respiratory carcinogens

    SciTech Connect

    Steele, V.E.; Arnold, J.T.; Arnold, J.V.; Mass, M.J. )

    1989-01-01

    To evaluate a short-term epithelial cell assay system to detect respiratory carcinogens, primary cultures of rat tracheal epithelial cells were exposed to a series of 17 compounds and scored for morphologically transformed cell colonies 28 days later. The test compounds included known carcinogens and noncarcinogens in volatile or liquid form. Tracheal epithelial cells were isolate from F344 rats, plated onto collagen-coated dishes, and exposed to the test compounds on day 1 for 24 hours. At day 30 the cultures were fixed, stained, and scored for colonies having a density greater than 1,300 cells/mm{sup 2}. With standardized protocols, such colonies are very infrequent in media and solvent control cultures. Concentration levels for each chemical were chosen over a range from nontoxic to toxic levels. Highly positive compounds in this assay included benzo(a)pyrene, benzo(l)aceanthrylene, 3-methylcholanthrene, and formaldehyde. Compounds which were negative in this assay included pyrene, benzo(e)pyrene, and 4-nitroquinoline-N-oxide. Examining the concordance of in vitro results with whole animal carcinogenesis studies revealed an accuracy of 88% with one false-positive and one false-negative compound. The results of these studies indicate that the rat tracheal epithelial cell assay may be useful in identifying potential respiratory carcinogens in our environment.

  14. Pseudomonas cepacia adherence to respiratory epithelial cells is enhanced by Pseudomonas aeruginosa

    SciTech Connect

    Saiman, L.; Cacalano, G.; Prince, A. )

    1990-08-01

    Pseudomonas aeruginosa and Pseudomonas cepacia are both opportunistic pathogens of patients with cystic fibrosis. The binding characteristics of these two species were compared to determine if they use similar mechanisms to adhere to respiratory epithelial cells. P. cepacia 249 was shown to be piliated, but there was no detectable homology between P. aeruginosa pilin gene probes and P. cepacia genomic DNA. P. cepacia and P. aeruginosa did not appear to compete for epithelial receptors. In the presence of purified P. aeruginosa pili, the adherence of 35S-labeled strain 249 to respiratory epithelial monolayers was unaffected, while that of P. aeruginosa PAO1 was decreased by 55%. The binding of P. cepacia 249 and 715j was increased by 2.4-fold and 1.5-fold, respectively, in the presence of an equal inoculum of PAO1. Interbacterial agglutination contributed to the increased adherence of P. cepacia, as the binding of 249 was increased twofold in the presence of irradiated PAO1. PAO1 exoproducts had a marked effect in enhancing the ability of the P. cepacia strains to adhere to the epithelial monolayers. A PAO1 supernatant increased the binding of 249 by eightfold and that of 715j by fourfold. Thus, there appears to be a synergistic relationship between P. aeruginosa and P. cepacia in which PAO1 exoproducts modify the epithelial cell surface, exposing receptors and facilitating increased P. cepacia attachment.

  15. Measles Virus Infection of Epithelial Cells in the Macaque Upper Respiratory Tract Is Mediated by Subepithelial Immune Cells

    PubMed Central

    Ludlow, Martin; Lemon, Ken; de Vries, Rory D.; McQuaid, Stephen; Millar, Emma L.; van Amerongen, Geert; Yüksel, Selma; Verburgh, R. Joyce; Osterhaus, Albert D. M. E.; Duprex, W. Paul

    2013-01-01

    Measles virus (MV), one of the most contagious viruses infecting humans, causes a systemic infection leading to fever, immune suppression, and a characteristic maculopapular rash. However, the specific mechanism or mechanisms responsible for the spread of MV into the respiratory epithelium in the late stages of the disease are unknown. Here we show the crucial role of PVRL4 in mediating the spread of MV from immune to epithelial cells by generating a PVRL4 “blind” recombinant wild-type MV and developing a novel in vitro coculture model of B cells with primary differentiated normal human bronchial epithelial cells. We utilized the macaque model of measles to analyze virus distribution in the respiratory tract prior to and at the peak of MV replication. Expression of PVRL4 was widespread in both the lower and upper respiratory tract (URT) of macaques, indicating MV transmission can be facilitated by more than only epithelial cells of the trachea. Analysis of tissues collected at early time points after experimental MV infection demonstrated the presence of MV-infected lymphoid and myeloid cells contacting respiratory tract epithelium in the absence of infected epithelial cells, suggesting that these immune cells seed the infection in vivo. Thereafter, lateral cell-to-cell spread of MV led to the formation of large foci of infected cells in the trachea and high levels of MV infection in the URT, particularly in the nasal cavity. These novel findings have important implications for our understanding of the high transmissibility of measles. PMID:23365435

  16. Biological effects of desert dust in respiratory epithelial cells and a murine model.

    PubMed

    Ghio, Andrew J; Kummarapurugu, Suryanaren T; Tong, Haiyan; Soukup, Joleen M; Dailey, Lisa A; Boykin, Elizabeth; Ian Gilmour, M; Ingram, Peter; Roggli, Victor L; Goldstein, Harland L; Reynolds, Richard L

    2014-04-01

    As a result of the challenge of recent dust storms to public health, we tested the postulate that desert dust collected in the southwestern United States imparts a biological effect in respiratory epithelial cells and an animal model. Two samples of surface sediment were collected from separate dust sources in northeastern Arizona. Analysis of the PM20 fraction demonstrated that the majority of both dust samples were quartz and clay minerals (total SiO₂ of 52 and 57%). Using respiratory epithelial and monocytic cell lines, the two desert dusts increased oxidant generation, measured by Amplex Red fluorescence, along with carbon black (a control particle), silica, and NIST 1649 (an ambient air pollution particle). Cell oxidant generation was greatest following exposures to silica and the desert dusts. Similarly, changes in RNA for superoxide dismutase-1, heme oxygenase-1, and cyclooxygenase-2 were also greatest after silica and the desert dusts supporting an oxidative stress after cell exposure. Silica, desert dusts, and the ambient air pollution particle NIST 1649 demonstrated a capacity to activate the p38 and ERK1/2 pathways and release pro-inflammatory mediators. Mice, instilled with the same particles, showed the greatest lavage concentrations of pro-inflammatory mediators, neutrophils, and lung injury following silica and desert dusts. We conclude that, comparable to other particles, desert dusts have a capacity to (1) influence oxidative stress and release of pro-inflammatory mediators in respiratory epithelial cells and (2) provoke an inflammatory injury in the lower respiratory tract of an animal model. The biological effects of desert dusts approximated those of silica.

  17. Biological effects of desert dust in respiratory epithelial cells and a murine model

    USGS Publications Warehouse

    Ghio, Andrew J.; Kummarapurugu, Suryanaren T.; Tong, Haiyan; Soukup, Joleen M.; Dailey, Lisa A.; Boykin, Elizabeth; Gilmour, M. Ian; Ingram, Peter; Roggli, Victor L.; Goldstein, Harland L.; Reynolds, Richard L.

    2014-01-01

    As a result of the challenge of recent dust storms to public health, we tested the postulate that desert dust collected in the southwestern United States imparts a biological effect in respiratory epithelial cells and an animal model. Two samples of surface sediment were collected from separate dust sources in northeastern Arizona. Analysis of the PM20 fraction demonstrated that the majority of both dust samples were quartz and clay minerals (total SiO2 of 52 and 57%). Using respiratory epithelial and monocytic cell lines, the two desert dusts increased oxidant generation, measured by Amplex Red fluorescence, along with carbon black (a control particle), silica, and NIST 1649 (an ambient air pollution particle). Cell oxidant generation was greatest following exposures to silica and the desert dusts. Similarly, changes in RNA for superoxide dismutase-1, heme oxygenase-1, and cyclooxygenase-2 were also greatest after silica and the desert dusts supporting an oxidative stress after cell exposure. Silica, desert dusts, and the ambient air pollution particle NIST 1649 demonstrated a capacity to activate the p38 and ERK1/2 pathways and release pro-inflammatory mediators. Mice, instilled with the same particles, showed the greatest lavage concentrations of pro-inflammatory mediators, neutrophils, and lung injury following silica and desert dusts. We conclude that, comparable to other particles, desert dusts have a capacity to (1) influence oxidative stress and release of pro-inflammatory mediators in respiratory epithelial cells and (2) provoke an inflammatory injury in the lower respiratory tract of an animal model. The biological effects of desert dusts approximated those of silica.

  18. Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

    PubMed Central

    Lin, C.; Agnes, J. T.; Behrens, N.; Tagawa, Y.; Gershwin, L. J.; Corbeil, L. B.

    2016-01-01

    Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo. PMID:26859677

  19. Group A streptococci efficiently invade human respiratory epithelial cells.

    PubMed

    LaPenta, D; Rubens, C; Chi, E; Cleary, P P

    1994-12-01

    Although infection by group A streptococci is a model of extracellular mucosal pathogenesis, these organisms can be associated with highly invasive infections resulting in sepsis and shock. Over the last 6 yr this species has renewed its reputation as a significant cause of sepsis and has piqued interest in the mechanism by which some strains are better able to breach mucosal barriers to gain access to the bloodstream than are others. An internalization assay was developed on the basis of resistance of intracellular streptococci to penicillin and gentamicin. Experiments showed that stationary-phase, as opposed to logarithmic-phase, bacteria are efficiently internalized and can persist in cultured human cells. Electron microscopy confirmed that streptococci were contained within intracellular vacuoles. Various strains of streptococci revealed significant differences in their capacity to be internalized. Two type M1 streptococci isolated from blood infections were internalized at frequencies equal to those reported for Salmonella and Listeria monocytogenes and greater than the frequency of a clonal variant from a case of pharyngitis.

  20. Particle retention by respiratory epithelial cells is associated with persistent biological effect.

    PubMed

    Soukup, Joleen M; Dailey, Lisa A; Ghio, Andrew J

    2015-01-01

    The biological effect of particles on respiratory epithelial cells involves, in part, the generation of an oxidative stress and a consequent cascade of reactions culminating in inflammatory mediator release. Whether there is either an immediate, transitory activation or a persistent response of the cells to the particles has not been established. We tested the postulate that respiratory epithelial cells exposed to wood smoke particle (WSP) would demonstrate increased oxidative stress and mediator release following re-seeding and propagation of the cells for two generations post-initial exposure. BEAS-2B cells grown to confluence (G0) in 75 cm(2) flasks were treated for 18 h with the WSP at 0, 25, 50 and 100 µg/ml. The flasks were then used to seed another set of flasks as well as 12- and 96-well plates (G1). These flasks were similarly grown to confluence and the process repeated (G2). Cell viability was assayed using trypan blue dye exclusion and was >85%. Dichlorohydrofluorescein fluorescence after exposure of BEAS-2B cells to 50 and 100 µg/ml WSP increased in all three generations when expressed as a ratio to unexposed cells. Similarly, IL-6 and IL-8 release following the initial exposure of cells to 100 µg/ml WSP increased in all three generations when expressed as a ratio to unexposed cells. The persistence of oxidative stress and inflammatory mediator release for two generations of cells beyond the initial exposure supports a postulate of continued cell response to retained particle.

  1. Development of an in vitro test to identify respiratory sensitizers in bronchial epithelial cells using gene expression profiling.

    PubMed

    Dik, Sander; Pennings, Jeroen L A; van Loveren, Henk; Ezendam, Janine

    2015-12-25

    Chemicals that induce asthma at the workplace are substances of concern. At present, there are no widely accepted methods to identify respiratory sensitizers, and classification of these substances is based on human occupational data. Several studies have contributed to understanding the mechanisms involved in respiratory sensitization, although uncertainties remain. One point of interest for respiratory sensitization is the reaction of the epithelial lung barrier to respiratory sensitizers. To elucidate potential molecular effects of exposure of the epithelial lung barrier, a gene expression profile was created based on a DNA microarray experiment using the bronchial epithelial cell line 16 HBE14o(-). The cells were exposed to 12 respiratory sensitizers and 10 non-sensitizers. For statistical analysis, we used a class prediction approach that combined three machine learning algorithms, leave-one-compound-out cross validation, and majority voting per tested compound. This approach allowed for a prediction accuracy of 95%. Identified predictive genes were mainly associated with the cytoskeleton and barrier function of the epithelial cell. Several of these genes were reported to be associated with asthma as well. Taken together, this indicates that pulmonary barrier function is an important target for respiratory sensitizers and associated genes can be used to predict the respiratory sensitization potential of chemicals.

  2. Epithelial cells, the “switchboard” of respiratory immune defense responses: effects of air pollutants

    PubMed Central

    Müller, Loretta; Jaspers, Ilona

    2015-01-01

    Summary “Epimmunome”, a term introduced recently by Swamy and colleagues, describes all molecules and pathways used by epithelial cells (ECs) to instruct immune cells. Today, we know that ECs are among the first sites within the human body to be exposed to pathogens (such as influenza viruses) and that the release of chemokine and cytokines by ECs is influenced by inhaled agents. The role of the ECs as a switchboard to initiate and regulate immune responses is altered through air pollutant exposure, such as ozone, tobacco smoke and diesel exhaust emissions. The details of the interplay between ECs and immune cells are not yet fully understood and need to be investigated further. Co-culture models, cell specific genetically-modified mice and the analysis of human biopsies provide great tools to gain knowledge about potential mechanisms. Increasing our understanding about the role of ECs in respiratory immunity may yield novel therapeutic targets to modulate downstream diseases. PMID:22851042

  3. Marked induction of matrix metalloproteinase-10 by respiratory syncytial virus infection in human nasal epithelial cells.

    PubMed

    Hirakawa, Satoshi; Kojima, Takashi; Obata, Kazufumi; Okabayashi, Tamaki; Yokota, Shin-Ichi; Nomura, Kazuaki; Obonai, Toshimasa; Fuchimoto, Jun; Himi, Tetsuo; Tsutsumi, Hiroyuki; Sawada, Norimasa

    2013-12-01

    Respiratory syncytial virus (RSV) is an important pathogen of bronchiolitis, asthma, and severe lower respiratory tract disease in infants and young children. Matrix metalloproteinases (MMPs) play key roles in viral infection, inflammation and remodeling of the airway. However, the roles and regulation of MMPs in human nasal epithelial cells (HNECs) after RSV infection remain unclear. To investigate the regulation of MMP induced after RSV infection in HNECs, an RSV-infected model of HNECs in vitro was used. It was found that mRNA of MMP-10 was markedly increased in HNECs after RSV infection, together with induction of mRNAs of MMP-1, -7, -9, and -19. The amount of MMP-10 released from HNECs was also increased in a time-dependent manner after RSV infection as was that of chemokine RANTES. The upregulation of MMP-10 in HNECs after RSV infection was prevented by inhibitors of NF-κB and pan-PKC with inhibition of RSV replication, whereas it was prevented by inhibitors of JAK/STAT, MAPK, and EGF receptors without inhibition of RSV replication. In lung tissue of an infant with severe RSV infection in which a few RSV antibody-positive macrophages were observed, MMP-10 was expressed at the apical side of the bronchial epithelial cells and alveolar epithelial cells. In conclusion, MMP-10 induced by RSV infection in HNECs is regulated via distinct signal transduction pathways with or without relation to RSV replication. MMP-10 may play an important role in the pathogenesis of RSV diseases and it has the potential to be a novel marker and therapeutic target for RSV infection.

  4. A Cross-Study Biomarker Signature of Human Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus.

    PubMed

    Gardinassi, Luiz Gustavo

    2016-01-01

    Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, elderly, and immunocompromised individuals. Despite of advances in diagnosis and treatment, biomarkers of RSV infection are still unclear. To understand the host response and propose signatures of RSV infection, previous studies evaluated the transcriptional profile of the human bronchial epithelial cell line-BEAS-2B-infected with different strains of this virus. However, the evolution of statistical methods and functional analysis together with the large amount of expression data provide opportunities to uncover novel biomarkers of inflammation and infections. In view of those facts publicly available microarray datasets from RSV-infected BEAS-2B cells were analyzed with linear model-based statistics and the platform for functional analysis InnateDB. The results from those analyses argue for the reevaluation of previously reported transcription patterns and biological pathways in BEAS-2B cell lines infected with RSV. Importantly, this study revealed a biosignature constituted by genes such as ABCC4, ARMC8, BCLAF1, EZH1, FAM118A, FAM208B, FUS, HSPH1, KAZN, MAP3K2, N6AMT1, PRMT2, S100PBP, SERPINA1, TLK2, ZNF322, and ZNF337 which should be considered in the development of new molecular diagnosis tools. PMID:27274726

  5. A Cross-Study Biomarker Signature of Human Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus

    PubMed Central

    Gardinassi, Luiz Gustavo

    2016-01-01

    Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, elderly, and immunocompromised individuals. Despite of advances in diagnosis and treatment, biomarkers of RSV infection are still unclear. To understand the host response and propose signatures of RSV infection, previous studies evaluated the transcriptional profile of the human bronchial epithelial cell line—BEAS-2B—infected with different strains of this virus. However, the evolution of statistical methods and functional analysis together with the large amount of expression data provide opportunities to uncover novel biomarkers of inflammation and infections. In view of those facts publicly available microarray datasets from RSV-infected BEAS-2B cells were analyzed with linear model-based statistics and the platform for functional analysis InnateDB. The results from those analyses argue for the reevaluation of previously reported transcription patterns and biological pathways in BEAS-2B cell lines infected with RSV. Importantly, this study revealed a biosignature constituted by genes such as ABCC4, ARMC8, BCLAF1, EZH1, FAM118A, FAM208B, FUS, HSPH1, KAZN, MAP3K2, N6AMT1, PRMT2, S100PBP, SERPINA1, TLK2, ZNF322, and ZNF337 which should be considered in the development of new molecular diagnosis tools. PMID:27274726

  6. INHIBITION OF RESPIRATORY SYNCYTIAL VIRUS (RSV)-INDUCED INFLAMMATION BY 3-NITROTYROSINE IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Inhibition of Respiratory Syncytial Virus (RSV)-Induced Inflammation by 3-Nitrotyrosine in Human Bronchial Epithelial Cells. J. M. Soukup, MPH 1, ZW. Li, MD 2 and YC. T. Huang, MD 1. 1 NHEERL, US Environmental Protection Agency, RTP, NC and 2 CEMALB, University of North Carolina,...

  7. Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway.

    PubMed

    Bian, Tao; Gibbs, John D; Örvell, Claes; Imani, Farhad

    2012-01-01

    Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.

  8. Respiratory Syncytial Virus Matrix Protein Induces Lung Epithelial Cell Cycle Arrest through a p53 Dependent Pathway

    PubMed Central

    Bian, Tao; Gibbs, John D.; Örvell, Claes; Imani, Farhad

    2012-01-01

    Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication. PMID:22662266

  9. Gene expressions changes in bronchial epithelial cells: markers for respiratory sensitizers and exploration of the NRF2 pathway.

    PubMed

    Remy, Sylvie; Verstraelen, Sandra; Van Den Heuvel, Rosette; Nelissen, Inge; Lambrechts, Nathalie; Hooyberghs, Jef; Schoeters, Greet

    2014-03-01

    For the classification of respiratory sensitizing chemicals, no validated in vivo nor in vitro tests are currently available. In this study, we evaluated whether respiratory sensitizers trigger specific signals in human bronchial epithelial (BEAS-2B) cells at the level of the transcriptome. The cells were exposed during 6, 10, and 24h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4×44K oligonucleotide arrays. A limited number of 11 transcripts could be identified as potential biomarkers to identify respiratory sensitizers. Three of these transcripts are associated to immune system processes (HSPA5, UPP1, and SEPRINE1). In addition, the transcriptome was screened for transcripts that are differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that - at the level of gene expression - this pathway shows no potential to discriminate between any of the three compound groups: respiratory sensitizers, skin sensitizers, or electrophilic respiratory irritants. PMID:24211530

  10. Bilateral Entry and Release of Middle East Respiratory Syndrome Coronavirus Induces Profound Apoptosis of Human Bronchial Epithelial Cells

    PubMed Central

    Tao, Xinrong; Hill, Terence E.; Morimoto, Chikao; Peters, Clarence J.; Ksiazek, Thomas G.

    2013-01-01

    The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) infects human bronchial epithelial Calu-3 cells. Unlike severe acute respiratory syndrome (SARS)-CoV, which exclusively infects and releases through the apical route, this virus can do so through either side of polarized Calu-3 cells. Infection results in profound apoptosis within 24 h irrespective of its production of titers that are lower than those of SARS-CoV. Together, our results provide new insights into the dissemination and pathogenesis of MERS-CoV and may indicate that the virus differs markedly from SARS-CoV. PMID:23824802

  11. Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    PubMed Central

    2009-01-01

    Background Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response. Results The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in

  12. Characterization of antibody inhibiting adherence of Bordetella pertussis to human respiratory epithelial cells.

    PubMed Central

    Tuomanen, E I; Zapiain, L A; Galvan, P; Hewlett, E L

    1984-01-01

    We have recently established the topographic specificity of the adherence of Bordetella pertussis to human ciliated respiratory epithelial cells. For this study, we employed the same quantitative, immunofluorescent adherence assay to test the possibility that sera of patients recovering from naturally acquired whooping cough or immunized with pertussis vaccine may contain activity capable of interfering with this specific adherence. Evaluation of paired sera from six children with culture-proven pertussis demonstrated that antiadherence activity appeared in serum during convalescence from disease. Nine children immunized with diptheria-pertussin-tetanus vaccine also showed activity against adherence, although it was significantly less than in those with clinical disease. Naturally acquired serum antiadherence activity was identified in both immunoglobulin G (IgG) and IgA antibody classes, whereas, as expected, only IgG antibody was present in children receiving the parenteral vaccine. The findings suggest that natural infection or vaccination are associated with the acquisition of serum activity inhibiting the adherence of B. pertussis to ciliated cells. Immunization may fail to elicit IgA antiadherence activity. PMID:6092416

  13. Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium

    PubMed Central

    Faron, Matthew; Fletcher, Joshua R.; Rasmussen, Jed A.; Apicella, Michael A.; Jones, Bradley D.

    2015-01-01

    Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our

  14. Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

    PubMed

    Schwagerus, Elena; Sladek, Svenja; Buckley, Stephen T; Armas-Capote, Natalia; Alvarez de la Rosa, Diego; Harvey, Brian J; Fischer, Horst; Illek, Beate; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

    2015-11-01

    Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

  15. A novel role for β2-microglobulin: a precursor of antibacterial chemokine in respiratory epithelial cells.

    PubMed

    Chiou, Shean-Jaw; Wang, Chan-Chi; Tseng, Yan-Shen; Lee, Yen-Jung; Chen, Shih-Chieh; Chou, Chi-Hsien; Chuang, Lea-Yea; Hong, Yi-Ren; Lu, Chi-Yu; Chiu, Chien-Chih; Chignard, Michel

    2016-01-01

    We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1β and different pathogen-associated molecular patterns. A 9 kDa fragment derived from β2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration. PMID:27503241

  16. A novel role for β2-microglobulin: a precursor of antibacterial chemokine in respiratory epithelial cells

    PubMed Central

    Chiou, Shean-Jaw; Wang, Chan-Chi; Tseng, Yan-Shen; Lee, Yen-Jung; Chen, Shih-Chieh; Chou, Chi-Hsien; Chuang, Lea-Yea; Hong, Yi-Ren; Lu, Chi-Yu; Chiu, Chien-Chih; Chignard, Michel

    2016-01-01

    We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1β and different pathogen-associated molecular patterns. A 9 kDa fragment derived from β2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration. PMID:27503241

  17. Roflumilast Inhibits Respiratory Syncytial Virus Infection in Human Differentiated Bronchial Epithelial Cells

    PubMed Central

    Mata, Manuel; Martinez, Isidoro; Melero, Jose A.; Tenor, Herman; Cortijo, Julio

    2013-01-01

    Respiratory syncytial virus (RSV) causes acute exacerbations in COPD and asthma. RSV infects bronchial epithelial cells (HBE) that trigger RSV associated lung pathology. This study explores whether the phosphodiesterase 4 (PDE4) inhibitor Roflumilast N-oxide (RNO), alters RSV infection of well-differentiated HBE (WD-HBE) in vitro. WD-HBE were RSV infected in the presence or absence of RNO (0.1-100 nM). Viral infection (staining of F and G proteins, nucleoprotein RNA level), mRNA of ICAM-1, ciliated cell markers (digital high speed videomicroscopy, β-tubulin immunofluorescence, Foxj1 and Dnai2 mRNA), Goblet cells (PAS), mRNA of MUC5AC and CLCA1, mRNA and protein level of IL-13, IL-6, IL-8, TNFα, formation of H2O2 and the anti-oxidative armamentarium (mRNA of Nrf2, HO-1, GPx; total antioxidant capacity (TAC) were measured at day 10 or 15 post infection. RNO inhibited RSV infection of WD-HBE, prevented the loss of ciliated cells and markers, reduced the increase of MUC5AC and CLCA1 and inhibited the increase of IL-13, IL-6, IL-8, TNFα and ICAM-1. Additionally RNO reversed the reduction of Nrf2, HO-1 and GPx mRNA levels and consequently restored the TAC and reduced the H2O2 formation. RNO inhibits RSV infection of WD-HBE cultures and mitigates the cytopathological changes associated to this virus. PMID:23936072

  18. Characterization of the Molecular Interplay between Moraxella catarrhalis and Human Respiratory Tract Epithelial Cells

    PubMed Central

    de Vries, Stefan P. W.; Eleveld, Marc J.; Hermans, Peter W. M.; Bootsma, Hester J.

    2013-01-01

    Moraxella catarrhalis is a mucosal pathogen that causes childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. During the course of infection, M. catarrhalis needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs, and consequently, efficient adhesion to epithelial cells is considered an important virulence trait of M. catarrhalis. By using Tn-seq, a genome-wide negative selection screenings technology, we identified 15 genes potentially required for adherence of M. catarrhalis BBH18 to pharyngeal epithelial Detroit 562 and lung epithelial A549 cells. Validation with directed deletion mutants confirmed the importance of aroA (3-phosphoshikimate 1-carboxyvinyl-transferase), ecnAB (entericidin EcnAB), lgt1 (glucosyltransferase), and MCR_1483 (outer membrane lipoprotein) for cellular adherence, with ΔMCR_1483 being most severely attenuated in adherence to both cell lines. Expression profiling of M. catarrhalis BBH18 during adherence to Detroit 562 cells showed increased expression of 34 genes in cell-attached versus planktonic bacteria, among which ABC transporters for molybdate and sulfate, while reduced expression of 16 genes was observed. Notably, neither the newly identified genes affecting adhesion nor known adhesion genes were differentially expressed during adhesion, but appeared to be constitutively expressed at a high level. Profiling of the transcriptional response of Detroit 562 cells upon adherence of M. catarrhalis BBH18 showed induction of a panel of pro-inflammatory genes as well as genes involved in the prevention of damage of the epithelial barrier. In conclusion, this study provides new insight into the molecular interplay between M. catarrhalis and host epithelial cells during the process of adherence. PMID:23936538

  19. Activation of respiratory epithelial cells by wood smoke particles persists beyond immediate exposure.

    EPA Science Inventory

    The biological effect of particles on epithelial cells involves, in part, oxidant generation and a cascade of reactions culminating in inflammatory mediator release. Whether there is an immediate short-lived activation or continued persistent response of the cells to the particle...

  20. Pulmonary Alveolar Type II Epithelial Cells and Adult Respiratory Distress Syndrome

    PubMed Central

    Mason, Robert J.

    1985-01-01

    During the past ten years, functions of alveolar type II cells have been well characterized with isolated cells in vitro. Some of the functions were well known from studies in vivo, but others such as transepithelial sodium transport were unsuspected. A better understanding of this important pulmonary cell type improves our knowledge of the pathophysiology of adult respiratory distress syndrome and may in time lead to new therapeutic strategies. ImagesFigure 1.Figure 2.Figure 3.Figure 4. PMID:3909639

  1. Identification of gene biomarkers for respiratory synctial virus infection in a bronchical epithelial cell line

    EPA Science Inventory

    Abstract: Respiratory syncytial virus (RSV) infection involves complex virus-host interplay. In this study, we analyzed gene expression in RSV-infected BEAS-2B cells to discover novel signaling pathways and biomarkers. We hybridized RNAs from RSV- or vehicle-treated BEAS-2B to ...

  2. Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response

    PubMed Central

    Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Groshong, Steve D.; Ito, Yoko; Travanty, Emily A.; Leete, Jennifer; Holmes, Kathryn V.; Mason, Robert J.

    2009-01-01

    The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6–8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host. PMID:19741068

  3. Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge.

    PubMed

    Hartwig, Stacey M; Ketterer, Margaret; Apicella, Michael A; Varga, Steven M

    2016-11-01

    Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Co-culture of 16HBE14o- cells with NTHi resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSV-specific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection.

  4. Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge.

    PubMed

    Hartwig, Stacey M; Ketterer, Margaret; Apicella, Michael A; Varga, Steven M

    2016-11-01

    Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Co-culture of 16HBE14o- cells with NTHi resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSV-specific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection. PMID:27573069

  5. Respiratory epithelial cysts of the orbit.

    PubMed

    Goh, Rachel L Z; Hardy, Thomas G; Williams, Richard A; McNab, Alan A

    2016-10-01

    To describe post-traumatic and congenital respiratory epithelial cysts in the orbit, which are rare lesions with only 5 and 13 published cases, respectively. We reviewed all cases of respiratory epithelial cysts diagnosed at three institutions (two tertiary referral hospitals, one private clinic) between 1995 and 2015. We describe 10 cases of post-traumatic respiratory epithelial cyst (age range 23 - 82), presenting a mean of 17.4 years after their original trauma; and 3 congenital cases (age range 17-34). All but one case underwent surgical excision of the cyst and its lining, along with any surgical implant within the cyst. Two were recurrent after incomplete excision. Three presented with acute infection within the cyst. Respiratory epithelial orbital cysts are probably commoner than the paucity of published reports would suggest. Post-traumatic cysts often present many years after trauma, and may become secondarily infected. Complete surgical removal is recommended to prevent future recurrence. PMID:27468088

  6. Respiratory epithelial cysts of the orbit.

    PubMed

    Goh, Rachel L Z; Hardy, Thomas G; Williams, Richard A; McNab, Alan A

    2016-10-01

    To describe post-traumatic and congenital respiratory epithelial cysts in the orbit, which are rare lesions with only 5 and 13 published cases, respectively. We reviewed all cases of respiratory epithelial cysts diagnosed at three institutions (two tertiary referral hospitals, one private clinic) between 1995 and 2015. We describe 10 cases of post-traumatic respiratory epithelial cyst (age range 23 - 82), presenting a mean of 17.4 years after their original trauma; and 3 congenital cases (age range 17-34). All but one case underwent surgical excision of the cyst and its lining, along with any surgical implant within the cyst. Two were recurrent after incomplete excision. Three presented with acute infection within the cyst. Respiratory epithelial orbital cysts are probably commoner than the paucity of published reports would suggest. Post-traumatic cysts often present many years after trauma, and may become secondarily infected. Complete surgical removal is recommended to prevent future recurrence.

  7. INFLUENZA-INDUCED UP-REGULATION OF TLR3 IN RESPIRATORY EPITHELIAL CELLS MAY OCCUR THROUGH A POSITIVE FEEDBACK LOOP INVOLVING TYPE I INTERFERON

    EPA Science Inventory

    Toll-like receptor 3 (TLR3) plays an important role in the host defense responses against viral infections, including Influenza virus infections. Based on our previous observations showing that Influenza infection of respiratory epithelial cells results in an up-regulation of Tol...

  8. Increased mucociliary differentiation and aquaporins formation of respiratory epithelial cells on retinoic acid-loaded hyaluronan-derivative membranes.

    PubMed

    Huang, Tsung-Wei; Chan, Yen-Hui; Su, Huang-Wei; Chou, Ya-Shuan; Young, Tai-Horng

    2013-06-01

    While playing a major role in maintaining the mucociliary phenotype of respiratory epithelial cells (RECs), retinoids are critical determinants of their normal function. However, despite being a powerful biological agent, retinoic acid (RA) is generally not used in regenerative medicine due to its scarce bioavailability via conventional administration. Therefore, the ability to incorporate RA into biomaterials allows for a combination of the biological effects of RA and biomaterials in influencing cellular behavior. This study attempts to develop RA-loaded hyaluronan-derivative membrane (RA-HAm) and investigates how this membrane affects the mucociliary differentiation and aquaporins (AQP) formation of RECs. In a simulated in vitro culture condition, the RA release from membranes is maintained for 7days. On the seventh day, the cumulative release rate of RA from supportive biomaterials is ~87% under detect limitation. RECs cultured on RA-HAm reveal numerous mature ciliated cells and microvilli compared to aggregated cilia-like structures on hyaluronan-derivative membrane (HAm). Moreover, the expression levels of MUC5AC and AQP on RA-HAm are higher than those on HAm. The proposed model elucidates the release of hydrophobic RA from hyaluronan-derivative biomaterials. We believe that RA-loaded hyaluronan biomaterials are highly promising biomaterials for use in sinonasal surgery and tissue engineering of the respiratory system.

  9. Respiratory Syncytial Virus Inhibits Ciliagenesis in Differentiated Normal Human Bronchial Epithelial Cells: Effectiveness of N-Acetylcysteine

    PubMed Central

    Mata, Manuel; Sarrion, Irene; Armengot, Miguel; Carda, Carmen; Martinez, Isidoro; Melero, Jose A.; Cortijo, Julio

    2012-01-01

    Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H2O2 levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD. PMID:23118923

  10. Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-acetylcysteine.

    PubMed

    Mata, Manuel; Sarrion, Irene; Armengot, Miguel; Carda, Carmen; Martinez, Isidoro; Melero, Jose A; Cortijo, Julio

    2012-01-01

    Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H(2)O(2) levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD. PMID:23118923

  11. Respiratory syncytial virus inhibits ciliagenesis in differentiated normal human bronchial epithelial cells: effectiveness of N-acetylcysteine.

    PubMed

    Mata, Manuel; Sarrion, Irene; Armengot, Miguel; Carda, Carmen; Martinez, Isidoro; Melero, Jose A; Cortijo, Julio

    2012-01-01

    Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H(2)O(2) levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD.

  12. ERK1/2 activation modulates pyocyanin-induced toxicity in A549 respiratory epithelial cells.

    PubMed

    Forbes, Amanda; Davey, Andrew K; Perkins, Anthony V; Grant, Gary D; McFarland, Amelia J; McDermott, Catherine M; Anoopkumar-Dukie, Shailendra

    2014-02-01

    Pyocyanin (PCN), a virulence factor produced by Pseudomonas aeruginosa, has many damaging effects on mammalian cells. Several lines of evidence suggest that this damage is primarily mediated by its ability to generate oxidative stress. However mechanisms underlying PCN-induced oxidative injury remain unclear. Although oxidative stress and subsequent MAPK signaling has been shown to modulate cell death in other models, its role in PCN-induced cytotoxicity remains unknown. Therefore the aim of this study was to investigate the role of redox-sensitive MAPK in PCN-induced toxicity in A549 cells. Here we show that PCN (50μM) rapidly increased ERK1/2 phosphorylation after 5min. Pre-treatment of A549 cells with the MEK1/2 inhibitor U0126 (10μM) decreased PCN-induced ERK1/2 phosphorylation and protected cells against apoptosis and cell injury suggesting a role for ERK signalling. In contrast, JNK and p38 MAPK phosphorylation remained unchanged following exposure to PCN and pretreatment with either the JNK or p38 MAPK inhibitors (10μM SP600125 and 10μM SB203580, respectively) did not afford protection against PCN toxicity. This would suggest that PCN-induced cytotoxicity appears to occur independently of JNK and p38 MAPK signaling pathways. Finally, although we confirm that oxidative stress contributes to PCN-induced toxicity, our data suggest the contribution of oxidative stress is independent of ERK1/2 signaling. These findings may provide insight for novel targeted therapies to reduce PCN-mediated lung injury in patients with chronic P. aeruginosa respiratory infections.

  13. Selective targeting of alveolar type II respiratory epithelial cells by anti-surfactant protein-C antibody-conjugated lipoplexes

    PubMed Central

    Wu, Yun; Ma, Junyu; Woods, Parker S.; Chesarino, Nicholas M.; Liu, Chang; Lee, L. James; Nana-Sinkam, Serge P.; Davis, Ian C.

    2015-01-01

    Alveolar type II (ATII) respiratory epithelial cells are essential to normal lung function. They may be also central to the pathogenesis of diseases such as acute lung injury, pulmonary fibrosis, and pulmonary adenocarcinoma. Hence, ATII cells are important therapeutic targets. However, effective ATII cell-specific drug delivery in vivo requires carriers of an appropriate size, which can cross the hydrophobic alveolar surfactant film and polar aqueous layer overlying ATII cells, and be taken up without inducing ATII cell dysfunction, pulmonary inflammation, lung damage, or excessive systemic spread and side-effects. We have developed lipoplexes as a versatile nanoparticle carrier system for drug/RNA delivery. To optimize their pulmonary localization and ATII cell specificity, lipoplexes were conjugated to an antibody directed against the ATII cell-specific antigen surfactant protein-C (SP-C) then administered to C57BL/6 mice via the nares. Intranasally-administered, anti-SP-C-conjugated lipoplexes targeted mouse ATII cells with >70% specificity in vivo, were retained within ATII cells for at least 48 hours, and did not accumulate at significant levels in other lung cell types or viscera. 48 hours after treatment with anti-SP-C-conjugated lipoplexes containing the test microRNA miR-486, expression of mature miR-486 was approximately 4-fold higher in ATII cells than whole lung by qRT-PCR, and was undetectable in other viscera. Lipoplexes induced no weight loss, hypoxemia, lung dysfunction, pulmonary edema, or pulmonary inflammation over a 6-day period. These findings indicate that ATII cell-targeted lipoplexes exhibit all the desired characteristics of an effective drug delivery system for treatment of pulmonary diseases that result primarily from ATII cell dysfunction. PMID:25687308

  14. DIFFERENTIAL GENE EXPRESSION INDUCED BY RESPIRATORY SYNCTIAL VIRUS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Respiratory syncytial virus (RSV), a negative-stranded RNA virus, is a common viral pathogen for respiratory infection in both children and immunocompromised adults. Early host defense may play a critical role in determining the severity of the infection. To gain further insight ...

  15. Effect of oral N-acetylcysteine (NAC) on volume and albumin content of respiratory tract fluid but not on epithelial secretory cell number in "smoking" rats.

    PubMed

    Robinson, N; Brattsand, R; Dahlbäck, M

    1990-03-01

    This study was designed to look at the effect of N-acetylcysteine (NAC) on epithelial secretory cells and the respiratory tract fluid volume and albumin content from the lower airways of "bronchitic" rats. Rats were exposed either to tobacco smoke (TS), TS and NAC, or NAC alone. TS caused a significant increase in epithelial secretory cell number which was not reduced by concomitant NAC administration; NAC alone had no effect on cell numbers. TS increased respiratory tract fluid volume and albumin content by a small but non-significant amount, whereas TS and NAC increased the volume and albumin content by a greater and significant amount; NAC alone was also shown to significantly increase both fluid volume and albumin content. PMID:2340888

  16. Effect of oral N-acetylcysteine (NAC) on volume and albumin content of respiratory tract fluid but not on epithelial secretory cell number in "smoking" rats.

    PubMed

    Robinson, N; Brattsand, R; Dahlbäck, M

    1990-03-01

    This study was designed to look at the effect of N-acetylcysteine (NAC) on epithelial secretory cells and the respiratory tract fluid volume and albumin content from the lower airways of "bronchitic" rats. Rats were exposed either to tobacco smoke (TS), TS and NAC, or NAC alone. TS caused a significant increase in epithelial secretory cell number which was not reduced by concomitant NAC administration; NAC alone had no effect on cell numbers. TS increased respiratory tract fluid volume and albumin content by a small but non-significant amount, whereas TS and NAC increased the volume and albumin content by a greater and significant amount; NAC alone was also shown to significantly increase both fluid volume and albumin content.

  17. EFFECTS OF DIESEL EXHAUST ON TLR3 SIGNALING IN RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    There are a variety of intrinsic as well as extrinsic factors, such as exposure to air pollution that can affect the pathogenesis of respiratory infections. Diesel exhaust (DE) emissions can significantly contribute to air pollution levels and exposure to DE can alter host defens...

  18. NITROTYROSINE INHIBITS RESPIRATORY SYNCTIAL VIRUS-INDUCED RANTES PRODUCTION IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    3-Nitrotyrosine (NO2Tyr) produced during inflammation can substitute the C-terminus tyrosine of a-tubulin post-translationally altering microtubular functions. Since propagation of respiratory syncytial virus (RSV) infection may require an intact microtubular activity, we tested ...

  19. CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells

    PubMed Central

    Chirkova, Tatiana; Lin, Songbai; Oomens, Antonius G. P.; Gaston, Kelsey A.; Boyoglu-Barnum, Seyhan; Meng, Jia; Stobart, Christopher C.; Cotton, Calvin U.; Hartert, Tina V.; Moore, Martin L.; Ziady, Assem G.

    2015-01-01

    Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection. PMID:26297201

  20. Use of a feline respiratory epithelial cell culture system grown at the air-liquid interface to characterize the innate immune response following feline herpesvirus 1 infection.

    PubMed

    Nelli, Rahul K; Maes, Roger; Kiupel, Matti; Hussey, Gisela Soboll

    2016-03-01

    Infection with feline herpesvirus-1 (FHV-1) accounts for 50% of viral upper respiratory diseases in domestic cats and is a significant cause of ocular diseases. Despite the clinical significance and high prevalence of FHV-1 infection, currently available vaccines cannot completely protect cats from infection and lifelong latency. FHV-1 infects via the mucous membranes and replicates in respiratory epithelial cells, but very little is known about the early innate immunity at this site. To address questions about immunity to FHV-1, feline respiratory epithelial cells cultured at air-liquid interface (ALI-FRECs) were established by collecting respiratory tracts from 6 healthy cats after euthanasia. Cells were isolated, cultured and characterized histologically and immunologically before infection with FHV-1. The expression of Toll-like receptors (TLRs), cytokine and chemokine responses were measured by real time PCR. ALI-FRECs morphologically resembled the natural airways of cats with multilayered columnar epithelial cells and cilia. Immunological properties of the natural airways were maintained in ALI-FRECs, as evidenced by the expression of TLRs, cytokines, chemokines, interferons, beta-defensins, and other regulatory genes. Furthermore, ALI-FRECs were able to support infection and replication of FHV-1, as well as modulate transcriptional regulation of various immune genes in response to infection. IL-1β and TNFα were increased in ALI-FRECs by 24hpi, whereas expression levels of IFN-α and TLR9 were not increased until 36hpi. In contrast, TLR3, GM-CSF and TGF-1β expression was down-regulated at 36hpi. The data presented show the development of a system ideal for investigating the molecular pathogenesis and immunity of FHV-1 or other respiratory pathogens.

  1. The Environment of “Mycobacterium avium subsp. hominissuis” Microaggregates Induces Synthesis of Small Proteins Associated with Efficient Infection of Respiratory Epithelial Cells

    PubMed Central

    Babrak, Lmar; Danelishvili, Lia; Rose, Sasha J.; Kornberg, Tiffany

    2014-01-01

    “Mycobacterium avium subsp. hominissuis” is an opportunistic environmental pathogen that causes respiratory illness in immunocompromised patients, such as those with cystic fibrosis as well as other chronic respiratory diseases. Currently, there is no efficient approach to prevent or treat M. avium subsp. hominissuis infection in the lungs. During initial colonization of the airways, M. avium subsp. hominissuis forms microaggregates composed of 3 to 20 bacteria on human respiratory epithelial cells, which provides an environment for phenotypic changes leading to efficient mucosal invasion in vitro and in vivo. DNA microarray analysis was employed to identify genes associated with the microaggregate phenotype. The gene encoding microaggregate-binding protein 1 (MBP-1) (MAV_3013) is highly expressed during microaggregate formation. When expressed in noninvasive Mycobacterium smegmatis, MBP-1 increased the ability of the bacteria to bind to HEp-2 epithelial cells. Using anti-MBP-1 immune serum, microaggregate binding to HEp-2 cells was significantly reduced. By far-Western blotting, and verified by coimmunoprecipitation, we observed that MBP-1 interacts with the host cytoskeletal protein vimentin. As visualized by confocal microscopy, microaggregates, as well as MBP-1, induced vimentin polymerization at the site of bacterium-host cell contact. Binding of microaggregates to HEp-2 cells was inhibited by treatment with an antivimentin antibody, suggesting that MBP-1 expression is important for M. avium subsp. hominissuis adherence to the host cell. MBP-1 immune serum significantly inhibited M. avium subsp. hominissuis infection throughout the respiratory tracts of mice. This study characterizes a pathogenic mechanism utilized by M. avium subsp. hominissuis to bind and invade the host respiratory epithelium, suggesting new potential targets for the development of antivirulence therapy. PMID:25422262

  2. Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia

    SciTech Connect

    Jumat, Muhammad Raihan; Yan, Yan; Ravi, Laxmi Iyer; Wong, Puisan; Huong, Tra Nguyen; Li, Chunwei; Tan, Boon Huan; Wang, De Yun; Sugrue, Richard J.

    2015-10-15

    The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5 dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss. - Highlights: • Respiratory syncytial virus (RSV) infects nasal ciliated epithelial cells. • Virus morphogenesis occurs within filamentous projections distinct from cilia. • The RSV N protein was not detected in the cilia at any time during infection. • Trafficking of the F protein into the cilia occurred early in infection. • Presence of the F protein in cilia correlated with impaired cilia function.

  3. Comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients.

    PubMed

    Daley, Peter; Castriciano, Santina; Chernesky, Max; Smieja, Marek

    2006-06-01

    Significantly more epithelial cells were collected by flocked swabs than by rayon swabs in parallel nasopharyngeal and nasal swabs taken from 16 volunteers. Nasopharyngeal sampling of 61 symptomatic patients also yielded more cells by flocked than rayon swabs, providing better clinical specimens for diagnosis.

  4. Model for assessing radiation dose to epithelial cells of the human respiratory tract from radon progeny

    SciTech Connect

    Fisher, D.R.; Hui, T.E.; James, A.C.

    1990-07-01

    A computational model was developed to evaluate radiation doses to sensitive cells from exposure to radon progeny throughout human bronchial epithelium. The model incorporated current information on nasal and oral filtration efficiencies for unattached radon progeny, characteristics of bronchial deposition by diffusive and inertial processes, mucous clearance and possible transfer of radon progeny to the airway epithelium, locations of target nuclei of secretory and basal cells in different regions of the bronchial tree epithelium, and other features. The model is useful for evaluating absorbed doses to various populations of target cell nuclei, the associated microdosimetric probability densities in specific energy, and the likelihood that target nuclei are hit one or more times by alpha-particle tracks. The model was applied to extrapolating lung cancer risks observed in underground miners to the general population exposed to low-level radon progeny in indoor home environments. The effect of increasing exposure rates by one and two orders of magnitude in both environments was modeled to determine the frequency of radiation events in target cell nuclei. The implications of dosimetric modeling for lung cancer risk analysis were also examined. 28 refs., 5 figs., 5 tabs.

  5. The roles of epithelial cell contact, respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae.

    PubMed

    Krishnamurthy, Ajay; Kyd, Jennelle

    2014-08-01

    Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) often share a common niche within the nasopharynx, both associated with infections such as bronchitis and otitis media. This study investigated how the association between NTHi and S. pneumoniae and the host affects their propensity to form biofilms. We investigated a selection of bacterial strain and serotype combinations on biofilm formation, and the effect of contact with respiratory epithelial cells. Measurement of biofilm showed that co-infection with NTHi and S. pneumoniae increased biofilm formation following contact with epithelial cells compared to no contact demonstrating the role of epithelial cells in biofilm formation. Additionally, the influence of phosphorylcholine (ChoP) on biofilm production was investigated using the licD mutant strain of NTHi 2019 and found that ChoP had a role in mixed biofilm formation but was not the only requirement. The study highlights the complex interactions between microbes and the host epithelium during biofilm production, suggesting the importance of understanding why certain strains and serotypes differentially influence biofilm formation. A key contributor to increased biofilm formation was the upregulation of biofilm formation by epithelial cell factors.

  6. Comparative in vitro study of interactions between particles and respiratory surface macrophages, erythrocytes, and epithelial cells of the chicken and the rat

    PubMed Central

    Kiama, S G; Adekunle, J S; Maina, J N

    2008-01-01

    Abstract In mammals, surface macrophages (SMs) play a foremost role in protecting the respiratory system by engulfing and destroying inhaled pathogens and harmful particulates. However, in birds, the direct defense role(s) that SMs perform remains ambiguous. Paucity and even lack of SMs have been reported in the avian respiratory system. It has been speculated that the pulmonary defenses in birds are inadequate and that birds are exceptionally susceptible to pulmonary diseases. In an endeavour to resolve the existing controversy, the phagocytic capacities of the respiratory SMs of the domestic fowl and the rat were compared under similar experimental conditions by exposure to polystyrene particles. In cells of equivalent diameters (8.5 µm in the chicken and 9.0 µm in the rat) and hence volumes, with the volume density of the engulfed polystyrene particles, i.e. the volume of the particles per unit volume of the cell (SM) of 23% in the chicken and 5% in the rat cells, the avian cells engulfed substantially more particles. Furthermore, the avian SMs phagocytized the particles more efficiently, i.e. at a faster rate. The chicken erythrocytes and the epithelial cells of the airways showed noteworthy phagocytic activity. In contrast to the rat cells that did not, 22% of the chicken erythrocytes phagocytized one to six particles. In birds, the phagocytic efficiencies of the SMs, erythrocytes, and epithelial cells may consolidate pulmonary defense. The assorted cellular defenses may explain how and why scarcity of SMs may not directly lead to a weak pulmonary defense. The perceived susceptibility of birds to respiratory diseases may stem from the human interventions that have included extreme genetic manipulation and intensive management for maximum productivity. The stress involved and the structural–functional disequilibria that have occurred from a ‘directed evolutionary process’, rather than weak immunological and cellular immunity, may explain the alleged

  7. Comparative in vitro study of interactions between particles and respiratory surface macrophages, erythrocytes, and epithelial cells of the chicken and the rat.

    PubMed

    Kiama, S G; Adekunle, J S; Maina, J N

    2008-10-01

    In mammals, surface macrophages (SMs) play a foremost role in protecting the respiratory system by engulfing and destroying inhaled pathogens and harmful particulates. However, in birds, the direct defense role(s) that SMs perform remains ambiguous. Paucity and even lack of SMs have been reported in the avian respiratory system. It has been speculated that the pulmonary defenses in birds are inadequate and that birds are exceptionally susceptible to pulmonary diseases. In an endeavour to resolve the existing controversy, the phagocytic capacities of the respiratory SMs of the domestic fowl and the rat were compared under similar experimental conditions by exposure to polystyrene particles. In cells of equivalent diameters (8.5 microm in the chicken and 9.0 microm in the rat) and hence volumes, with the volume density of the engulfed polystyrene particles, i.e. the volume of the particles per unit volume of the cell (SM) of 23% in the chicken and 5% in the rat cells, the avian cells engulfed substantially more particles. Furthermore, the avian SMs phagocytized the particles more efficiently, i.e. at a faster rate. The chicken erythrocytes and the epithelial cells of the airways showed noteworthy phagocytic activity. In contrast to the rat cells that did not, 22% of the chicken erythrocytes phagocytized one to six particles. In birds, the phagocytic efficiencies of the SMs, erythrocytes, and epithelial cells may consolidate pulmonary defense. The assorted cellular defenses may explain how and why scarcity of SMs may not directly lead to a weak pulmonary defense. The perceived susceptibility of birds to respiratory diseases may stem from the human interventions that have included extreme genetic manipulation and intensive management for maximum productivity. The stress involved and the structural-functional disequilibria that have occurred from a 'directed evolutionary process', rather than weak immunological and cellular immunity, may explain the alleged

  8. Effect of respiratory syncytial virus (RSV) infection on the adherence of pathogenic bacteria to human epithelial cells

    SciTech Connect

    Faden, H.; Hong, J.J.; Ogra, P.L.

    1986-03-01

    The effect of RSV infection on the adherence of Streptococcus pneumoniae (SP), Haemophilus influenzae (HI) and Staphylococcus aureus (SA) to human epithelial cells was determined. RSV-infected Hep-2 cell cultures at different stages of expression of surface viral antigens and bacteria labeled with /sup 3/H-thymidine were employed to examine the kinetics of bacterial adherence to virus-infected cells. RSV infection did not alter the magnitude of adherence of HI or SA to HEp-2 cells. However, adherence of SP to HEp-2 cells was significantly (P < 0.01) enhanced by prior RSV infection. The degree of adherence was directly related to the amount of viral antigen expressed on the cell surface. The adherence was temperature dependent, with maximal adherence observed at 37/sup 0/C. Heat-inactivation of SP did not alter adherence characteristics. These data suggest that RSV infection increases adherence of SP to the surface of epithelial cells in vitro. Since attachment of bacteria to mucosal surfaces is the first step in many infections, it is suggested that viral infections of epithelial cells render them more susceptible to bacterial adherence. Thus, RSV infection in vivo may predispose children to SP infections, such as in otitis media, by increasing colonization with SP.

  9. A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens.

    PubMed

    Hiono, Takahiro; Okamatsu, Masatoshi; Nishihara, Shoko; Takase-Yoden, Sayaka; Sakoda, Yoshihiro; Kida, Hiroshi

    2014-05-01

    Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR.

  10. Breakdown of Epithelial Barrier Integrity and Overdrive Activation of Alveolar Epithelial Cells in the Pathogenesis of Acute Respiratory Distress Syndrome and Lung Fibrosis

    PubMed Central

    Yanagi, Shigehisa; Tsubouchi, Hironobu; Miura, Ayako; Matsumoto, Nobuhiro; Nakazato, Masamitsu

    2015-01-01

    Individual alveolar epithelial cells (AECs) collaboratively form a tight barrier between atmosphere and fluid-filled tissue to enable normal gas exchange. The tight junctions of AECs provide intercellular sealing and are integral to the maintenance of the AEC barrier integrity. Disruption and failure of reconstitution of AEC barrier result in catastrophic consequences, leading to alveolar flooding and subsequent devastating fibrotic scarring. Recent evidences reveal that many of the fibrotic lung diseases involve AECs both as a frequent target of injury and as a driver of ongoing pathological processes. Aberrantly activated AECs express most of the growth factors and chemokines responsible for the proliferation, migration, and activation of fibroblasts. Current evidences suggest that AECs may acquire overdrive activation in the initial step of fibrosis by several mechanisms, including abnormal recapitulation of the developmental pathway, defects of the molecules essential for epithelial integrity, and acceleration of aging-related properties. Among these initial triggering events, epithelial Pten, a multiple phosphatase that negatively regulates the PI3K/Akt pathway and is crucial for lung development, is essential for the prevention of alveolar flooding and lung fibrosis through the regulation of AEC barrier integrity after injury. Reestablishment of AEC barrier integrity also involves the deployment of specialized stem/progenitor cells. PMID:26523279

  11. Lung epithelial NOX/DUOX and respiratory virus infections.

    PubMed

    Grandvaux, Nathalie; Mariani, Mélissa; Fink, Karin

    2015-03-01

    Determining the role of NADPH oxidases in the context of virus infection is an emerging area of research and our knowledge is still sparse. The expression of various isoforms of NOX/DUOX (NADPH oxidase/dual oxidase) in the epithelial cells (ECs) lining the respiratory tract renders them primary sites from which to orchestrate the host defence against respiratory viruses. Accumulating evidence reveals distinct facets of the involvement of NOX/DUOX in host antiviral and pro-inflammatory responses and in the control of the epithelial barrier integrity, with individual isoforms mediating co-operative, but surprisingly also opposing, functions. Although in vivo studies in mice are in line with some of these observations, a complete understanding of the specific functions of epithelial NOX/DUOX awaits lung epithelial-specific conditional knockout mice. The goal of the present review is to summarize our current knowledge of the role of individual NOX/DUOX isoforms expressed in the lung epithelium in the context of respiratory virus infections so as to highlight potential opportunities for therapeutic intervention.

  12. A comprehensive proteomic view of responses of A549 type II alveolar epithelial cells to human respiratory syncytial virus infection.

    PubMed

    Dave, Keyur A; Norris, Emma L; Bukreyev, Alexander A; Headlam, Madeleine J; Buchholz, Ursula J; Singh, Toshna; Collins, Peter L; Gorman, Jeffrey J

    2014-12-01

    Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious

  13. A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection*

    PubMed Central

    Dave, Keyur A.; Norris, Emma L.; Bukreyev, Alexander A.; Headlam, Madeleine J.; Buchholz, Ursula J.; Singh, Toshna; Collins, Peter L.; Gorman, Jeffrey J.

    2014-01-01

    Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious

  14. Preventive Activity against Influenza (H1N1) Virus by Intranasally Delivered RNA-Hydrolyzing Antibody in Respiratory Epithelial Cells of Mice.

    PubMed

    Cho, Seungchan; Youn, Ha-Na; Hoang, Phuong Mai; Cho, Sungrae; Kim, Kee-Eun; Kil, Eui-Joon; Lee, Gunsup; Cho, Mun-Ju; Hong, Juhyun; Byun, Sung-June; Song, Chang-Seon; Lee, Sukchan

    2015-09-21

    The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.

  15. Knockdown of HMGN2 increases the internalization of Klebsiella pneumoniae by respiratory epithelial cells through the regulation of α5β1 integrin expression

    PubMed Central

    Wang, Xinyuan; Li, Jingyu; Chen, Shanze; Shen, Xiaofei; Yang, Xiaolong; Teng, Yan; Deng, Luxia; Wang, Yi; Chen, Junli; Wang, Xiaoying; Huang, Ning

    2016-01-01

    Integrin receptors, a large family of adhesion receptors, are involved in the attachment of Klebsiella pneumoniae to respiratory epithelial cells, and subsequently cause the internalization of K. pneumoniae by host cells. Although a number of molecules have been reported to regulate the expression and activity of integrin receptors in respiratory epithelial cells, the specific underlying molecular mechanisms remain largely unknown. High mobility group nucleosomal binding domain 2 (HMGN2), a non-histone nuclear protein, is present in eukaryotic cells as a ubiquitous nuclear protein. Our previous studies have demonstrated that HMGN2 affects chromatin function and modulates the expression of antibacterial peptide in A549 cells exposed to lipopolysaccharide, which indicates the critical role of HMGN2 in innate immune responses. In addition, our cDNA microarray analysis suggested that HMGN2 knockdown induced the enhanced expression of α5β1 integrin in A549 cells. Therefore, we hypothesized that intercellular HMGN2 may mediate the internalization of K. pneumoniae by altering the expression of α5β1 integrin. Using the A549 cell line, we demonstrated that HMGN2 knockdown induced the increased expression of α5β1 integrin on cell membranes, which resulted in a significant increase in K. pneumoniae internalization. Further results revealed that HMGN2 silencing induced the expression of talin and the activation of α5β1 integrin, which led to actin polymerization following the phosphorylation of FAK and Src. This study suggests a possible therapeutic application for bacterial internalization by targeting HMGN2 in order to treat K. pneumoniae infection. PMID:27460641

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

    PubMed

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

    2016-11-01

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

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

    PubMed

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

    2016-11-01

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

  18. Nicotine enhances expression of the alpha 3, alpha 4, alpha 5, and alpha 7 nicotinic receptors modulating calcium metabolism and regulating adhesion and motility of respiratory epithelial cells.

    PubMed

    Zia, S; Ndoye, A; Nguyen, V T; Grando, S A

    1997-09-01

    The purpose of this study was to investigate the possibility of direct toxic effects of nicotine (Nic) on human bronchial epithelial cells (BEC) suggested by our previous findings of functional nicotinic acetylcholine receptors (nAChRs) in the epithelial cells lining mucocutaneous membranes. We now demonstrate for the first time that human and murine BEC both in vivo and in vitro express functional nAChRs, and that classic alpha 3, alpha 4, alpha 5 and alpha 7 subunits can contribute to formation of these acetylcholine-gated ion channels. In human bronchial and mouse lung tissues, and in cultures of human BEC, the nAChRs were visualized by subunit-specific antibodies on the cell membranes, particularly at the sites of cell-to-cell contacts. The epithelial cells of submucosal glands abundantly expressed alpha 7 nAChRs. Smoking significantly (p < 0.05) increased the relative numbers of nAChRs, and this effects could be reproduced in cultures of BEC exposed to 10 microM Nic. At a higher dose, Nic decreased the relative numbers of alpha 5-containing nAChRs, suggesting a role for receptor desensitization. The function of the nAChR channels expressed by BEC was demonstrated by biphasic increase in the concentrations of intracellular calcium ([Ca++]i) in response to activation of the channel by Nic and fluctuations of [Ca++]i due to channel blockade by mecamylamine (Mec). Long-term exposure to milimolar concentrations of Nic resulted in a steady increase of [Ca++]i, which may lead to cell damage. The biological roles of epithelial nAChRs apparently involve regulation of cell-to-cell communications, adhesion and motility, because Mec caused rapid and profound changes in these cell functions which were reversible by Nic. An over exposure of BEC to Nic, however, produced an antagonist-like effect, suggesting that the pathobiological effects of Nic toxicity might result from both activation of nAChR channels and nAChR desensitization. We conclude that medical consequences of

  19. Bacterial siderophores that evade or overwhelm lipocalin 2 induce hypoxia inducible factor 1α and proinflammatory cytokine secretion in cultured respiratory epithelial cells.

    PubMed

    Holden, Victoria I; Lenio, Steven; Kuick, Rork; Ramakrishnan, Sadeesh K; Shah, Yatrik M; Bachman, Michael A

    2014-09-01

    Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself and also can be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1α (HIF-1α), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1α was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production.

  20. The transcriptional responses of respiratory epithelial cells to Bordetella pertussis reveal host defensive and pathogen counter-defensive strategies

    PubMed Central

    Belcher, Christopher E.; Drenkow, Jörg; Kehoe, Bettina; Gingeras, Thomas R.; McNamara, Nancy; Lemjabbar, Hassan; Basbaum, Carol; Relman, David A.

    2000-01-01

    Bordetella pertussis, the causative agent of whooping cough, has many well-studied virulence factors and a characteristic clinical presentation. Despite this information, it is not clear how B. pertussis interaction with host cells leads to disease. In this study, we examined the interaction of B. pertussis with a human bronchial epithelial cell line (BEAS-2B) and measured host transcriptional profiles by using high-density DNA microarrays. The early transcriptional response to this pathogen is dominated by altered expression of cytokines, DNA-binding proteins, and NFκB-regulated genes. This previously unrecognized response to B. pertussis was modified in similar but nonidentical fashions by the antiinflammatory agents dexamethasone and sodium salicylate. Cytokine protein expression was confirmed, as was neutrophil chemoattraction. We show that B. pertussis induces mucin gene transcription by BEAS-2B cells then counters this defense by using mucin as a binding substrate. A set of genes is described for which the catalytic activity of pertussis toxin is both necessary and sufficient to regulate transcription. Host genomic transcriptional profiling, in combination with functional assays to evaluate subsequent biological events, provides insight into the complex interaction of host and pathogen. PMID:11087813

  1. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  2. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells.

    PubMed

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na(+) channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  3. Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

    PubMed

    Le Goffic, Ronan; Bouguyon, Edwige; Chevalier, Christophe; Vidic, Jasmina; Da Costa, Bruno; Leymarie, Olivier; Bourdieu, Christiane; Decamps, Laure; Dhorne-Pollet, Sophie; Delmas, Bernard

    2010-10-15

    The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-β gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-β expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-β expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-β expression and found that PB1-F2-mediated IFN-β induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

  4. Respiratory Syncytial Virus Infection Upregulates NLRC5 and Major Histocompatibility Complex Class I Expression through RIG-I Induction in Airway Epithelial Cells

    PubMed Central

    Guo, Xuancheng; Liu, Taixiang; Shi, Hengfei; Wang, Jingjing; Ji, Ping; Wang, Hongwei; Hou, Yayi; Tan, Ren Xiang

    2015-01-01

    ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of acute respiratory tract viral infection in infants, causing bronchiolitis and pneumonia. The host antiviral response to RSV acts via retinoic acid-inducible gene I (RIG-I). We show here that RSV infection upregulates major histocompatibility complex class I (MHC-I) expression through the induction of NLRC5, a NOD-like, CARD domain-containing intracellular protein that has recently been identified as a class I MHC transactivator (CITA). RSV infection of A549 cells promotes upregulation of NLRC5 via beta interferon (IFN-β) production, since the NLRC5-inducing activity in a conditioned medium from RSV-infected A549 cells was removed by antibody to IFN-β, but not by antibody to IFN-γ. RSV infection resulted in RIG-I upregulation and induction of NLRC5 and MHC-I. Suppression of RIG-I induction significantly blocked NLRC5, as well as MHC-I, upregulation and diminished IRF3 activation. Importantly, Vero cells deficient in interferon production still upregulated MHC-I following introduction of the RSV genome by infection or transfection, further supporting a key role for RIG-I. A model is therefore proposed in which the host upregulates MHC-I expression during RSV infection directly via the induction of RIG-I and NLRC5 expression. Since elevated expression of MHC-I molecules can sensitize host cells to T lymphocyte-mediated cytotoxicity or immunopathologic damage, the results have significant implications for the modification of immunity in RSV disease. IMPORTANCE Human respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants and young children worldwide. Infection early in life is linked to persistent wheezing and allergic asthma in later life, possibly related to upregulation of major histocompatibility class I (MHC-I) on the cell surface, which facilitates cytotoxic T cell activation and antiviral immunity. Here, we show that RSV infection of lung epithelial

  5. Effects of sulfite on the uptake and binding of benzo[a]pyrene diol epoxide in cultured murine respiratory epithelial cells.

    PubMed Central

    Green, J L; Jones, B C; Reed, G A

    1994-01-01

    Sulfur dioxide (SO2) may act as a cocarcinogen with benzo[a]pyrene (BaP) in the respiratory tract. We have modeled this effect by examining the interactions of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with sulfite, the physiological form of SO2, in a murine respiratory epithelial cell line (C10). We exposed C10 cells to [3H]-anti-BPDE and determined the effects of 1 and 10 mM sulfite on the uptake and subcellular localization of labeled products. Autoradiographic analysis showed that sulfite doubled the nuclear localization of anti-BPDE-derived materials after a 4-hr incubation period. The net nuclear localization of anti-BPDE-derived materials was not affected by sulfite during the first 60 min, but nuclear localization continued to increase in the sulfite-containing incubations throughout the 4-hr incubation period. Little increase in nuclear localization of anti-BPDE-derived material was noted in the incubations without sulfite after 60 min. Subcellular fractionation was performed to determine the amount of label associated with cytosolic and nuclear fractions and to determine covalent binding to protein and DNA. Sulfite produced a modest increase in the amount of [3H]-anti-BPDE-derived products bound to protein; however, binding to nuclear DNA increased by more than 200% with 10 mM sulfite. Analysis of the supernatants from the cytosolic and nuclear fractions of cells exposed to anti-BPDE and sulfite demonstrated the presence of 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10c-su lfonate (BPT-10-sulfonate). [3H]-BPT-10-sulfonate was unable to enter C10 cells, suggesting that it is formed intracellularly.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 3. Figure 4. PMID:8033853

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

    SciTech Connect

    Arnold, Ralf . E-mail: ralf.arnold@medizin.uni-magdeburg.de; Koenig, Wolfgang

    2006-07-05

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

  7. Efficient suilysin-mediated invasion and apoptosis in porcine respiratory epithelial cells after streptococcal infection under air-liquid interface conditions

    PubMed Central

    Meng, Fandan; Wu, Nai-Huei; Seitz, Maren; Herrler, Georg; Valentin-Weigand, Peter

    2016-01-01

    Streptococci may colonize the epithelium in the airways and other entry sites. While local infection often remains asymptomatic, severe or even fatal diseases occur when streptococci become invasive and spread to different sites in the infected host. We have established porcine respiratory air-liquid interface cultures (ALI) from the porcine lung to analyze the interaction of streptococci with their primary target cells. As representative of the streptococcal family we chose Streptococcus suis (S. suis) that is not only a major swine respiratory pathogen but can also infect humans. Suilysin, a cholesterol-dependent cytolysin (CDC), is an important virulence factor. By comparing a S. suis wt strain with a suilysin-deficient mutant, we demonstrate that suilysin contributes to (i) adherence to airway cells (ii) loss of ciliated cells (iii) apoptosis, and (iv) invasion. Furthermore, we show that cytolytic activity of suilysin is crucial for these effects. A striking result of our analysis was the high efficiency of S. suis-induced apoptosis and invasion upon infection under ALI conditions. These properties have been reported to be less efficient when analyzed with immortalized cells. We hypothesize that soluble effectors such as suilysin are present at higher concentrations in cells kept at ALI conditions and thus more effective. These results should be relevant also for infection of the respiratory tract by other respiratory pathogens. PMID:27229328

  8. Feasibility of a 3D human airway epithelial model to study respiratory absorption.

    PubMed

    Reus, Astrid A; Maas, Wilfred J M; Jansen, Harm T; Constant, Samuel; Staal, Yvonne C M; van Triel, Jos J; Kuper, C Frieke

    2014-03-01

    The respiratory route is an important portal for human exposure to a large variety of substances. Consequently, there is an urgent need for realistic in vitro strategies for evaluation of the absorption of airborne substances with regard to safety and efficacy assessment. The present study investigated feasibility of a 3D human airway epithelial model to study respiratory absorption, in particular to differentiate between low and high absorption of substances. Bronchial epithelial models (MucilAir™), cultured at the air-liquid interface, were exposed to eight radiolabeled model substances via the apical epithelial surface. Absorption was evaluated by measuring radioactivity in the apical compartment, the epithelial cells and the basolateral culture medium. Antipyrine, caffeine, naproxen and propranolol were highly transported across the epithelial cell layer (>5%), whereas atenolol, mannitol, PEG-400 and insulin were limitedly transported (<5%). Results indicate that the 3D human airway epithelial model used in this study is able to differentiate between substances with low and high absorption. The intra-experimental reproducibility of the results was considered adequate based on an average coefficient of variation (CV) of 15%. The inter-experimental reproducibility of highly absorbed compounds was in a similar range (CV of 15%), but this value was considerably higher for those compounds that were limitedly absorbed. No statistical significant differences between different donors and experiments were observed. The present study provides a simple method transposable in any lab, which can be used to rank the absorption of chemicals and pharmaceuticals, and is ready for further validation with respect to reproducibility and capacity of the method to predict respiratory transport in humans.

  9. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  10. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  11. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  12. Retinal pigment epithelial cell proliferation

    PubMed Central

    Temple, Sally

    2015-01-01

    The human retinal pigment epithelium forms early in development and subsequently remains dormant, undergoing minimal proliferation throughout normal life. Retinal pigment epithelium proliferation, however, can be activated in disease states or by removing retinal pigment epithelial cells into culture. We review the conditions that control retinal pigment epithelial proliferation in culture, in animal models and in human disease and interpret retinal pigment epithelium proliferation in context of the recently discovered retinal pigment epithelium stem cell that is responsible for most in vitro retinal pigment epithelial proliferation. Retinal pigment epithelial proliferation-mediated wound repair that occurs in selected macular diseases is contrasted with retinal pigment epithelial proliferation-mediated fibroblastic scar formation that underlies proliferative vitreoretinopathy. We discuss the role of retinal pigment epithelial proliferation in age-related macular degeneration which is reparative in some cases and destructive in others. Macular retinal pigment epithelium wound repair and regression of choroidal neovascularization are more pronounced in younger than older patients. We discuss the possibility that the limited retinal pigment epithelial proliferation and latent wound repair in older age-related macular degeneration patients can be stimulated to promote disease regression in age-related macular degeneration. PMID:26041390

  13. A Fine-Tuned Interaction between Trimeric Autotransporter Haemophilus Surface Fibrils and Vitronectin Leads to Serum Resistance and Adherence to Respiratory Epithelial Cells

    PubMed Central

    Singh, Birendra; Su, Yu-Ching; Al-Jubair, Tamim; Mukherjee, Oindrilla; Hallström, Teresia; Mörgelin, Matthias; Blom, Anna M.

    2014-01-01

    Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin, which inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We reported previously that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in the inhibition of MAC formation and the invasion of lung epithelial cells. The vitronectin-binding region of Hsf was defined at the N-terminal region comprising Hsf amino acids 429 to 652. Moreover, the Hsf recognition site on vitronectin consisted of the C-terminal amino acids 352 to 374. H. influenzae was killed more rapidly in vitronectin-depleted serum than in normal human serum (NHS), and increased MAC deposition was observed at the surface of an Hsf-deficient H. influenzae mutant. In parallel, Hsf-expressing Escherichia coli selectively acquired vitronectin from serum, resulting in significant inhibition of the MAC. Moreover, when vitronectin was bound to Hsf, increased bacterial adherence and internalization into epithelial cells were observed. Taking our findings together, we have defined a fine-tuned protein-protein interaction between Hsf and vitronectin that may contribute to increased Hib virulence. PMID:24664511

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

    PubMed

    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.

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

    PubMed

    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. PMID:25673693

  16. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  17. Ozone exposure increases respiratory epithelial permeability in humans

    SciTech Connect

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

    1987-05-01

    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.

  18. The NS1 protein of a human influenza virus inhibits type I interferon production and the induction of antiviral responses in primary human dendritic and respiratory epithelial cells.

    PubMed

    Haye, Kester; Burmakina, Svetlana; Moran, Thomas; García-Sastre, Adolfo; Fernandez-Sesma, Ana

    2009-07-01

    The NS1 protein of the influenza A virus is a potent virulence factor that inhibits type I interferon (IFN) synthesis, allowing the virus to overcome host defenses and replicate efficiently. However, limited studies have been conducted on NS1 function using human virus strains and primary human cells. We used NS1 truncated mutant influenza viruses derived from the human isolate influenza A/TX/91 (TX WT, where WT is wild type) to study the functions of NS1 in infected primary cells. Infection of primary differentiated human tracheo-bronchial epithelial cells with an NS1 truncated mutant demonstrated limited viral replication and enhanced type I IFN induction. Additionally, human dendritic cells (DCs) infected with human NS1 mutant viruses showed higher levels of activation and stimulated naïve T-cells better than TX WT virus-infected DCs. We also compared infections of DCs with TX WT and our previously characterized laboratory strain A/PR/8/34 (PR8) and its NS1 knockout strain, deltaNS1. TX WT-infected DCs displayed higher viral replication than PR8 but had decreased antiviral gene expression at late time points and reduced naïve T-cell stimulation compared to PR8 infections, suggesting an augmented inhibition of IFN production and human DC activation. Our findings show that human-derived influenza A viruses have a high capacity to inhibit the antiviral state in a human system, and here we have evaluated the possible mechanism of this inhibition. Lastly, C-terminal truncations in the NS1 protein of human influenza virus are sufficient to make the virus attenuated and more immunogenic, supporting its use as a live attenuated influenza vaccine in humans.

  19. Heme Oxygenase-1 Protects Corexit 9500A-Induced Respiratory Epithelial Injury across Species

    PubMed Central

    Oliva, Octavio M.; Karki, Suman; Surolia, Ranu; Wang, Zheng; Watson, R. Douglas; Thannickal, Victor J.; Powell, Mickie; Watts, Stephen; Kulkarni, Tejaswini; Batra, Hitesh; Bolisetty, Subhashini; Agarwal, Anupam; Antony, Veena B.

    2015-01-01

    The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its

  20. Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

    PubMed

    Li, Fu Jun; Duggal, Ryan N; Oliva, Octavio M; Karki, Suman; Surolia, Ranu; Wang, Zheng; Watson, R Douglas; Thannickal, Victor J; Powell, Mickie; Watts, Stephen; Kulkarni, Tejaswini; Batra, Hitesh; Bolisetty, Subhashini; Agarwal, Anupam; Antony, Veena B

    2015-01-01

    The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its

  1. Relation of smoking to immunoreactive endothelin in the bronchiolar epithelial cells.

    PubMed Central

    Shokeir, M. O.; Paré, P.; Wright, J. L.

    1994-01-01

    BACKGROUND--Endothelin is a potent bronchoconstrictor which appears to be important in asthma. To ascertain whether cigarette smoking is associated with any alteration in the proportion of bronchiolar epithelial cells which express endothelin immunoreactivity, the airways in the lungs of non-smokers and smokers were analysed. Since an increase in immunoreactivity has been found in the bronchial epithelial cells of asthmatic subjects, cigarette smokers with and without evidence of airway hyperresponsiveness were also selected. METHODS--A point counting method which examined the proportion of endothelin immunoreactive epithelial cells in membranous and respiratory bronchioles was used. RESULTS--Neither smoking itself nor evidence of airway hyperresponsiveness altered the percentage of endothelin immunoreactive epithelial cells in the membraneous and respiratory bronchioles. CONCLUSIONS--Cigarette smoke does not induce endothelin production in bronchiolar epithelial cells, and the airway hyperresponsiveness seen in some patients with lung disease induced by cigarette smoking is not related to exaggerated endothelin production in epithelial cells. PMID:8091324

  2. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

    Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens. PMID:23097097

  3. Nipah Virus Entry and Egress from Polarized Epithelial Cells

    PubMed Central

    Lamp, Boris; Dietzel, Erik; Kolesnikova, Larissa; Sauerhering, Lucie; Erbar, Stephanie; Weingartl, Hana

    2013-01-01

    Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal. PMID:23283941

  4. Secretory component: a glandular epithelial cell marker.

    PubMed Central

    Harris, J. P.; South, M. A.

    1981-01-01

    Secretory component (SC) has been demonstrated to be produced by both normal and malignantly transformed glandular epithelial cells. By an indirect immunofluorescent technique, this study surveys tumors of varied cellular origin in order to determine the reliability of SC as a marker for tumor cells derived from glandular epithelium. Both primary and metastatic tumors of glandular epithelial origin demonstrated SC fluorescence, while nonglandular epithelial tumors did not. This observation was extended to live single-cell preparations, which demonstrated intense cell-surface fluorescence only when glandular epithelial tumors cells were examined. Additionally, fixed, cytocentrifuged, single-cell preparations of glandular epithelial tumors demonstrated cytoplasmic SC fluorescence. When breast carcinoma was examined, all cases demonstrated SC, regardless of the degree of differentiation. This assay appears to have useful clinical application in that the finding of SC provides indication of the glandular epithelial origin of a malignantly transformed cell. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6271014

  5. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

  6. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    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.

  7. Mechanobiology in Lung Epithelial Cells: Measurements, Perturbations, and Responses

    PubMed Central

    Waters, Christopher M.; Roan, Esra; Navajas, Daniel

    2015-01-01

    Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis. PMID:23728969

  8. Measles virus breaks through epithelial cell barriers to achieve transmission

    PubMed Central

    Takeda, Makoto

    2008-01-01

    Measles is a highly contagious disease that causes immunosuppression in patients. Measles virus infection has been thought to begin in the respiratory epithelium and then spread to lymphoid tissue. In this issue of the JCI, Leonard et al. provide data to suggest an alternative model of measles virus pathogenesis (see the related article beginning on page 2448). In human primary epithelial cells and rhesus monkeys in vivo, the authors show that initial infection of respiratory epithelium is not necessary for the virus to enter the host but that viral entry into epithelial cells via interaction of the virus with a receptor located on the basolateral side of the epithelium is required for viral shedding into the airway and subsequent transmission. PMID:18568081

  9. Respiratory epithelial cytotoxicity and membrane damage (holes) caused by amine-modified nanoparticles.

    PubMed

    Ruenraroengsak, Pakatip; Novak, Pavel; Berhanu, Deborah; Thorley, Andrew J; Valsami-Jones, Eugenia; Gorelik, Julia; Korchev, Yuri E; Tetley, Teresa D

    2012-02-01

    The respiratory epithelium is a significant target of inhaled, nano-sized particles, the biological reactivity of which will depend on its physicochemical properties. Surface-modified, 50 and 100 nm, polystyrene latex nanoparticles (NPs) were used as model particles to examine the effect of particle size and surface chemistry on transformed human alveolar epithelial type 1-like cells (TT1). Live images of TT1 exposed to amine-modified NPs taken by hopping probe ion conductance microscopy revealed severe damage and holes on cell membranes that were not observed with other types of NPs. This paralleled induction of cell detachment, cytotoxicity and apoptotic (caspase-3/7 and caspase-9) cell death, and increased release of CXCL8 (IL-8). In contrast, unmodified, carboxyl-modified 50 nm NPs and the 100 nm NPs did not cause membrane damage, and were less reactive. Thus, the susceptibility and membrane damage to respiratory epithelium following inhalation of NPs will depend on both surface chemistry (e.g., cationic) and nano-size. PMID:21352086

  10. Particle complexation of mitochondrial iron produces superoxide generation and activates MAP kinases, NF-kappa B, nrf-2 in human respiratory epithelial cell

    EPA Science Inventory

    The biological effect of particles is associated with a disruption in cell iron homeostasis. We tested the postulate that complexation of cell iron by silica (Si02) results in both an oxidative stress and biological effect. BEAS-2B cells were exposed to either media or 100 ug/ml....

  11. Clara epithelial cell depletion in the lung.

    PubMed

    Sonar, Sanchaita S; Dudda, Jan C

    2013-01-01

    The bronchial epithelium has been increasingly recognized as an important immunomodulatory compartment in asthma and other lung diseases. Clara cells, which comprise the nonciliated secretory epithelial cells, are an important epithelial cell type with functions in the regulation of lung homeostasis and inflammation. Using naphthalene, Clara cells can be depleted within 24 h and regenerate by 1 month, hence, providing an easy method to study the impact of Clara cells on lung inflammation.

  12. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  13. Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3.

    PubMed

    Yamamoto, Keisuke; Yamamoto, Soh; Ogasawara, Noriko; Takano, Kenichi; Shiraishi, Tsukasa; Sato, Toyotaka; Miyata, Ryo; Kakuki, Takuya; Kamekura, Ryuta; Kojima, Takashi; Tsutsumi, Hiroyuki; Himi, Tetsuo; Yokota, Shin-Ichi

    2016-09-01

    Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-β and -λ production. Furthermore, IFN-β promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role. PMID:27468646

  14. Human glomerular epithelial cell proteoglycans

    SciTech Connect

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

    1990-04-01

    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.

  15. Polymerase discordance in novel swine influenza H3N2v constellations is tolerated in swine but not human respiratory epithelial cells.

    PubMed

    Powell, Joshua D; Dlugolenski, Daniel; Nagy, Tamas; Gabbard, Jon; Lee, Christopher; Tompkins, Stephen M; Tripp, Ralph A

    2014-01-01

    Swine-origin H3N2v, a variant of H3N2 influenza virus, is a concern for novel reassortment with circulating pandemic H1N1 influenza virus (H1N1pdm09) in swine because this can lead to the emergence of a novel pandemic virus. In this study, the reassortment prevalence of H3N2v with H1N1pdm09 was determined in swine cells. Reassortants evaluated showed that the H1N1pdm09 polymerase (PA) segment occurred within swine H3N2 with ∼ 80% frequency. The swine H3N2-human H1N1pdm09 PA reassortant (swH3N2-huPA) showed enhanced replication in swine cells, and was the dominant gene constellation. Ferrets infected with swH3N2-huPA had increased lung pathogenicity compared to parent viruses; however, swH3N2-huPA replication in normal human bronchoepithelial cells was attenuated - a feature linked to expression of IFN-β and IFN-λ genes in human but not swine cells. These findings indicate that emergence of novel H3N2v influenza constellations require more than changes in the viral polymerase complex to overcome barriers to cross-species transmission. Additionally, these findings reveal that while the ferret model is highly informative for influenza studies, slight differences in pathogenicity may not necessarily be indicative of human outcomes after infection.

  16. Short-term exposure of nontumorigenic human bronchial epithelial cells to carcinogenic chromium(VI) compromises their respiratory capacity and alters their bioenergetic signature.

    PubMed

    Cerveira, Joana F; Sánchez-Aragó, María; Urbano, Ana M; Cuezva, José M

    2014-01-01

    Previous studies on the impact of hexavalent chromium [Cr(VI)] on mammalian cell energetics revealed alterations suggestive of a shift to a more fermentative metabolism. Aiming at a more defined understanding of the metabolic effects of Cr(VI) and of their molecular basis, we assessed the impact of a mild Cr(VI) exposure on critical bioenergetic parameters (lactate production, oxygen consumption and intracellular ATP levels). Cells derived from normal human bronchial epithelium (BEAS-2B cell line), the main in vivo target of Cr(VI) carcinogenicity, were subjected for 48 h to 1 μM Cr(VI). We could confirm a shift to a more fermentative metabolism, resulting from the simultaneous inhibition of respiration and stimulation of glycolysis. This shift was accompanied by a decrease in the protein levels of the catalytic subunit (subunit β) of the mitochondrial H(+)-ATP synthase (β-F1-ATPase) and a concomitant marked increase in those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The corresponding alteration in the β-F1-ATPase/GAPDH protein ratio (viewed as a bioenergetic signature) upon Cr(VI) exposure was in agreement with the observed attenuation of cellular respiration and enhancement of glycolytic flux. Altogether, these results constitute a novel finding in terms of the molecular mechanisms of Cr(VI) effects. PMID:25161867

  17. Short-term exposure of nontumorigenic human bronchial epithelial cells to carcinogenic chromium(VI) compromises their respiratory capacity and alters their bioenergetic signature

    PubMed Central

    Cerveira, Joana F.; Sánchez-Aragó, María; Urbano, Ana M.; Cuezva, José M.

    2014-01-01

    Previous studies on the impact of hexavalent chromium [Cr(VI)] on mammalian cell energetics revealed alterations suggestive of a shift to a more fermentative metabolism. Aiming at a more defined understanding of the metabolic effects of Cr(VI) and of their molecular basis, we assessed the impact of a mild Cr(VI) exposure on critical bioenergetic parameters (lactate production, oxygen consumption and intracellular ATP levels). Cells derived from normal human bronchial epithelium (BEAS-2B cell line), the main in vivo target of Cr(VI) carcinogenicity, were subjected for 48 h to 1 μM Cr(VI). We could confirm a shift to a more fermentative metabolism, resulting from the simultaneous inhibition of respiration and stimulation of glycolysis. This shift was accompanied by a decrease in the protein levels of the catalytic subunit (subunit β) of the mitochondrial H+-ATP synthase (β-F1-ATPase) and a concomitant marked increase in those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The corresponding alteration in the β-F1-ATPase/GAPDH protein ratio (viewed as a bioenergetic signature) upon Cr(VI) exposure was in agreement with the observed attenuation of cellular respiration and enhancement of glycolytic flux. Altogether, these results constitute a novel finding in terms of the molecular mechanisms of Cr(VI) effects. PMID:25161867

  18. Short-term exposure of nontumorigenic human bronchial epithelial cells to carcinogenic chromium(VI) compromises their respiratory capacity and alters their bioenergetic signature.

    PubMed

    Cerveira, Joana F; Sánchez-Aragó, María; Urbano, Ana M; Cuezva, José M

    2014-01-01

    Previous studies on the impact of hexavalent chromium [Cr(VI)] on mammalian cell energetics revealed alterations suggestive of a shift to a more fermentative metabolism. Aiming at a more defined understanding of the metabolic effects of Cr(VI) and of their molecular basis, we assessed the impact of a mild Cr(VI) exposure on critical bioenergetic parameters (lactate production, oxygen consumption and intracellular ATP levels). Cells derived from normal human bronchial epithelium (BEAS-2B cell line), the main in vivo target of Cr(VI) carcinogenicity, were subjected for 48 h to 1 μM Cr(VI). We could confirm a shift to a more fermentative metabolism, resulting from the simultaneous inhibition of respiration and stimulation of glycolysis. This shift was accompanied by a decrease in the protein levels of the catalytic subunit (subunit β) of the mitochondrial H(+)-ATP synthase (β-F1-ATPase) and a concomitant marked increase in those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The corresponding alteration in the β-F1-ATPase/GAPDH protein ratio (viewed as a bioenergetic signature) upon Cr(VI) exposure was in agreement with the observed attenuation of cellular respiration and enhancement of glycolytic flux. Altogether, these results constitute a novel finding in terms of the molecular mechanisms of Cr(VI) effects.

  19. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  20. Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

    PubMed Central

    Ren, Xiaoyun

    2014-01-01

    Neisseria meningitidis is the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen, Neisseria meningitidis is frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using an in vitro assay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis. PMID:25225250

  1. Disease-associated Neisseria meningitidis isolates inhibit wound repair in respiratory epithelial cells in a type IV pilus-independent manner.

    PubMed

    Ren, Xiaoyun; MacKichan, Joanna K

    2014-12-01

    Neisseria meningitidis is the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen, Neisseria meningitidis is frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using an in vitro assay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis. PMID:25225250

  2. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

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

  3. Lung epithelial cell death induced by oil-dispersant mixtures.

    PubMed

    Wang, He; Shi, Yongli; Major, Danielle; Yang, Zhanjun

    2012-08-01

    The dispersants used in oil spill disasters are claimed to be safe, but increased solubility of high-molecular-weight components in crude oil is of public health concern. The water-accommodated fractions (WAF) of crude oil mixed with dispersants may become airborne and cause lung epithelial damage when inhaled. This study was designed to examine the cell death and related death pathways of lung epithelial cells in response to WAF. Cultured A549 cells were treated for 2 or 24h with different concentrations of WAF. The WAF was prepared by mixing each of the dispersants (Corexit EC9527A, Corexit EC9500A and Corexit EC9580A) with crude oil for extraction with PBS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay, lactate dehydrogenase assay, morphology and cleaved caspase 9 protein, and microtubule-associated protein 1 light chain 3 were all used to measure cell viability, necrosis, apoptosis and autophagy quantitation, respectively. Results showed that the WAF of oil-dispersant mixtures caused cell death in the lung epithelial cells, in a dose-dependent manner, with the major cellular pathways of necrosis and apoptosis involved. Autophagy also occurred in cells exposed to WAF mixtures at lower concentrations before any detectable cell death, indicating greater sensitivity to WAF exposure. The three types of cell behavior, namely necrosis, apoptosis and autophagy, may play different roles in oil spill-related respiratory disorders. PMID:22504303

  4. Origin of the phagocytic respiratory burst and its role in gut epithelial phagocytosis in a basal chordate.

    PubMed

    Yang, Ping; Huang, Shengfeng; Yan, Xinyu; Huang, Guangrui; Dong, Xiangru; Zheng, Tingting; Yuan, Dongjuan; Wang, Ruihua; Li, Rui; Tan, Ying; Xu, Anlong

    2014-05-01

    The vertebrate phagocytic respiratory burst (PRB) is a highly specific and efficient mechanism for reactive oxygen species (ROS) production. This mechanism is mediated by NADPH oxidase 2 (NOX2) and used by vertebrate phagocytic leukocytes to destroy internalized microbes. Here we demonstrate the presence of the PRB in a basal chordate, the amphioxus Branchiostoma belcheri tsingtauense (bbt). We show that using the antioxidant NAC to scavenge the production of ROS significantly decreased the survival rates of infected amphioxus, indicating that ROS are indispensable for efficient antibacterial responses. Amphioxus NOX enzymes and cytosolic factors were found to colocalize in the epithelial cells of the gill, intestine, and hepatic cecum and could be upregulated after exposure to microbial pathogens. The ROS production in epithelial cell lysates could be reconstructed by supplementing recombinant cytosolic factors, including bbt-p47phox, bbt-p67phox, bbt-p47phox, and bbt-Rac; the restored ROS production could be inhibited by anti-bbt-NOX2 and anti-bbt-p67phox antibodies. We also reveal that the gut epithelial lining cells of the amphioxus are competent at bacterial phagocytosis, and there is evidence that the PRB machinery could participate in the initiation of this phagocytic process. In conclusion, we report the presence of the classical PRB machinery in nonvertebrates and provide the first evidence for the possible role of PRB in epithelial cell immunity and phagocytosis.

  5. Origin of the phagocytic respiratory burst and its role in gut epithelial phagocytosis in a basal chordate.

    PubMed

    Yang, Ping; Huang, Shengfeng; Yan, Xinyu; Huang, Guangrui; Dong, Xiangru; Zheng, Tingting; Yuan, Dongjuan; Wang, Ruihua; Li, Rui; Tan, Ying; Xu, Anlong

    2014-05-01

    The vertebrate phagocytic respiratory burst (PRB) is a highly specific and efficient mechanism for reactive oxygen species (ROS) production. This mechanism is mediated by NADPH oxidase 2 (NOX2) and used by vertebrate phagocytic leukocytes to destroy internalized microbes. Here we demonstrate the presence of the PRB in a basal chordate, the amphioxus Branchiostoma belcheri tsingtauense (bbt). We show that using the antioxidant NAC to scavenge the production of ROS significantly decreased the survival rates of infected amphioxus, indicating that ROS are indispensable for efficient antibacterial responses. Amphioxus NOX enzymes and cytosolic factors were found to colocalize in the epithelial cells of the gill, intestine, and hepatic cecum and could be upregulated after exposure to microbial pathogens. The ROS production in epithelial cell lysates could be reconstructed by supplementing recombinant cytosolic factors, including bbt-p47phox, bbt-p67phox, bbt-p47phox, and bbt-Rac; the restored ROS production could be inhibited by anti-bbt-NOX2 and anti-bbt-p67phox antibodies. We also reveal that the gut epithelial lining cells of the amphioxus are competent at bacterial phagocytosis, and there is evidence that the PRB machinery could participate in the initiation of this phagocytic process. In conclusion, we report the presence of the classical PRB machinery in nonvertebrates and provide the first evidence for the possible role of PRB in epithelial cell immunity and phagocytosis. PMID:24560860

  6. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

    Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs. PMID:23241529

  7. Cholinergic epithelial cell with chemosensory traits in murine thymic medulla.

    PubMed

    Panneck, Alexandra Regina; Rafiq, Amir; Schütz, Burkhard; Soultanova, Aichurek; Deckmann, Klaus; Chubanov, Vladimir; Gudermann, Thomas; Weihe, Eberhard; Krasteva-Christ, Gabriela; Grau, Veronika; del Rey, Adriana; Kummer, Wolfgang

    2014-12-01

    Specialized epithelial cells with a tuft of apical microvilli ("brush cells") sense luminal content and initiate protective reflexes in response to potentially harmful substances. They utilize the canonical taste transduction cascade to detect "bitter" substances such as bacterial quorum-sensing molecules. In the respiratory tract, most of these cells are cholinergic and are approached by cholinoceptive sensory nerve fibers. Utilizing two different reporter mouse strains for the expression of choline acetyltransferase (ChAT), we observed intense labeling of a subset of thymic medullary cells. ChAT expression was confirmed by in situ hybridization. These cells showed expression of villin, a brush cell marker protein, and ultrastructurally exhibited lateral microvilli. They did not express neuroendocrine (chromogranin A, PGP9.5) or thymocyte (CD3) markers but rather thymic epithelial (CK8, CK18) markers and were immunoreactive for components of the taste transduction cascade such as Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel (TRPM5), and phospholipase Cβ2. Reverse transcription and polymerase chain reaction confirmed the expression of Gα-gustducin, TRPM5, and phospholipase Cβ2. Thymic "cholinergic chemosensory cells" were often in direct contact with medullary epithelial cells expressing the nicotinic acetylcholine receptor subunit α3. These cells have recently been identified as terminally differentiated epithelial cells (Hassall's corpuscle-like structures in mice). Contacts with nerve fibers (identified by PGP9.5 and CGRP antibodies), however, were not observed. Our data identify, in the thymus, a previously unrecognized presumptive chemosensitive cell that probably utilizes acetylcholine for paracrine signaling. This cell might participate in intrathymic infection-sensing mechanisms.

  8. The secretome of alginate-encapsulated limbal epithelial stem cells modulates corneal epithelial cell proliferation.

    PubMed

    Wright, Bernice; Hopkinson, Andrew; Leyland, Martin; Connon, Che J

    2013-01-01

    Limbal epithelial stem cells may ameliorate limbal stem cell deficiency through secretion of therapeutic proteins, delivered to the cornea in a controlled manner using hydrogels. In the present study the secretome of alginate-encapsulated limbal epithelial stem cells is investigated. Conditioned medium was generated from limbal epithelial stem cells encapsulated in 1.2% (w/v) calcium alginate gels. Conditioned medium proteins separated by 1-D gel electrophoresis were visualized by silver staining. Proteins of interest including secreted protein acidic and rich in cysteine, profilin-1, and galectin-1 were identified by immunoblotting. The effect of conditioned medium (from alginate-encapsulated limbal epithelial stem cells) on corneal epithelial cell proliferation was quantified and shown to significantly inhibit (P≤0.05) their growth. As secreted protein acidic and rich in cysteine was previously reported to attenuate proliferation of epithelial cells, this protein may be responsible, at least in part, for inhibition of corneal epithelial cell proliferation. We conclude that limbal epithelial stem cells encapsulated in alginate gels may regulate corneal epithelialisation through secretion of inhibitory proteins.

  9. The influence of nasal airflow on respiratory and olfactory epithelial distribution in felids.

    PubMed

    Pang, Benison; Yee, Karen K; Lischka, Fritz W; Rawson, Nancy E; Haskins, Mark E; Wysocki, Charles J; Craven, Brent A; Van Valkenburgh, Blaire

    2016-06-15

    The surface area of the maxilloturbinals and fronto-ethmoturbinals is commonly used as an osteological proxy for the respiratory and the olfactory epithelium, respectively. However, this assumption does not fully account for animals with short snouts in which these two turbinal structures significantly overlap, potentially placing fronto-ethmoturbinals in the path of respiratory airflow. In these species, it is possible that anterior fronto-ethmoturbinals are covered with non-sensory (respiratory) epithelium instead of olfactory epithelium. In this study, we analyzed the distribution of olfactory and non-sensory, respiratory epithelia on the turbinals of two domestic cats (Felis catus) and a bobcat (Lynx rufus). We also conducted a computational fluid dynamics simulation of nasal airflow in the bobcat to explore the relationship between epithelial distribution and airflow patterns. The results showed that a substantial amount of respiratory airflow passes over the anterior fronto-ethmoturbinals, and that contrary to what has been observed in caniform carnivorans, much of the anterior ethmoturbinals are covered by non-sensory epithelium. This confirms that in short-snouted felids, portions of the fronto-ethmoturbinals have been recruited for respiration, and that estimates of olfactory epithelial coverage based purely on fronto-ethmoturbinal surface area will be exaggerated. The correlation between the shape of the anterior fronto-ethmoturbinals and the direction of respiratory airflow suggests that in short-snouted species, CT data alone are useful in assessing airflow patterns and epithelium distribution on the turbinals. PMID:27045093

  10. Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions.

    PubMed

    Short, Kirsty R; Kasper, Jennifer; van der Aa, Stijn; Andeweg, Arno C; Zaaraoui-Boutahar, Fatiha; Goeijenbier, Marco; Richard, Mathilde; Herold, Susanne; Becker, Christin; Scott, Dana P; Limpens, Ronald W A L; Koster, Abraham J; Bárcena, Montserrat; Fouchier, Ron A M; Kirkpatrick, Charles James; Kuiken, Thijs

    2016-03-01

    A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection.

  11. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  12. Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties.

    PubMed

    Tsunematsu, Takaaki; Fujiwara, Natsumi; Yoshida, Maki; Takayama, Yukihiro; Kujiraoka, Satoko; Qi, Guangying; Kitagawa, Masae; Kondo, Tomoyuki; Yamada, Akiko; Arakaki, Rieko; Miyauchi, Mutsumi; Ogawa, Ikuko; Abiko, Yoshihiro; Nikawa, Hiroki; Murakami, Shinya; Takata, Takashi; Ishimaru, Naozumi; Kudo, Yasusei

    2016-10-01

    Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of the Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. ERM cells are unique epithelial cells that remain in periodontal tissues throughout adult life. They have a functional role in the repair/regeneration of cement or enamel. Here, we isolated odontogenic epithelial cells from ERM in the periodontal ligament, and the cells were spontaneously immortalized. Immortalized odontogenic epithelial (iOdE) cells had the ability to form spheroids and expressed stem cell-related genes. Interestingly, iOdE cells underwent osteogenic differentiation, as demonstrated by the mineralization activity in vitro in mineralization-inducing media and formation of calcification foci in iOdE cells transplanted into immunocompromised mice. These findings suggest that a cell population with features similar to stem cells exists in ERM and that this cell population has a differentiation capacity for producing calcifications in a particular microenvironment. In summary, iOdE cells will provide a convenient cell source for tissue engineering and experimental models to investigate tooth growth, differentiation, and tumorigenesis. PMID:27479086

  13. Wound healing of intestinal epithelial cells

    PubMed Central

    Iizuka, Masahiro; Konno, Shiho

    2011-01-01

    The intestinal epithelial cells (IECs) form a selective permeability barrier separating luminal content from underlying tissues. Upon injury, the intestinal epithelium undergoes a wound healing process. Intestinal wound healing is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of epithelial cells adjacent to the wounded area. Previous studies have shown that various regulatory peptides, including growth factors and cytokines, modulate intestinal epithelial wound healing. Recent studies have revealed that novel factors, which include toll-like receptors (TLRs), regulatory peptides, particular dietary factors, and some gastroprotective agents, also modulate intestinal epithelial wound repair. Among these factors, the activation of TLRs by commensal bacteria is suggested to play an essential role in the maintenance of gut homeostasis. Recent studies suggest that mutations and dysregulation of TLRs could be major contributing factors in the predisposition and perpetuation of inflammatory bowel disease. Additionally, studies have shown that specific signaling pathways are involved in IEC wound repair. In this review, we summarize the function of IECs, the process of intestinal epithelial wound healing, and the functions and mechanisms of the various factors that contribute to gut homeostasis and intestinal epithelial wound healing. PMID:21633524

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

    EPA Science Inventory

    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...

  15. Replication of cultured lung epithelial cells

    SciTech Connect

    Guzowski, D.; Bienkowski, R.

    1986-03-05

    The authors have investigated the conditions necessary to support replication of lung type 2 epithelial cells in culture. Cells were isolated from mature fetal rabbit lungs (29d gestation) and cultured on feeder layers of mitotically inactivated 3T3 fibroblasts. The epithelial nature of the cells was demonstrated by indirect immunofluorescent staining for keratin and by polyacid dichrome stain. Ultrastructural examination during the first week showed that the cells contained myofilaments, microvilli and lamellar bodies (markers for type 2 cells). The following changes were observed after the first week: increase in cell size; loss of lamellar bodies and appearance of multivesicular bodies; increase in rough endoplasmic reticulum and golgi; increase in tonafilaments and well-defined junctions. General cell morphology was good for up to 10 wk. Cells cultured on plastic surface degenerated after 1 wk. Cell replication was assayed by autoradiography of cultures exposed to (/sup 3/H)-thymidine and by direct cell counts. The cells did not replicate during the first week; however, between 2-10 wk the cells incorporated the label and went through approximately 6 population doublings. They have demonstrated that lung alveolar epithelial cells can replicate in culture if they are maintained on an appropriate substrate. The coincidence of ability to replicate and loss of markers for differentiation may reflect the dichotomy between growth and differentiation commonly observed in developing systems.

  16. RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction

    PubMed Central

    Liesman, Rachael M.; Buchholz, Ursula J.; Luongo, Cindy L.; Yang, Lijuan; Proia, Alan D.; DeVincenzo, John P.; Collins, Peter L.; Pickles, Raymond J.

    2014-01-01

    Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease. PMID:24713657

  17. Control of the epithelial stem cell epigenome: the shaping of epithelial stem cell identity.

    PubMed

    Iglesias-Bartolome, Ramiro; Callejas-Valera, Juan Luis; Gutkind, J Silvio

    2013-04-01

    The squamous epithelium covering the skin and oral mucosa relies on epithelial stem cells for tissue renewal. Dynamic changes in DNA methylation, histone methylation and acetylation, and higher order chromatin structure are required to preserve their self-renewal capacity while orchestrating the timely execution of cell differentiation programs. This complex network of epigenetic modifications shapes the epithelial stem cell identity and fate. Pathological alterations can be perceived by aberrant chromatin sensors, such as the INK4/ARF locus, which initiate tumor suppressive cell senescence programs, and can often result in epithelial stem cell exhaustion. Unveiling the mechanisms controlling the epigenome in epithelial stem cells may help protect against the loss of their tissue regenerative capacity, thereby preventing premature aging without increasing cancer risk. PMID:23434069

  18. Evaluation of the ability of tilmicosin to prevent adherence of Mycoplasma hyopneumoniae to cilia using a differentiated swine respiratory epithelial culture system.

    PubMed

    Thacker, E L; Young, T F; Erickson, B Z; DeBey, M C

    2001-01-01

    Mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae is a serious economic problem for swine producers in the United States. Mycoplasma hyopneumoniae colonizes ciliated respiratory epithelial cells. The organism has been shown to be sensitive to tilmicosin, a synthetic macrolide, in antibiotic sensitivity assays. The efficacy of tilmicosin to inhibit adherence of M. hyopneumoniae to ciliated epithelial cells without direct contact between the antibiotic and the organism was evaluated using in vitro methods. The study demonstrated that tilmicosin inhibited the adherence of M. hyopneumoniae at the highest level tested in the system (2 microg/ml) suggesting that tilmicosin may be efficacious against mycoplasmal pneumonia.

  19. Protons sensitize epithelial cells to mesenchymal transition.

    PubMed

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M; Pluth, Janice M; Cucinotta, Francis A

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1.

  20. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  1. Localization of type I interferon receptor limits interferon-induced TLR-3 in epithelial cells

    EPA Science Inventory

    This study aimed to expand on the role of type I IFNs in the influenza-induced upregulation of TLR3 and determine whether and how the localization of the IFN-alpha/beta receptor (IFNAR) in respiratory epithelial cells could modify IFN-induced responses. Using differentiated prima...

  2. Branching Morphogenesis of Immortalized Human Bronchial Epithelial Cells in Three-Dimensional Culture

    PubMed Central

    Kaisani, Aadil; Delgado, Oliver; Fasciani, Gail; Kim, Sang Bum; Wright, Woodring E.; Minna, John D.; Shay, Jerry W.

    2014-01-01

    While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases. PMID:24830354

  3. Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

    PubMed Central

    Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

  4. Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.; Carson, Johnny L.; Fischer, William A.; Jaspers, Ilona

    2013-01-01

    In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial

  5. Culturing of human nasal epithelial cells at the air liquid interface.

    PubMed

    Müller, Loretta; Brighton, Luisa E; Carson, Johnny L; Fischer, William A; Jaspers, Ilona

    2013-10-08

    In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial

  6. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

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

    SciTech Connect

    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.

  8. Growth inhibition of Candida by human oral epithelial cells.

    PubMed

    Steele, C; Leigh, J; Swoboda, R; Fidel, P L

    2000-11-01

    Oropharyngeal candidiasis (OPC) caused by Candida albicans is a significant problem in human immunodeficiency virus (HIV)-infected persons. Recognizing the paucity of information on innate and/or adaptive mucosal host defenses against C. albicans, we recently reported that human and nonhuman primate and mouse vaginal epithelial cells inhibit the growth of C. albicans in vitro. In the present study, oral epithelial cells collected from saliva of healthy volunteers and a purified oral epithelial cell line were found to inhibit blastoconidia and/or hyphal growth of several Candida species. Cell contact was a strict requirement for the epithelial cell anti-Candida activity; neither saliva nor culture supernatants alone inhibited Candida growth, and addition of saliva to the coculture did not modulate the epithelial cell activity. Finally, epithelial cell anti-Candida activity was significantly lower in HIV-infected persons with OPC. Together, these results suggest that oral epithelial cells may play a role in innate resistance against OPC.

  9. Interleukin-13 interferes with CFTR and AQP5 expression and localization during human airway epithelial cell differentiation

    SciTech Connect

    Skowron-zwarg, Marie; Boland, Sonja; Caruso, Nathalie; Coraux, Christelle; Marano, Francelyne; Tournier, Frederic . E-mail: f-tournier@paris7.jussieu.fr

    2007-07-15

    Interleukin-13 (IL-13) is a central regulator of Th2-dominated respiratory disorders such as asthma. Lesions of the airway epithelial barrier frequently observed in chronic respiratory inflammatory diseases are repaired through proliferation, migration and differentiation of epithelial cells. Our work is focused on the effects of IL-13 in human cellular models of airway epithelial cell regeneration. We have previously shown that IL-13 altered epithelial cell polarity during mucociliary differentiation of human nasal epithelial cells. In particular, the cytokine inhibited ezrin expression and interfered with its apical localization during epithelial cell differentiation in vitro. Here we show that CFTR expression is enhanced in the presence of the cytokine, that two additional CFTR protein isoforms are expressed in IL-13-treated cells and that part of the protein is retained within the endoplasmic reticulum. We further show that aquaporin 5 expression, a water channel localized within the apical membrane of epithelial cells, is completely abolished in the presence of the cytokine. These results show that IL-13 interferes with ion and water channel expression and localization during epithelial regeneration and may thereby influence mucus composition and hydration.

  10. Chronic inflammatory cells with epithelial cell characteristics in teleost fishes.

    PubMed

    Noga, E J; Dykstra, M J; Wright, J F

    1989-09-01

    Certain cells that participate in the chronic inflammatory response of teleost fishes have many features typical of epithelioid cells of mammals. Such features include high metabolic activity, frequent phagolysosomes, and cytoplasmic interdigitations between adjacent cells; however, the epithelioid granulomas formed in response to certain diseases in teleost fishes also have several features associated with epithelial cells. Cases of ulcerative mycosis or acid-fast bacterial infection in Atlantic menhaden (Brevoortia tyrannus), fungal infection in silver perch (Bairdiella chrysoura), and mycobacteriosis in Mozambique tilapia (Oreochromis mossambicus) had epithelioid cells that were joined together by well-formed desmosomes with tonofilaments. "Mature granulomas" of the ulcerative mycosis-infected menhaden stained positively for cytokeratin, a cytoskeletal protein that is considered to be highly specific for epithelial cells. The consistent presence of these heretofore unrecognized epithelial features suggest that they may be characteristic of certain types of cells participating in piscine chronic inflammation. PMID:2686148

  11. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

    The dog kidney epithelial cell line (MDCK) has been shown to exhibit a density-correlated inhibition of growth at approxmately 6.6 X 10(5) cells per cm2. When a confluent monolayer at its maximal density was wounded by removal of a wide swath of cells, migration of the cell sheet into the denuded area occurred. Precise measurements of the rate of migration for 5 day showed that the cells accelerated at a uniform rate of 0.24 micrometer . hr-2 and, by extrapolation, possessed an apparent initial velocity of 2.8 micrometer . hr-1 at the time of wounding. The apparent initial velocity was considered to be the result of a brief (< 10 hr) and rapid acceleration dependent on cell density. To verify this, wounds were made at different densities below the maximum. In these experiments, the cells did not migrate until a "threshold" density of 2.0 X 10(5) cells per cm2 was reached regardless of the density at the time of wounding. At the threshold density, the cell sheet began to accelerate at the previously measured rate (0.24 micrometer . hr-2). Any increase in density by cell division was balanced by cell migration, so that the same threshold density was maintained by the migrating cells. Each migrating cell sustained the movement of the cell sheet at a constant rate of acceleration. It is proposed that an acceleration is, in general, characteristic of the vectorial movement of an epithelial cell sheet. Images PMID:6933523

  12. Airway Epithelial Cell Integrity Protects from Cytotoxicity of Pseudomonas aeruginosa Quorum-Sensing Signals.

    PubMed

    Losa, Davide; Köhler, Thilo; Bacchetta, Marc; Saab, Joanna Bou; Frieden, Maud; van Delden, Christian; Chanson, Marc

    2015-08-01

    Cell-to-cell communication via gap junctions regulates airway epithelial cell homeostasis and maintains the epithelium host defense. Quorum-sensing molecules produced by Pseudomonas aeruginosa coordinate the expression of virulence factors by this respiratory pathogen. These bacterial signals may also incidentally modulate mammalian airway epithelial cell responses to the pathogen, a process called interkingdom signaling. We investigated the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). C12 degradation and its effects on cells were monitored in various airway epithelial cell models grown under nonpolarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. C12 rapidly altered epithelial integrity and decreased GJIC in nonpolarized airway epithelial cells, whereas other quorum-sensing molecules had no effect. The effects of C12 were dependent on [Ca(2+)]i and could be prevented by inhibitors of Src tyrosine family and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12 except when undergoing repair after wounding. In vivo during colonization of intubated patients, C12 did not accumulate, but it paralleled bacterial densities. In vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in nonpolarized cells, whereas PON2 expression was increased during epithelial polarization. The cytotoxicity of C12 on nonpolarized epithelial cells, combined with its impaired degradation allowing its accumulation, provides an additional pathogenic mechanism for P. aeruginosa infections.

  13. Detection of Bone Marrow Derived Lung Epithelial Cells

    PubMed Central

    Kassmer, Susannah H.; Krause, Diane S.

    2010-01-01

    Studies on the ability of bone marrow derived cells to adopt the morphology and protein expression of epithelial cells in vivo have expanded rapidly over the last decade, and hundreds of publications report that bone marrow derived cells can become epithelial cells of multiple organs including lung, liver, GI tract, skin, pancreas and others. In this review, we critically evaluate the literature related to engraftment of bone marrow derived cells as epithelial cells in the lung. Over 40 manuscripts focused on whether bone marrow cells can differentiate into lung epithelial cells have been published, nearly all of which claim to identify marrow derived epithelial cells. A few investigations have concluded that no such cells are present and that the phenomenon of marrow derived epithelial cells is based on detection artifacts. Here we discuss the problems that exist in published papers identifying marrow derived epithelial cells, and propose standards for detection methods that provide the most definitive data. Identification of BM derived epithelial cells requires reliable and sensitive techniques for their detection, which must include cell identification based on the presence of an epithelial marker and the absence of blood cell markers as well as a marker for donor BM origin. In order for these studies to be rigorous, they must also use approaches to rule out cell overlap by microscopy or single cell isolation. Once these stringent criteria for identification of marrow derived epithelial cells are used universally, then the field can move forward to address the critical questions regarding which bone marrow derived cells are responsible for engraftment as epithelial cells, the mechanisms by which this occurs, whether these cells play a role in normal tissue repair, and whether specific cell subsets can be used for therapeutic benefit. PMID:20447442

  14. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens.

    PubMed

    Meliopoulos, Victoria A; Van de Velde, Lee-Ann; Van de Velde, Nicholas C; Karlsson, Erik A; Neale, Geoff; Vogel, Peter; Guy, Cliff; Sharma, Shalini; Duan, Susu; Surman, Sherri L; Jones, Bart G; Johnson, Michael D L; Bosio, Catharine; Jolly, Lisa; Jenkins, R Gisli; Hurwitz, Julia L; Rosch, Jason W; Sheppard, Dean; Thomas, Paul G; Murray, Peter J; Schultz-Cherry, Stacey

    2016-08-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival.

  15. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens

    PubMed Central

    Van de Velde, Nicholas C.; Karlsson, Erik A.; Neale, Geoff; Vogel, Peter; Sharma, Shalini; Duan, Susu; Surman, Sherri L.; Jones, Bart G.; Johnson, Michael D. L.; Bosio, Catharine; Jolly, Lisa; Jenkins, R. Gisli; Hurwitz, Julia L.; Rosch, Jason W.; Sheppard, Dean; Thomas, Paul G.; Murray, Peter J.; Schultz-Cherry, Stacey

    2016-01-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival. PMID:27505057

  16. An Epithelial Integrin Regulates the Amplitude of Protective Lung Interferon Responses against Multiple Respiratory Pathogens.

    PubMed

    Meliopoulos, Victoria A; Van de Velde, Lee-Ann; Van de Velde, Nicholas C; Karlsson, Erik A; Neale, Geoff; Vogel, Peter; Guy, Cliff; Sharma, Shalini; Duan, Susu; Surman, Sherri L; Jones, Bart G; Johnson, Michael D L; Bosio, Catharine; Jolly, Lisa; Jenkins, R Gisli; Hurwitz, Julia L; Rosch, Jason W; Sheppard, Dean; Thomas, Paul G; Murray, Peter J; Schultz-Cherry, Stacey

    2016-08-01

    The healthy lung maintains a steady state of immune readiness to rapidly respond to injury from invaders. Integrins are important for setting the parameters of this resting state, particularly the epithelial-restricted αVβ6 integrin, which is upregulated during injury. Once expressed, αVβ6 moderates acute lung injury (ALI) through as yet undefined molecular mechanisms. We show that the upregulation of β6 during influenza infection is involved in disease pathogenesis. β6-deficient mice (β6 KO) have increased survival during influenza infection likely due to the limited viral spread into the alveolar spaces leading to reduced ALI. Although the β6 KO have morphologically normal lungs, they harbor constitutively activated lung CD11b+ alveolar macrophages (AM) and elevated type I IFN signaling activity, which we traced to the loss of β6-activated transforming growth factor-β (TGF-β). Administration of exogenous TGF-β to β6 KO mice leads to reduced numbers of CD11b+ AMs, decreased type I IFN signaling activity and loss of the protective phenotype during influenza infection. Protection extended to other respiratory pathogens such as Sendai virus and bacterial pneumonia. Our studies demonstrate that the loss of one epithelial protein, αVβ6 integrin, can alter the lung microenvironment during both homeostasis and respiratory infection leading to reduced lung injury and improved survival. PMID:27505057

  17. Effects of retinoic acid receptor-selective agonists on human nasal epithelial cell differentiation.

    PubMed

    Million, K; Tournier, F; Houcine, O; Ancian, P; Reichert, U; Marano, F

    2001-12-01

    Retinoids play a critical role in the maintenance of the mucociliary phenotype of epithelial cells in the upper respiratory tract. To determine the role of retinoic acid receptors (RARs) in the regulation of epithelial differentiation, we tested the effect of the synthetic retinoids CD336, CD2019, and CD666, selective agonists for RARalpha, RARbeta, and RARgamma, respectively, during differentiation of human nasal epithelial (HNE) cells in vitro. Using glutamylated tubulin and transglutaminase I (Tg I) as markers of ciliated cell and squamous cell differentiation, respectively, we showed that retinoic acid (RA) stimulated mucociliary differentiation and, in parallel, inhibited squamous cell differentiation. The agonists of the three RARs independently induced ciliogenesis and inhibited squamous cell differentiation by downregulating Tg I expression in a dose- and time-dependent manner. Antagonists specific for the three RARs abolished the effects of the corresponding agonists, demonstrating an RAR-specific mediated effect. Moreover, treatment of retinoid-deficient cultures with RAR agonists induced conversion of the squamous-like phenotype into a ciliated phenotype. In conclusion, all three RARs are potentially involved in the differentiating effects of RA in respiratory epithelial cells.

  18. Isolated Epithelial Cells of the Toad Bladder

    PubMed Central

    Gatzy, J. T.; Berndt, W. O.

    1968-01-01

    Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. QOO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O2 at a rate of 4 µl hr-1 dry wt-1. QOO2 was increased 50% by ADH (100 U/liter) or by cyclic 3',5'-AMP (10 mM/liter). Na+-free Ringer's depressed the QOO2 by 40%. The QOO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na+-free Ringer's but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na+ lack or ADH. The intracellular Na+ and K+ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H2O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na+ concentrations. Cells unresponsive to ADH and Na+ lack had high sucrose spaces and low transcellular membrane gradients of Na+, K+, and Cl-. The results suggest that trypsin and EDTA disaggregation damage the active Na+ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally. PMID:4300150

  19. Oxidative stress in Nipah virus-infected human small airway epithelial cells

    PubMed Central

    Escaffre, Olivier; Halliday, Hailey; Borisevich, Viktoriya; Casola, Antonella

    2015-01-01

    Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development. PMID:26297489

  20. Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

    PubMed Central

    Johnson, Sara M.; McNally, Beth A.; Ioannidis, Ioannis; Flano, Emilio; Teng, Michael N.; Oomens, Antonius G.; Walsh, Edward E.; Peeples, Mark E.

    2015-01-01

    Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization. PMID:26658574

  1. Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells.

    PubMed

    Ulm, Ashley; Mayhew, Christopher N; Debley, Jason; Khurana Hershey, Gurjit K; Ji, Hong

    2016-01-01

    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. PMID:27022951

  2. Scrib is Required for Epithelial Cell Identity and Prevents Epithelial To Mesenchymal Transition in the Mouse

    PubMed Central

    Yamben, Idella F.; Rachel, Rivka A.; Shatadal, Shalini; Copeland, Neal G.; Jenkins, Nancy A.; Warming, Soren; Griep, Anne E.

    2013-01-01

    The integrity and function of epithelial tissues depends on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling. PMID:24095903

  3. Interaction with epithelial cells modifies airway macrophage response to ozone.

    PubMed

    Bauer, Rebecca N; Müller, Loretta; Brighton, Luisa E; Duncan, Kelly E; Jaspers, Ilona

    2015-03-01

    The initial innate immune response to ozone (O3) 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 signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

  4. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G.; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-01-01

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  5. [Research progress of corneal epithelial basal cells and basement membrane].

    PubMed

    Qu, J H; Sun, X G

    2016-09-11

    The cylinder cells at the bottom of corneal epithelial cells are basal cells. Their cytoplasm contains keratin intermediate filament which is important in secretion of basement membrane. Corneal epithelial dysfunction due to diabetes or ocular surgery is intimately related with basal cell abnormality. Corneal epithelial basement membrane is a highly specific extracellular matrix which is made up of lamina lucida and lamina densa. It plays an extremely important role in renewal and restoration. Many ocular abnormalities and diseases have been described to relate to the corneal epithelial basement membrane, such as traumatic recurrent corneal erosion, corneal dystrophy and keratoconus. (Chin J Ophthalmol, 2016, 52: 703-707). PMID:27647251

  6. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  7. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells.

    PubMed

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C; Liu, Jinsong

    2016-03-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  8. Characterization of glomerular epithelial cell matrix receptors.

    PubMed Central

    Adler, S.

    1992-01-01

    Integrin matrix receptors on glomerular epithelial cells (GEC) may play an important role in adhesion of GEC to the glomerular basement membrane (GBM) and in the maintenance of normal glomerular permeability. Therefore, the author determined the types of matrix receptors present on cultured rat GEC and examined their interactions with several components of the extracellular matrix. Beta 1 integrin matrix receptors were detected on all three glomerular cell types in rat kidney in vivo and at areas of cell-cell contact on cultured GEC. Glomerular epithelial cell adhesion to types I and IV collagen was slightly greater than to laminin and fibronectin. Adhesion to fibronectin was significantly inhibited by a synthetic peptide containing the RGD adhesion sequence. Immunoprecipitation of lysates of surface-iodinated GEC showed the presence of alpha 3 beta 1 integrin. Chromatography of lysates on immobilized collagen showed alpha 3 beta 1 integrin and a 70- to 75-kd protein band as the collagen receptors on GEC. Chromatography on the 120-kd cell-binding fragment of fibronectin disclosed only alpha 3 beta 1 as a specific fibronectin receptor. Antibody to the beta 1 integrin chain inhibited adhesion to laminin and collagen. These studies demonstrate that in vitro, as in vivo, GEC appear to express only alpha 3 beta 1 integrin. Furthermore, this matrix receptor is capable of mediating GEC adhesion to collagen, fibronectin, and laminin, components of the GBM, and presumably plays a similar role in promoting GEC adhesion to GBM in vivo. Images Figure 1 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1325740

  9. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  10. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  11. Anthrax Toxin Entry into Polarized Epithelial Cells

    PubMed Central

    Beauregard, Kathryn E.; Wimer-Mackin, Susan; Collier, R. John; Lencer, Wayne I.

    1999-01-01

    We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl− secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl− secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease. PMID:10338515

  12. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

    The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

  13. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  14. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  15. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

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

    PubMed Central

    Bauer, Rebecca N.; Müller, Loretta; Brighton, Luisa E.; Duncan, Kelly E.

    2015-01-01

    The initial innate immune response to ozone (O3) 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 signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell–Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell–derived signals are important determinants of Mac immunophenotype and response to O3. PMID:25054807

  17. Eikenella corrodens adherence to human buccal epithelial cells.

    PubMed Central

    Yamazaki, Y; Ebisu, S; Okada, H

    1981-01-01

    The mechanism of Eikenella corrodens adherence to human buccal epithelial cells in vitro was studied. Initial experiments to determine the optimal conditions for adherence of E. corrodens to buccal epithelial cells showed that adherence was dependent on time, temperature, bacterial concentration, and pH. Different strains of E. corrodens varied in their ability to adhere, and strain 1073 showed the greatest ability in adherence. Strain 1073 was selected for studies of adherence mechanisms. Trypsin treatment or heating (100 degrees C, 10 min) of the bacterial cells abolished their capacity to adhere to buccal epithelial cells. Treatment of buccal epithelial cells with trypsin also abolished adherence of E. corrodens 1073, whereas neuraminidase treatment of buccal epithelial cells enhanced the adherence. The adherence was inhibited by ethylenediaminetetraacetic acid and restored by adding Ca2+. The adherence was remarkably inhibited by sugars containing D-galactose and n-acetyl-D-galactosamine. Treatment of neuraminidase-treated epithelial cells with sodium metaperiodate or alpha- and beta-galactosidase did not decrease the adherence. These data suggest that adherence of E. corrodens 1073 to human buccal epithelial cells may require the interaction of lectin-like proteins on the bacterial surface with galactose-like receptors on the surface of epithelial cells. PMID:6260661

  18. Ouabain modulates epithelial cell tight junction

    PubMed Central

    Larre, Isabel; Lazaro, Amparo; Contreras, Ruben G.; Balda, Maria S.; Matter, Karl; Flores-Maldonado, Catalina; Ponce, Arturo; Flores-Benitez, David; Rincon-Heredia, Ruth; Padilla-Benavides, Teresita; Castillo, Aída; Shoshani, Liora; Cereijido, Marcelino

    2010-01-01

    Epithelial cells treated with high concentrations of ouabain (e.g., 1 μM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell–cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K+ pumping nor disturb the K+ balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (JDEX). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, JDEX, and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell–cell contacts. PMID:20534449

  19. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.

    PubMed

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. PMID:27431614

  20. STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION

    PubMed Central

    Loewenstein, Werner R.; Kanno, Yoshinobu

    1964-01-01

    Membrane permeability of an epithelial cell junction (Drosophila salivary gland) was examined with intracellular microelectrodes and with fluorescent tracers. In contrast to the non-junctional cell membrane surface, which has a low permeability to ions (10-4 mho/cm2), the junctional membrane surface is highly permeable. In fact, it introduces no substantial restriction to ion flow beyond that in the cytoplasm; the resistance through a chain of cells (150 Ω cm) is only slightly greater than in extruded cytoplasm (100 Ω cm). The diffusion resistance along the intercellular space to the exterior, on the other hand, is very high. Here, there exists an ion barrier of, at least, 104Ω cm2. As a result, small ions and fluorescein move rather freely from one cell to the next, but do not leak appreciably through the intercellular space to the exterior. The organ here, rather than the single cell, appears to be the unit of ion environment. The possible underlying structural aspects are discussed. PMID:14206423

  1. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  2. Multi-functionality and plasticity characterize epithelial cells in Hydra

    PubMed Central

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  3. Sialomucin complex in the rat respiratory tract: a model for its role in epithelial protection.

    PubMed Central

    McNeer, R R; Huang, D; Fregien, N L; Carraway, K L

    1998-01-01

    The pulmonary epithelium has a multitude of specialized functions, which depend on regulated growth and differentiation of several cell types. One such function is the synthesis and secretion of mucins, which offer the epithelium protection from and a means for removal of noxious environmental factors. Sialomucin complex (SMC) is a heterodimeric glycoprotein consisting of a mucin subunit (ASGP-1, ascites sialoglycoprotein-1) and a transmembrane protein (ASGP-2) with two epidermal-growth-factor-like domains. SMC was originally discovered in a highly metastatic rat mammary adenocarcinoma and has been implicated in metastasis and in the protection of the tumour cells from natural killer cells. It can also act as a ligand for the receptor tyrosine kinase 185(neu), suggesting that it is bifunctional as well as heterodimeric. SMC is expressed on the epithelium of rat conducting airways, with the highest levels occurring in the proximal trachea and progressively decreasing into the bronchioles. Airway SMC consists of two forms: a soluble form that lacks the C-terminal cytoplasmic and transmembrane domains and accounts for about 70% of the total, and a membrane-associated form that has the C-terminal domains. Immunocytochemical analyses show that SMC is predominantly present on the apical surfaces of the airway epithelium, but not in goblet cells. Soluble form can be removed from the trachea by rinsing, suggesting that a fraction of the protein is adsorbed to the apical surface. Based on these results, we propose a protective mechanism in which membrane and soluble forms of SMC are produced by airway luminal epithelial cells to provide a cell-associated epithelial glycoprotein barrier that also serves as an interface with flowing mucus. In support of this mechanism, we demonstrated secretion of soluble SMC by primary cultures of tracheal epithelial cells. This model suggests that SMC is a critical element in the protective barrier of the airway epithelium. PMID:9480884

  4. Cloned kids derived from caprine mammary gland epithelial cells.

    PubMed

    Yuan, Y-G; Cheng, Y; Guo, L; Ding, G-L; Bai, Y-J; Miao, M-X; An, L-Y; Zhao, J-H; Cao, Y-J

    2009-09-01

    The use of nucleus transfer techniques to generate transgenic dairy goats capable of producing recombinant therapeutic proteins in milk could have a major impact on the pharmaceutical industry. However, transfection or gene targeting of nucleus transfer donor cells requires a long in vitro culture period and the selection of marker genes. In the current study, we evaluated the potential for using caprine mammary gland epithelial cells (CMGECs), isolated from udders of lactating F1 hybrid goats (Capra hircus) and cryopreserved at Passages 24 to 26, for nucleus transfer into enucleated in vivo-matured oocytes. Pronuclear-stage reconstructed embryos were transferred into the oviducts of 31 recipient goats. Twenty-three (74%), 21 (72%), and 14 (48%) recipients were confirmed pregnant by ultrasonography on Days 30, 60, and 90, respectively. Four recipients aborted between 35 and 137 d of gestation. Five recipients carried the pregnancies to term and delivered one goat kid each, one of which subsequently died due to respiratory difficulties. The remaining four goat kids were healthy and well. Single-strand conformation polymorphism analysis confirmed that all kids were clones of the donor cells. In conclusion, the CMGECs remained totipotent for nucleus transfer.

  5. Effect of epithelial cell type on in vitro invasion of non-typeable Haemophilus influenzae.

    PubMed

    Singh, Neeraj Kumar; Kunde, Dale A; Tristram, Stephen G

    2016-10-01

    Non-typeable Haemophilus influenzae (NTHi) have been shown to have variable ability for in vitro invasion with a range of epithelial cells, and increased invasion of BEAS-2B cells has been associated with altered penicillin binding protein3 (PBP3), which is concerning as these strains are increasing worldwide. The aim of the study was to investigate the effect of respiratory cell type and the presence of altered PBP3 on the in vitro invasion of NTHi. A collection of 16 clinical NTHi isolates was established, 7 had normal PBP3, and 9 had altered PBP3 as defined by an N526K substitution. The isolates were tested for invasion of BEAS-2B, NHBE, A549 and NCI-H292 respiratory epithelial cells in vitro using a gentamicin survival assay, with invasion measured as the percentage of intracellular organisms relative to the initial inoculum. The overall median invasion for the 16 NTHi isolates for cell types BEAS-2B, NHBE, A549 and NCI-H292 cells were 3.17, 2.31, 0.11 and 1.52 respectively. The differences were statistically significant for BEAS-2B compared to A549 (P=0.015) and A549 compared to NCI-H292 (P=0.015), and there were also very marked differences in invasion for some individual isolates depending on the cell type used. There was a consistent bias for invasion of isolates with normal versus abnormal PBP3: and this was statistically significant for BEAS-2B (0.07 to 9.90, P=0.031) and A549 cells (0.02 to 1.68, P=0.037). These results show that NTHi invasion of respiratory epithelial cells in vitro is both strain dependant and influenced significantly by the cell line used, and that the association between altered PBP3 and increased invasion is conserved across multiple cell lines.

  6. COMPARISON OF SUSCEPTIBILITY TO INFLUENZA INFECTION IN NASAL EPITHELIAL CELLS OBTAINED FROM SMOKERS AND NON-SMOKERS

    EPA Science Inventory

    Several studies have demonstrated that individuals who smoke have greater susceptibility to influenza infections, as well as other respiratory virus infections, than non-smokers, yet the role of airway epithelial cells in this response is not clear. To determine whether in vivo t...

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

    PubMed

    Eckerle, Isabella; Ehlen, Lukas; Kallies, René; Wollny, Robert; Corman, Victor M; Cottontail, Veronika M; Tschapka, Marco; Oppong, Samuel; Drosten, Christian; Müller, Marcel A

    2014-01-01

    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. PMID:24454736

  8. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow. PMID:26641038

  9. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

    PubMed

    Le Guen, Ludovic; Marchal, Stéphane; Faure, Sandrine; de Santa Barbara, Pascal

    2015-10-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  10. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    PubMed

    Knodler, Leigh A; Nair, Vinod; Steele-Mortimer, Olivia

    2014-01-01

    Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol.

  11. Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells.

    PubMed

    Pickering, Janessa L; Prosser, Amy; Corscadden, Karli J; de Gier, Camilla; Richmond, Peter C; Zhang, Guicheng; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-01-01

    Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic

  12. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    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. PMID:25506353

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

    PubMed

    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. PMID:25506353

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

    PubMed

    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.

  15. Immunochemical Detection of Metallothionein in Specific Epithelial Cells of Rat Organs

    NASA Astrophysics Data System (ADS)

    Danielson, Keith G.; Ohi, Seigo; Huang, P. C.

    1982-04-01

    The distribution of a heavy metal binding protein, metallothionein, was studied immunocytochemically by using antimetallothionein antibody and the immunoperoxidase staining technique on histological sections of liver, kidney, intestine, lung, and testis from cadmium-treated rats. These tissues either accumulate heavy metals (e.g., liver, kidney, and testis) or are exposed to metal by ingestion or inhalation (intestine and lung). Staining for metallothionein was observed intracellularly in epithelial parenchymal cells of the liver and kidney; all hepatocytes and most renal tubular cells stained for the protein. Accumulation of metallothionein was not seen in connective tissue cells surrounding either blood vessels or renal tubules. Extracellular localization of metallothionein was also observed in the liver sinusoids and within the lumina of the renal tubules, suggesting a metal transport or excretory function for this protein. Surface columnar epithelial cells of the intestinal villi indicated the presence of metallothionein but connective tissue cells of the lamina propria were negative for the protein. The granular secretory Paneth cells of the small intestine also stained strongly for metallothionein as did respiratory epithelial cells of the lung. In the testis, metallothionein was detected in the Sertoli cells and interstitial cells but not within the spermatogonia. Sertoli cells are closely associated with the developing spermatogonia and appear to serve a nutritive role in spermatogenesis. Because of the secretory, absorptive, or nutritive function of the metallothionein-localizing cells in the organs studied, we suggest that metallothionein may be involved in metal storage or transport in addition to its commonly proposed detoxification role.

  16. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  17. Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells.

    PubMed

    Koo, Michael Jakun; Rooney, Kristen T; Choi, Mary E; Ryter, Stefan W; Choi, Augustine M K; Moon, Jong-Seok

    2015-08-28

    Cellular metabolism can impact cell life or death outcomes. While metabolic dysfunction has been linked to cell death, the mechanisms by which metabolic dysfunction regulates the cell death mode called necroptosis remain unclear. Our study demonstrates that mitochondrial oxidative phosphorylation (OXPHOS) activates programmed necrotic cell death (necroptosis) in human lung epithelial cells. Inhibition of mitochondrial respiration and ATP synthesis induced the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and necroptotic cell death. Furthermore, we demonstrate that the activation of AMP-activated protein kinase (AMPK), resulting from impaired mitochondrial OXPHOS, regulates necroptotic cell death. These results suggest that impaired mitochondrial OXPHOS contributes to necroptosis in human lung epithelial cells.

  18. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

    This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

  19. Growth of corneal epithelial cells over in situ therapeutic contact lens after simple limbal epithelial transplantation (SLET)

    PubMed Central

    Bhalekar, Swapnil; Sangwan, Virender S; Basu, Sayan

    2013-01-01

    An 11-year-old boy underwent simple limbal epithelial transplantation (SLET) from the healthy right eye to his left eye for total limbal stem cell deficiency. One month later, corneal surface epithelialised and whitish plaques overlying the transplants were seen inferiorly. Those plaques were adherent to the surface of the contact lens and underlying corneal surface had smooth elevations. Similar findings were noted in a 23-year man following cyanoacrylate glue application for corneal perforation. On histological and immunohistochemical analysis, cells lining the contact lenses were identified as corneal epithelial cells. These cases illustrate epithelial cell growth on the contact lens and epithelial hyperplasia on corresponding surface of the cornea. Exorbitant proliferation of the epithelial cells may be owing to young age; therefore, early contact lens removal after SLET in young age, can possibly avoid epithelial hyperplasia. This also reiterates the possibility of using contact lens as a scaffold to grow epithelial cells. PMID:23814196

  20. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  1. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  2. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis.

  3. Bilateral respiratory epithelial adenomatoid hamartoma of the olfactory cleft penetrating into the endocranium.

    PubMed

    Mladina, Ranko; Skitarelić, Neven; Poje, Gorazd; Vuković, Katarina

    2011-09-01

    Respiratory epithelial adenomatoid hamartomas (REAHs) of the nose and paranasal sinuses are relatively rare. These tumors usually do not extend over the boundaries of the nose and sinuses. The authors presented a 65-year-old man experiencing progressive hyposmia, followed by intermittent stubborn headache. The symptoms lasted for almost 2 years and were getting worse very slowly. Fiberendoscopy showed relatively discrete polypoid tissue occupying the olfactory cleft bilaterally. The computed tomography and magnetic resonance imaging suggested the possible lack of the cribriform plate and the unity and uniformity of the tissues located both in the endocranium and high in the nasal cavity. The clinical picture resembled very much a esthesineuroblastoma.The patient underwent endoscopic sinus surgery under the general hypotensive anesthesia. Frozen sections during the surgery showed REAH. The entire tumor was removed in a piece meal way, including both olfactory bulbs because they were involved within the pathologic tissue as well.This case showed that REAH could also be a locally aggressive process, penetrating even into the endocranium. PMID:21959462

  4. Crystal violet staining to quantify Candida adhesion to epithelial cells.

    PubMed

    Negri, M; Gonçalves, V; Silva, S; Henriques, M; Azeredo, J; Oliveira, R

    2010-01-01

    In vitro studies of adhesion capability are essential to characterise the virulence of Candida species. However, the assessment of adhesion by traditional methods is time-consuming. The aim of the present study is the development of a simple methodology using crystal violet staining to quantify in vitro adhesion of different Candida species to epithelial cells. The experiments are performed using Candida albicans (ATCC 90028), C. glabrata (ATCC 2001), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750). A human urinary bladder epithelial cell line (TCC-SUP) is used. Yeast and epithelial cells were stained with crystal violet, epithelial cells were then destained using intermediate washing, and the dye in the yeast cells was extracted with acetic acid. The method was validated for the different Candida reference species by comparison with traditional microscope observation and enumeration. The method was then used to assess Candida adhesion to epithelial cells and also to silicone. For all Candida spp. high correlation values (r2= 0.9724-0.9997) between the number of adherent yeasts (microscope enumeration) and absorbance values were obtained for an inoculum concentration >10(6) cells/mL. The proposed technique was easy to perform and reproducible, enabling the determination of adhesion ability of Candida species to an epithelial cell line. PMID:20973406

  5. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Eule, Stephan; Metzger, Jakob; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Grosshans, Joerg; Wolf, Fred

    2015-03-01

    We found that the constriction of epithelial cell contacts during intercalation in germ band extension in Drosophila embryos follows intriguingly simple quantitative laws. The mean contact length < L > follows < L > (t) ~(T - t) α , where T is the finite collapse time; the time dependent variance of contact length is proportional to the square of the mean; finally the time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations suggest that the dynamics of contact collapse can be captured by a stochastic differential equation analytically tractable in small noise approximation. Here, we present such a model, providing an effective description of the non-equilibrium statistical mechanics of contact collapse. All model parameters are fixed by measurements of time dependent mean and variance of contact lengths. The model predicts the contact length covariance function that we obtain in closed form. The contact length covariance function closely matches experimental observations suggesting that the model well captures the dynamics of contact collapse.

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

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    2009-07-01

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

  7. mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells

    PubMed Central

    2014-01-01

    Background Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist. Methods Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses. Results Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP. Conclusions Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs. PMID:24678619

  8. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line. PMID:20400167

  9. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line.

  10. Intranasal Immunization Strategy To Impede Pilin-Mediated Binding of Pseudomonas aeruginosa to Airway Epithelial Cells

    PubMed Central

    Hsieh, Jennifer C.; Tham, Doris M.; Feng, Weijun; Huang, Fan; Embaie, Selamawit; Liu, Keyi; Dean, Deborah; Hertle, Ralf; FitzGerald, David J.; Mrsny, Randall J.

    2005-01-01

    Prevention of pulmonary Pseudomonas aeruginosa infections represents a critical unmet medical need for cystic fibrosis (CF) patients. We have examined the tenet that a mucosal immunization approach can reduce interactions of a piliated form of this opportunistic pathogen with respiratory epithelial cells. Vaccinations were performed using ntPEpilinPAK, a protein chimera composed of a nontoxic form of P. aeruginosa exotoxin A (ntPE), where the C-terminal loop amino acid sequence of the PAK strain pilin protein was inserted in place of the ntPE Ib domain. Intranasal (i.n.) immunization of BALB/c mice with ntPEpilinPAK generated both serum and saliva immune responses. A series of in vitro studies showed that diluted samples of saliva obtained from immunized mice reduced pilin-dependent P. aeruginosa binding to polarized human tracheal epithelial cells, protected human pulmonary epithelial cells from cytotoxic actions associated with bacterial challenge, and reduced exotoxin A toxicity. Overall, i.n. administration of ntPEpilinPAK induced mucosal and systemic immune responses that may be beneficial for blocking early stage adhesion and/or infection events of epithelial cell-P. aeruginosa interactions at oropharyngeal surfaces. PMID:16239575

  11. Gene expression in TGFbeta-induced epithelial cell differentiation in a three-dimensional intestinal epithelial cell differentiation model

    PubMed Central

    Juuti-Uusitalo, Kati M; Kaukinen, Katri; Mäki, Markku; Tuimala, Jarno; Kainulainen, Heikki

    2006-01-01

    Background The TGFβ1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFβ1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. Results The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFβ1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. Conclusion Our results reveal potential new signaling pathways and several new genes affected by TGFβ in epithelial cell differentiation. The differentiation induced by TGFβ1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells. PMID:17074098

  12. Influence of the mesenchymal cell source on oral epithelial development.

    PubMed

    Kinikoglu, Beste; Rovere, Marie Rose; Haftek, Marek; Hasirci, Vasif; Damour, Odile

    2012-03-01

    The extent of the influence of mesenchymal tissue on epithelial development is still debated, and elucidation of epithelial-mesenchymal interactions should be of relevance for controlling normal as well as pathological growth and development. The aim of the present study was to elucidate the influence of the mesenchymal cell type on oral mucosa epithelial development in vitro, using tissue-engineering principles, by including three different sources for mesenchymal cell type, viz. oral mucosa, skin and cornea, each of them presenting a distinct type of epithelium in situ. We investigated epithelial-mesenchymal interactions, considering both morphological criteria and protein expression (filaggrin, keratin 10, keratin 12, keratin 13 and laminin 5). The results of the histology, immunohistochemistry and transmission electron microscopy of the three types of tissue-engineered constructs composed of mesenchymal cells of different sources (oral, dermal and corneal fibroblasts) and of the same oral epithelial cells showed that the mesenchymal cell source had a significant influence on the thickness and ultrastructure of the epithelium, but not on the differentiation of oral epithelial cells, which might be an intrinsic property of these cells due to their genetic programming. PMID:21548135

  13. Isolation and characterization of cutaneous epithelial stem cells

    PubMed Central

    Jensen, Uffe B.; Ghazizadeh, Soosan; Owens, David M.

    2014-01-01

    SUMMARY During homeostasis, adult mammalian skin turnover is maintained by a number of multipotent and unipotent epithelial progenitors located either in the epidermis, hair follicle or sebaceous gland. Recent work has illustrated that these various progenitor populations reside in regionalized niches and are phenotypically distinct from one another. This degree of heterogeneity within the progenitor cell landscape in the cutaneous epithelium complicates our ability to target, purify and manipulate cutaneous epithelial stem cell subpopulations in adult skin. The techniques outlined in this chapter describe basic procedures for the isolation and purification of murine epithelial progenitors and assessing their capacity for ex vivo propagation. PMID:23483387

  14. TSLP in Epithelial Cell and Dendritic Cell Cross Talk

    PubMed Central

    Liu, Yong-Jun

    2013-01-01

    Dendritic cells (DCs) are professional antigen-presenting cells that have the ability to sense infection and tissue stress, sample and present antigen to T lymphocytes, and instruct the initiation of different forms of immunity and tolerance. The functional versatility of DCs depends on their remarkable ability to translate collectively the information from the invading microbes, as well as their resident tissue microenvironments. Recent progress in understanding Toll-like receptor (TLR) biology has illuminated the mechanisms by which DCs link innate and adaptive antimicrobial immune responses. However, how tissue microenvironments shape the function of DCs has remained elusive. Recent studies of TSLP (thymic stromal lymphopoietin), an epithelial cell-derived cytokine that strongly activates DCs, provide strong evidence at a molecular level that epithelial cells/tissue microenvironments directly communicate with DCs, the professional antigen-presenting cells of the immune system. We review recent progress on how TSLP expressed within thymus and peripheral lymphoid and nonlymphoid tissues regulates DC-mediated central tolerance, peripheral T cell homeostasis, and inflammatory Th2 responses. PMID:19231591

  15. A dynamic system for single and repeated exposure of airway epithelial cells to gaseous pollutants.

    PubMed

    Kastner, Pierre Edouard; Le Calvé, Stéphane; Zheng, Wuyin; Casset, Anne; Pons, Françoise

    2013-03-01

    In vitro models are promising approaches to investigate the adverse effects and the mode of action of air pollutants on the respiratory tract. We designed a dynamic system that allows the single or repeated exposure of cultured cells to two major indoor air gaseous pollutants, formaldehyde (HCHO) and nitrogen dioxide (NO2), alone or as a mixture. In this system, the Calu-3 human bronchial epithelial cell line was exposed at the air-liquid interface (ALI) or submerged by culture medium to synthetic air or to target concentrations of HCHO and/or NO2 once or on 4 consecutive days before assessment of cell viability and necrosis, IL-6 and IL-8 release and trans-epithelial electrical resistance. Our data showed that whereas the ALI method can be used for single short-term exposures only, the submerged method provides the possibility to expose Calu-3 cells in a repeated manner. As well, we found that repeated exposures of the cells to HCHO and NO2 at concentrations that can be found indoors triggered a significant decrease in cell metabolism and an increase in IL-8 release that were not evoked by a single exposure. Thus, our work highlights the fact that the development of systems and methods that allow repeated exposures of cultured cells to gaseous compounds in mixtures is of major interest to evaluate the impact of air pollution on the respiratory tract.

  16. Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation during Acinar Differentiation

    PubMed Central

    Williams, Courtney M.; Engler, Adam J.; Slone, R. Daniel; Galante, Leontine L.; Schwarzbauer, Jean E.

    2009-01-01

    The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland. PMID:18451144

  17. Biliary epithelial cells proliferate during oxygenated ex situ liver culture

    PubMed Central

    Bian, Congwen; Du, Yiqi; Ding, Rui; Huang, Jun; Dai, Yan; Bao, Sujin; Zhao, Lijuan; Shen, Hefang; Dong, Jing; Xu, Jianjian; Xiong, Qiru; Xu, Lili

    2016-01-01

    Biliary complications remain a major source of morbidity in liver transplant patients. Among these complications, nonanastomotic biliary strictures (NAS) are especially common and they are frequently therapy resistant in part because biliary epithelial cells are more sensitive to warm ischemic injury than hepatocytes. It has been a challenge to maintain the physiological function of biliary epithelial cells during liver transplantation. In this work, we have examined the effect of oxygen on proliferation of biliary epithelial cells in the rat livers obtained from donation after circulatory death (DCD). Twelve rat livers from DCD were divided into two groups. Livers in the control group were isolated following a standard procedure without oxygen supply. Livers in the experimental group were isolated with a constant supply of oxygen. All livers were then connected to an ex situ liver culture system in the presence of bromodeoxyuridine (BrdU), a thymidine analogue and a marker for cell proliferation. After 6 hours of normothermic ex situ liver culture, morphology and DNA replication in hepatocytes and biliary epithelial cells were assessed and compared between the two groups. We found that about 4.5% of the biliary epithelial cells in the experimental group proliferated compared with only 0.4% of cells in the control based on BrdU staining. No significant change in cell morphology was observed in those cells between the two groups. Thus, our results indicate that oxygen supply is required for maintenance of the physiological function of biliary epithelial cells during liver transplant and suggest that a constant oxygen supply during liver isolation along with ex situ liver organ culture can enhance the repair of biliary epithelial cell injury during liver transplantation. PMID:27725875

  18. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    SciTech Connect

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  19. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  20. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    SciTech Connect

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with {sup 3}H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-{beta} did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 {times} 10{sup 6} sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined.

  1. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-12-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction.

  2. Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

    PubMed Central

    Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2013-01-01

    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. PMID:24349408

  3. Role of autophagy in the regulation of epithelial cell junctions.

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  4. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. PMID:20113446

  5. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing.

  6. In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

    PubMed

    Broadbent, Lindsay; Villenave, Remi; Guo-Parke, Hong; Douglas, Isobel; Shields, Michael D; Power, Ultan F

    2016-01-01

    The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis. PMID:27464691

  7. DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

    PubMed

    Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

    2014-04-01

    DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

  8. Expression of Transcription Factor GATA-6 in Alveolar Epithelial Cells Is Linked to Neonatal Lung Disease

    PubMed Central

    Vähätalo, Riika; Asikainen, Tiina M.; Karikoski, Riitta; Kinnula, Vuokko L.; White, Carl W.; Andersson, Sture; Heikinheimo, Markku; Myllärniemi, Marjukka

    2011-01-01

    Background Premature birth and respiratory distress syndrome (RDS) are risk factors for disturbed lung development and bronchopulmonary dysplasia (BPD). The molecular mechanisms related to prematurity and BPD remain largely unknown. Epithelial expression of the transcription factor GATA-6 has been implicated in normal and abnormal murine lung development. Objectives The possible involvement of GATA-6 in the normal development and in RDS and BPD was investigated in the human and baboon lung. Methods Immunohistochemistry was used to study the expression of GATA-6 and thyroid transcription factor 1 in lung specimens from different age groups of human and baboon fetuses and newborns with lung disease. Furthermore, the regulatory role of TGF-β1 in GATA-6 expression was investigated in human pulmonary epithelial cell lines using RT-PCR. Results GATA-6 expression increased in the developing human airway epithelium along with advancing gestation, but diminished to negligible at birth. In RDS, GATA-6 expression was enhanced at 5–7 days after birth, and decreased thereafter. In BPD, the expression of GATA-6 in alveolar epithelial cells was low. These results were confirmed and extended using an established baboon model of prematurity. The in vitro experiments revealed that TGF-β1 induces GATA-6 and thyroid transcription factor 1 expression in lung epithelial cells. Conclusions Our results suggest that the expression of GATA-6 at the early stages of the preterm lung may be related to impaired postnatal alveolar development. PMID:21071980

  9. Mechanisms of Disease: Host-Pathogen Interactions between Burkholderia Species and Lung Epithelial Cells

    PubMed Central

    David, Jonathan; Bell, Rachel E.; Clark, Graeme C.

    2015-01-01

    Members of the Burkholderia species can cause a range of severe, often fatal, respiratory diseases. A variety of in vitro models of infection have been developed in an attempt to elucidate the mechanism by which Burkholderia spp. gain entry to and interact with the body. The majority of studies have tended to focus on the interaction of bacteria with phagocytic cells with a paucity of information available with regard to the lung epithelium. However, the lung epithelium is becoming more widely recognized as an important player in innate immunity and the early response to infections. Here we review the complex relationship between Burkholderia species and epithelial cells with an emphasis on the most pathogenic species, Burkholderia pseudomallei and Burkholderia mallei. The current gaps in knowledge in our understanding are highlighted along with the epithelial host-pathogen interactions that offer potential opportunities for therapeutic intervention. PMID:26636042

  10. NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence

    PubMed Central

    Kostadinova, Elena; Chaput, Catherine; Gutbier, Birgitt; Lippmann, Juliane; Sander, Leif E.; Mitchell, Timothy J.; Suttorp, Norbert; Witzenrath, Martin; Opitz, Bastian

    2016-01-01

    Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL-1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes. PMID:27476670

  11. Gastrin stimulates MMP-1 expression in gastric epithelial cells: putative role in gastric epithelial cell migration

    PubMed Central

    Kumar, J. Dinesh; Steele, Islay; Moore, Andrew R.; Murugesan, Senthil V.; Rakonczay, Zoltan; Venglovecz, Viktoria; Pritchard, D. Mark; Dimaline, Rodney; Tiszlavicz, Laszlo; Varro, Andrea

    2015-01-01

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling. PMID:25977510

  12. Trefoil peptides promote restitution of wounded corneal epithelial cells.

    PubMed

    Göke, M N; Cook, J R; Kunert, K S; Fini, M E; Gipson, I K; Podolsky, D K

    2001-04-01

    The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

  13. The response of guinea pig airway epithelial cells and alveolar macrophages to environmental stress

    SciTech Connect

    Cohen, D.S.; Palmer, E.; Welch, W.J.; Sheppard, D. )

    1991-08-01

    Cells lining the respiratory tract form an interface between the organism and the external environment and are repeatedly exposed to physical, chemical, and metabolic stresses. The authors examined the response of cultured guinea pig tracheal epithelial cells and alveolar macrophages to various forms of stress, including clinically and environmentally relevant metabolic stresses such as ozone and acid exposure. Classic stress treatments such as heat shock and sodium arsenite treatment induced the synthesis of 28, 32, 72, 73, 90, and 110 kD stress proteins similar to those observed in other cell types. In contrast, no significant changes in the pattern of protein synthesis were detected after exposure to ambient concentrations of ozone, although ozone exposure caused significant cytotoxicity to both cell types. Another potent oxidant, hydrogen peroxide, similarly did not induce appreciable stress protein synthesis. However, surface acidification of tracheal epithelial cells and alveolar macrophages caused the induction of 72 and 78 kD stress proteins. While stress proteins may play a role in the response of respiratory cells to certain injuries such as hyperthermia and surface acidification, they may not be important in the defense against ozone or other forms of oxidative injury.

  14. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  15. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

    PubMed

    Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

    2015-11-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  16. Lingual Epithelial Stem Cells and Organoid Culture of Them.

    PubMed

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-28

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine.

  17. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  18. Development of human epithelial cell systems for radiation risk assessment.

    PubMed

    Yang, C H; Craise, L M

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells. PMID:11538024

  19. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  20. Autophagic and apoptotic cell death in amniotic epithelial cells.

    PubMed

    Shen, Z-Y; Li, E-M; Lu, S-Q; Shen, J; Cai, Y-M; Wu, Y-E; Zheng, R-M; Tan, L-J; Xu, L-Y

    2008-11-01

    The aim of this paper is to determine if autophagic cell death is associated with apoptosis and whether it participates in the process of term amniotic rupture. Forty pieces of fresh term amnions, including twenty from a position near the margin of the placentas and twenty from the margin of the naturally ruptured part of the placentas in term gestation were collected, respectively. The amnions were examined by scanning electron microscopy (SEM) and amniotic epithelial (AE) cells were examined by transmission electron microscopy (TEM). Autophagic and apoptotic cell death (PCD) were assayed by laser scanning confocal microscopy (LSCM) or flow cytometry using monodansylcadaverin (MDC) and propidium iodide (PI) stain. BCL(2) and BAX were examined by immunoblotting. Under SEM the amniotic epithelia appeared normal in the position near the placenta. They had an atrophied appearance in the margin of their natural broken parts. In the AE cells PCD was divided into three subtypes by TEM: autophagic cell death with positive stains of MDC and PI; apoptotic cell death; and the mixed type. Quantitative detection showed that there were more death cells, including autophagic and apoptotic, in the AE cells near the ruptured parts than near the placentas. An increased expression of BAX and a decreased expression of BCL(2) protein in the AE cells near the broken margin were observed. Apoptotic and autophagic cell death by the intrinsic pathway are the basic event in the AE cell and they are involved in the cause of membrane rupture of the human amnion in term gestation.

  1. Microaggregate-associated protein involved in invasion of epithelial cells by Mycobacterium avium subsp. hominissuis

    PubMed Central

    Babrak, Lmar; Danelishvili, Lia; Rose, Sasha J; Bermudez, Luiz E

    2015-01-01

    The environmental opportunistic pathogen Mycobacterium avium subsp hominissuis (MAH), a member of the nontuberculous mycobacteria (NTM) cluster, causes respiratory as well as disseminated disease in patients such as those with chronic respiratory illnesses or AIDS. Currently, there is no effective method to prevent NTM respiratory infections. The formation of mycobacterial microaggregates comprises of phenotypic changes that lead to efficient adherence and invasion of the respiratory mucosa in vitro and in vivo. Microaggregate adhesion to the respiratory epithelium is mediated in part through the mycobacterial protein, MAV_3013 (MBP-1). Through DNA microarray analysis, the small hypothetical gene MAV_0831 (Microaggregate Invasion Protein-1, MIP-1) was identified as being upregulated during microaggregate formation. When MIP-1 was overexpressed in poorly-invasive Mycobacterium smegmatis, it provided the bacterium the ability to bind and enter epithelial cells. In addition, incubating microaggregates with recombinant MIP-1 protein enhanced the ability of microaggregates to invade HEp-2 cells, and exposure to anti-MIP-1 immune serum reduced the invasion of the host epithelium. Through protein-protein interaction assays, MIP-1 was found to bind to the host protein filamin A, a cytoskeletal actin-binding protein integral to the modulation of host cell shape and migration. As visualized by immunofluorescence, filamin A was able to co-localize with microaggregates and to a lesser extent planktonic bacteria. Invasion of HEp-2 cells by microaggregates and planktonic bacteria was also inhibited by the addition of anti-filamin A antibody suggesting that filamin A plays an important role during infection. In addition, at earlier time points binding and invasion assay results suggest that MBP-1 participates significantly during the first interactions with the host cell while MIP-1 becomes important once the bacteria adhere to the host epithelium. In summary, we have unveiled

  2. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    PubMed

    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy.

  3. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling

    PubMed Central

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. DOI: http://dx.doi.org/10.7554/eLife.15034.001 PMID:27431614

  4. Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549.

    PubMed

    Salvador-Membreve, Daile Meek C; Jacinto, Sonia D; Rivera, Windell L

    2014-12-01

    Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association.

  5. The distribution of intermediate-conductance, calcium-activated, potassium (IK) channels in epithelial cells.

    PubMed

    Thompson-Vest, Nichola; Shimizu, Yasutake; Hunne, Billie; Furness, John B

    2006-02-01

    Intermediate-conductance, calcium-activated, potassium (IK) channels were first identified by their roles in cell volume regulation, and were later shown to be involved in control of proliferation of lymphocytes and to provide a K+ current for epithelial secretory activity. Until now, there has been no systematic investigation of IK channel localization within different epithelia. IK channel immunoreactivity was present in most epithelia, where it occurred in surface membranes of epithelial cells. It was found in all stratified epithelia, including skin, cornea, oral mucosa, vaginal mucosa, urothelium and the oesophageal lining. It occurred in the ducts of fluid-secreting glands, the salivary glands, lacrimal glands and pancreas, and in the respiratory epithelium. A low level of expression was seen in serous acinar cells. It was also found in other epithelia with fluid-exchange properties, the choroid plexus epithelium, the ependyma, visceral pleura and peritoneum, bile ducts and intestinal lining epithelium. However, there was little or no expression in vascular endothelial cells, kidney tubules or collecting ducts, lung alveoli, or in sebaceous glands. It is concluded that the channel is present in surface epithelia (e.g. skin) where it has a cell-protective role against osmotic challenge, and in epithelia where there is anion secretion that is facilitated by a K+ current-dependent hyperpolarization. It was also in some epithelial cells where its roles are as yet unknown. PMID:16441566

  6. Methemoglobin-induced signaling and chemokine responses in human alveolar epithelial cells

    PubMed Central

    Mumby, Sharon; Ramakrishnan, Latha; Evans, Timothy W.; Griffiths, Mark J. D.

    2013-01-01

    Diffuse alveolar hemorrhage is characterized by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron- and oxidant-mediated activation of the transcription factor NF-κB. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-κB pathway; additionally, IL-8 required ERK activation and MCP-1 required JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary, we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron- and redox-mediated signaling involving the activation of the NF-κB and MAPK pathways. Decompartmentalization of hemoglobin may be a significant proinflammatory stimulus in a variety of lung diseases. PMID:24142518

  7. Cholera toxin stimulation of human mammary epithelial cells in culture

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

    Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system.

  8. Trans-sialidase Stimulates Eat Me Response from Epithelial Cells

    PubMed Central

    Butler, Claire E; de Carvalho, Tecia M U; Grisard, Edmundo C; Field, Robert A; Tyler, Kevin M

    2013-01-01

    Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake of microparticles into a perinuclear compartment in a manner which may emulate entosis. PMID:23601193

  9. Cell division and the maintenance of epithelial order

    PubMed Central

    Ragkousi, Katerina

    2014-01-01

    Epithelia are polarized layers of adherent cells that are the building blocks for organ and appendage structures throughout animals. To preserve tissue architecture and barrier function during both homeostasis and rapid growth, individual epithelial cells divide in a highly constrained manner. Building on decades of research focused on single cells, recent work is probing the mechanisms by which the dynamic process of mitosis is reconciled with the global maintenance of epithelial order during development. These studies reveal how symmetrically dividing cells both exploit and conform to tissue organization to orient their mitotic spindles during division and establish new adhesive junctions during cytokinesis. PMID:25349258

  10. Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.

    PubMed

    Nakahashi-Oda, Chigusa; Udayanga, Kankanam Gamage Sanath; Nakamura, Yoshiyuki; Nakazawa, Yuta; Totsuka, Naoya; Miki, Haruka; Iino, Shuichi; Tahara-Hanaoka, Satoko; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira

    2016-04-01

    Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-β (IFN-β), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-β production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis. PMID:26855029

  11. Angiomyolipoma with epithelial cysts: mimic of renal cell carcinoma.

    PubMed

    Rosenkrantz, Andrew B; Hecht, Elizabeth M; Taneja, Samir S; Melamed, Jonathan

    2010-01-01

    Angiomyolipoma with epithelial cysts (AMLEC) is a rare variant of angiomyolipoma with minimal fat that contains epithelial-lined cysts and may mimic a cystic renal cell carcinoma. While 17 cases have been described in the pathology literature since this entity was first described in 2006, the radiologic appearance was not demonstrated in any of these cases. We report the CT and MRI appearance of AMLEC found incidentally in a patient with lupus nephritis.

  12. Change in Cell Shape Is Required for Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition of Mammary Epithelial Cells

    PubMed Central

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2010-01-01

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a “cuboidal” epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-β-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents. PMID:18506791

  13. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  14. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation.

  15. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation. PMID:26276826

  16. Primary human bronchial epithelial cells grown from explants.

    PubMed

    Yaghi, Asma; Zaman, Aisha; Dolovich, Myrna

    2010-01-01

    Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and < or =1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm(3) pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 microg/ml), fibronectin (10 microg/ml), and BSA (10 microg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37 degrees C in 5% CO(2) humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly

  17. Diesel exhaust particles induce aberrant alveolar epithelial directed cell movement by disruption of polarity mechanisms.

    PubMed

    LaGier, Adriana J; Manzo, Nicholas D; Dye, Janice A

    2013-01-01

    Disruption of the respiratory epithelium contributes to the progression of a variety of respiratory diseases that are aggravated by exposure to air pollutants, specifically traffic-based pollutants such as diesel exhaust particles (DEP). Recognizing that lung repair following injury requires efficient and directed alveolar epithelial cell migration, this study's goal was to understand the mechanisms underlying alveolar epithelial cells response to DEP, particularly when exposure is accompanied with comorbid lung injury. Separate mechanistic steps of directed migration were investigated in confluent murine LA-4 cells exposed to noncytotoxic concentrations (0-100 μg/cm(2)) of either automobile-emitted diesel exhaust particles (DEP(A)) or carbon black (CB) particles. A scratch wound model ascertained how DEP(A) exposure affected directional cell migration and BCECF ratio fluorimetry-monitored intracellular pH (pHi). Cells were immunostained with giantin to assess cell polarity, and with paxillin to assess focal cell adhesions. Cells were immunoblotted for ezrin/radixin/moesin (ERM) to assess cytoskeletal anchoring. Data demonstrate herein that exposure of LA-4 cells to DEP(A) (but not CB) resulted in delayed directional cell migration, impaired de-adhesion of the trailing edge cell processes, disrupted regulation of pHi, and altered Golgi polarity of leading edge cells, along with modified focal adhesions and reduced ERM levels, indicative of decreased cytoskeletal anchoring. The ability of DEP(A) to disrupt directed cell migration at multiple levels suggests that signaling pathways such as ERM/Rho are critical for transduction of ion transport signals into cytoskeletal arrangement responses. These results provide insights into the mechanisms by which chronic exposure to traffic-based emissions may result in decrements in lung capacity. PMID:23294296

  18. Molecular mechanisms of asbestos-induced lung epithelial cell apoptosis.

    PubMed

    Liu, Gang; Beri, Rohinee; Mueller, Amanda; Kamp, David W

    2010-11-01

    Asbestos causes pulmonary fibrosis (asbestosis) and malignancies (bronchogenic lung cancer and mesothelioma) by mechanisms that are not fully elucidated. Accumulating evidence show that alveolar epithelial cell (AEC) apoptosis is a crucial initiating and perpetuating event in the development of pulmonary fibrosis following exposure to a wide variety of noxious stimuli, including asbestos. We review the important molecular mechanisms underlying asbestos-induced AEC apoptosis. Specifically, we focus on the role of asbestos in augmenting AEC apoptosis by the mitochondria- and p53-regulated death pathways that result from the production of iron-derived reactive oxygen species (ROS) and DNA damage. We summarize emerging evidence implicating the endoplasmic reticulum (ER) stress response in AEC apoptosis in patients with idiopathic pulmonary fibrosis (IPF), a disease with similarities to asbestosis. Finally, we discuss a recent finding that a mitochondrial oxidative DNA repair enzyme (8-oxoguanine DNA glycosylase; Ogg1) acts as a mitochondrial aconitase chaperone protein to prevent oxidant (asbestos and H(2)O(2))-induced AEC mitochondrial dysfunction and intrinsic apoptosis. The coupling of mitochondrial Ogg1 to mitochondrial aconitase is a novel mechanism linking metabolism to mitochondrial DNA that may be important in the pathophysiologic events resulting in oxidant-induced toxicity as seen in tumors, aging, and respiratory disorders (e.g. asbestosis, IPF). Collectively, these studies are illuminating the molecular basis of AEC apoptosis following asbestos exposure that may prove useful for developing novel therapeutic strategies. Importantly, the asbestos paradigm is elucidating pathophysiologic insights into other more common pulmonary diseases, such as IPF and lung cancer, for which better therapy is required. PMID:20380827

  19. Tracheobronchial epithelium of the sheep: IV. Lectin histochemical characterization of secretory epithelial cells.

    PubMed

    Mariassy, A T; Plopper, C G; St George, J A; Wilson, D W

    1988-09-01

    Conventional histochemical characterization of the mucus secretory apparatus is often difficult to reconcile with the biochemical analysis of respiratory secretions. This study was designed to examine the secretory glycoconjugates in airways using lectins with biochemically defined affinities for main sugar residues of mucus. We used five biotinylated lectins--DBA (Dolichos biflorus) and SBA (Glycine max) for N-acetyl galactosamine (galNAc), BSA I (Bandeiraea simplicifolia) and PNA (Arachis hypogea) for galactose (gal), and UEA I (Ulex europeus)--for detection of fucose (fuc) in HgCl2-fixed, paraffin-embedded, serially sectioned trachea, lobar and segmental bronchi and bronchioles of nine sheep. Lectins selectively localized the carbohydrate residues in luminal secretions, on epithelial cell surfaces, and in secretory cells. In proximal airways, the major carbohydrate residues in luminal secretions, cell surfaces, goblet cells, and glands were fuc and gal-NAc. PNA reacted mainly with apical granules of less than 10% of goblet cells, and gal residues were only detected in some of the mucous cells and on basolateral cell surfaces. Distal airways contained sparse secretion in the lumen, mucous cells contained weakly reactive fuc and gal-NAc, and the epithelial surfaces of Clara cells contained gal. Sugars abundant in the airway secretions were also the major component of cells in glands. We conclude that there is a correlation between specific sugar residues in secretory cells, glycocalyx, and luminal secretions in proximal and distal airways. This suggests that lectins may be used to obtain information about airway secretory cell composition from respiratory secretions. PMID:3189886

  20. Tracheobronchial epithelium of the sheep: IV. Lectin histochemical characterization of secretory epithelial cells.

    PubMed

    Mariassy, A T; Plopper, C G; St George, J A; Wilson, D W

    1988-09-01

    Conventional histochemical characterization of the mucus secretory apparatus is often difficult to reconcile with the biochemical analysis of respiratory secretions. This study was designed to examine the secretory glycoconjugates in airways using lectins with biochemically defined affinities for main sugar residues of mucus. We used five biotinylated lectins--DBA (Dolichos biflorus) and SBA (Glycine max) for N-acetyl galactosamine (galNAc), BSA I (Bandeiraea simplicifolia) and PNA (Arachis hypogea) for galactose (gal), and UEA I (Ulex europeus)--for detection of fucose (fuc) in HgCl2-fixed, paraffin-embedded, serially sectioned trachea, lobar and segmental bronchi and bronchioles of nine sheep. Lectins selectively localized the carbohydrate residues in luminal secretions, on epithelial cell surfaces, and in secretory cells. In proximal airways, the major carbohydrate residues in luminal secretions, cell surfaces, goblet cells, and glands were fuc and gal-NAc. PNA reacted mainly with apical granules of less than 10% of goblet cells, and gal residues were only detected in some of the mucous cells and on basolateral cell surfaces. Distal airways contained sparse secretion in the lumen, mucous cells contained weakly reactive fuc and gal-NAc, and the epithelial surfaces of Clara cells contained gal. Sugars abundant in the airway secretions were also the major component of cells in glands. We conclude that there is a correlation between specific sugar residues in secretory cells, glycocalyx, and luminal secretions in proximal and distal airways. This suggests that lectins may be used to obtain information about airway secretory cell composition from respiratory secretions.

  1. Extracellular matrix proteins regulate epithelial-mesenchymal transition in mammary epithelial cells

    PubMed Central

    Chen, Qike K.; Lee, KangAe; Radisky, Derek C.; Nelson, Celeste M.

    2013-01-01

    Mouse mammary epithelial cells undergo transdifferentiation via epithelial-mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland. PMID:23660532

  2. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells.

    PubMed Central

    Paris, S; Boisvieux-Ulrich, E; Crestani, B; Houcine, O; Taramelli, D; Lombardi, L; Latgé, J P

    1997-01-01

    The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. PMID:9119494

  3. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  4. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

    Plikus, Maksim V.; Gay, Denise L.; Treffeisen, Elsa; Wang, Anne; Supapannachart, Rarinthip June; Cotsarelis, George

    2012-01-01

    Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis hasa mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new inter-follicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover cellular and signaling basis of this remarkable adult wound regeneration phenomenon. PMID:23085626

  5. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

    PubMed Central

    St Johnston, Daniel

    2016-01-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside of the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  6. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  7. Characterization of discrete equine intestinal epithelial cell lineages

    PubMed Central

    Gonzalez, Liara M.; Kinnin, Leslie A.; Blikslager, Anthony T.

    2015-01-01

    OBJECTIVE To characterize epithelial cells of the small intestine and colon in horses without clinical gastrointestinal abnormalities with an emphasis on the stem cell niche constituents. SAMPLE Mucosal biopsy specimens from small and large intestines obtained from 12 horses euthanized for reasons unrelated to gastrointestinal disease or systemic disease. PROCEDURES Intestinal biopsy specimens were collected by sharp dissection immediately following euthanasia. Specimens were prepared for immunohistochemical, immunofluorescence, and transmission electron microscopic imaging to detect and characterize each epithelial cell type. Antibodies against protein biomarkers for cellular identification were selected on the basis of expression in other mammalian species. RESULTS Intestinal epithelial cell types were identified by means of immunostaining and morphological characterization with transmission electron microscopy. Some differences in biomarker expression and antibody cross-reactivity were identified in equine tissue, compared with other species. However, each known type of mucosal epithelial cell was identified in equine tissue. CONCLUSIONS AND CLINICAL RELEVANCE The methodology used can enhance detection of stem cells and progenitor cells as well as postmitotic cell lineages in equine intestinal tissues. Results may have relevance to regenerative potential of intestinal mucosa and survival in horses with colic. PMID:25815577

  8. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  9. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  10. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

    PubMed Central

    West, John D; Dorà, Natalie J; Collinson, J Martin

    2015-01-01

    In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed

  11. Porphyromonas gingivalis genes isolated by screening for epithelial cell attachment.

    PubMed Central

    Duncan, M J; Emory, S A; Almira, E C

    1996-01-01

    Porphyromonas gingivalis is associated with chronic and severe periodontitis in adults. P. gingivalis and the other periodontal pathogens colonize and interact with gingival epithelial cells, but the genes and molecular mechanisms involved are unknown. To dissect the first steps in these interactions, a P. gingivalis expression library was screened for clones which bound human oral epithelial cells. Insert DNA from the recombinant clones did not contain homology to the P. gingivalis fimA gene, encoding fimbrillin, the subunit protein of fimbriae, but showed various degrees of homology to certain cysteine protease-hemagglutinin genes. The DNA sequence of one insert revealed three putative open reading frames which appeared to be in an operon. The relationship between P. gingivalis attachment to epithelial cells and the activities identified by the screen is discussed. PMID:8751909

  12. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  13. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    PubMed

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  14. Insect cells respiratory activity in bioreactor

    PubMed Central

    Jorge, Soraia Athie Calil; Santos, Mariza Gerdulo; Yokomizo, Adriana Yurie; Pereira, Carlos Augusto; Tonso, Aldo

    2008-01-01

    Specific respiration rate ( \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} }} $$\\end{document}) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} }} $$\\end{document}. Spodoptera frugiperda (Sf9) cells have been used through virus infection as host for foreign protein expression and bioinsecticide production. Transfected Drosophila melanogaster (S2) cells can be used to produce different proteins. The objective of this work is to investigate respiratory activity and oxygen transfer during the growth of different insect cells lines as Spodoptera frugiperda (Sf9), Drosophila melanogaster (S2) wild and transfected for the expression of GPV and EGFP. All experiments were performed in a well-controlled 1-L bioreactor, with SF900II serum free medium. Spodoptera frugiperda (Sf9) cells reached 10.7 × 106 cells/mL and maximum specific respiration rate (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$ Q_{{{\\text{O}}_{2} \\max }} $$\\end{document}) of 7.3 × 10−17 molO2/cell s. Drosophila melanogaster (S2) cells achieved 51.2 × 106 cells/mL and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage

  15. Interactions of Aspergillus fumigatus Conidia with Airway Epithelial Cells: A Critical Review

    PubMed Central

    Croft, Carys A.; Culibrk, Luka; Moore, Margo M.; Tebbutt, Scott J.

    2016-01-01

    Aspergillus fumigatus is an environmental filamentous fungus that also acts as an opportunistic pathogen able to cause a variety of symptoms, from an allergic response to a life-threatening disseminated fungal infection. The infectious agents are inhaled conidia whose first point of contact is most likely to be an airway epithelial cell (AEC). The interaction between epithelial cells and conidia is multifaceted and complex, and has implications for later steps in pathogenesis. Increasing evidence has demonstrated a key role for the airway epithelium in the response to respiratory pathogens, particularly at early stages of infection; therefore, elucidating the early stages of interaction of conidia with AECs is essential to understand the establishment of infection in cohorts of at-risk patients. Here, we present a comprehensive review of the early interactions between A. fumigatus and AECs, including bronchial and alveolar epithelial cells. We describe mechanisms of adhesion, internalization of conidia by AECs, the immune response of AECs, as well as the role of fungal virulence factors, and patterns of fungal gene expression characteristic of early infection. A clear understanding of the mechanisms involved in the early establishment of infection by A. fumigatus could point to novel targets for therapy and prophylaxis. PMID:27092126

  16. Experimental impact of aspirin exposure on rat intestinal bacteria, epithelial cells and cell line.

    PubMed

    Upreti, Raj K; Kannan, A; Pant, A B

    2010-10-01

    Aspirin, a commonly used therapeutic non-steroidal anti-inflammatory drug (NSAID) is known to cause gastric mucosal damage. Intestinal bacteria having a regulatory effect on intestinal homeostasis play significant role in NSAID-induced intestinal injury. Bacteria and specific cell lines are considered to be suitable for toxicity screening and testing of chemicals. Therefore, to evaluate and compare in vitro toxicity, cultures of rat intestinal epithelial cells (IEC), isolated bacteria and IEC-6 cell line were assessed for viability, morphometric analysis, membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producing processes and esterase activity test for intra- and extra-cellular degradation, following the post exposure to aspirin (0-50 µg mL(- 1)). Similar pattern of dose-dependent changes in these parameters were observed in three types of cells. Similar in situ effects on IEC validated the in vitro findings. These findings indicate that higher aspirin concentrations may alter cellular functions of IEC and gut bacteria. Furthermore, results suggest that gut bacteria and IEC-6 cell line can be used for the initial screening of gastrointestinal cellular toxicity caused by NSAIDs. PMID:20167629

  17. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

    PubMed Central

    Aoshiba, K; Nagai, A

    2003-01-01

    Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke. PMID:19570263

  18. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

    PubMed Central

    Aoshiba, K; Nagai, A

    2003-01-01

    Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke.

  19. Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells.

    PubMed

    Cho, Kyoung Bin; Cho, Min Kyong; Lee, Won Young; Kang, Keon Wook

    2010-07-28

    The c-myc gene is frequently overexpressed in human breast cancer and its target genes are involved in tumorigenesis. Epithelial mesenchymal transitions (EMT), where cells undergo a developmental switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype, are associated with invasion and motility of cancer cells. Basal E-cadherin expression was down-regulated in c-myc overexpressing MCF10A (c-myc-MCF10A) cells compared to GFP-overexpressing MCF10A (GFP-MCF10A) cells, while N-cadherin was distinctly increased in c-myc-MCF10A cells. Given that glycogen synthase kinase-3beta (GSK-3beta) and the snail axis have key roles in E-cadherin deregulation during EMT, we investigated the role of GSK-3beta/snail signaling pathways in the induction of EMT by c-myc overexpression. In contrast to GFP-MCF10A cells, both the transcriptional activity and the ubiquitination-dependent protein stability of snail were enhanced in c-myc-MCF10A cells, and this was reversed by GSK-3beta overexpression. We also found that c-myc overexpression inhibits GSK-3beta activity through activation of extracellular signal-regulated kinase (ERK). Inhibition of ERK by dominant negative mutant transfection or chemical inhibitor significantly suppressed snail gene transcription. These results suggest that c-myc overexpression during transformation of mammary epithelial cells (MEC) is involved in EMTs via ERK-dependent GSK-3beta inactivation and subsequent snail activation.

  20. Chronic air-flow limitation does not increase respiratory epithelial permeability assessed by aerosolized solute, but smoking does

    SciTech Connect

    Huchon, G.J.; Russell, J.A.; Barritault, L.G.; Lipavsky, A.; Murray, J.F.

    1984-09-01

    To determine the separate influences of smoking and severe air-flow limitation on aerosol deposition and respiratory epithelial permeability, we studied 26 normal nonsmokers, 12 smokers without airway obstruction, 12 nonsmokers with chronic obstructive pulmonary disease (COPD), and 11 smokers with COPD. We aerosolized 99mTc-labeled diethylene triamine pentaacetic acid to particles approximately 1 micron activity median aerodynamic diameter. Levels of radioactivity were plotted semilogarithmically against time to calculate clearance as percent per minute. The distribution of radioactivity was homogeneous in control subjects and in smokers, but patchy in both groups with COPD. No difference was found between clearances of the control group (1.18 +/- 0.31% min-1), and nonsmoker COPD group (1.37 +/- 0.82% min-1), whereas values in smokers without COPD (4.00 +/- 1.70% min-1) and smokers with COPD (3.62 +/- 2.88% min-1) were significantly greater than in both nonsmoking groups. We conclude that (1) small particles appear to deposit peripherally, even with severe COPD; (2) respiratory epithelial permeability is normal in nonsmokers with COPD; (3) smoking increases permeability by a mechanism unrelated to air-flow limitation.

  1. Emergence of an Apical Epithelial Cell Surface In Vivo.

    PubMed

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

    2016-01-11

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological, and laser-dissection experiments with theoretical modeling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  2. Sheep stromal-epithelial cell interactions and ovarian tumor progression.

    PubMed

    Wang-Johanning, Feng; Huang, Miao; Liu, Jinsong; Rycaj, Kiera; Plummer, Joshua B; Barnhart, Kirstin F; Satterfield, William C; Johanning, Gary L

    2007-11-15

    Previous studies suggest that underlying ovarian stromal cues may regulate the ovarian surface epithelium. However, little is known about the interaction between ovarian stromal cells (OSC) and ovarian surface epithelial cells (OSE) under normal physiologic and pathologic conditions, largely because of the lack of a suitable model. In the current study, the OSC obtained from a sheep were immortalized with SV-40 T/t antigen (designated IOSC) and telomerase reverse transcriptase (designated IOSCH), followed by transfection with the oncogenic allele of the human H-Ras oncogene (designated IOSChR). IOSC cells transfected with H-Ras before immortalization with telomerase were designated IOSCRH. These sheep OSCs were used in both in vitro and in vivo model systems to evaluate mechanisms by which OSCs influence ovarian tumor progression. Normal sheep OSCs were found to inhibit the growth of SKOV3 and OVCAR3 human ovarian cancer cells, but not normal sheep OSE and human OSE cells (hOSE137 cells). In contrast, IOSChR and IOSCRH cells stimulated the growth of normal sheep and human OSE cells, as well as cancer cells. These findings were confirmed by in vivo studies. Our data provide compelling support for the importance of stromal-epithelial cell interactions during tumor progression, and show for the first time that immortalized and transformed OSCs promote growth of ovarian epithelial tumors.

  3. [Methotrexate as inducer of proinflammatory cytokines by epithelial cells].

    PubMed

    Morón-Medina, Alejandra; Viera, Ninoska; de Morales, Thaís Rojas; Alcocer, Sirley; Bohorquez, Dinorath

    2014-03-01

    Methotrexate (MTX), a drug commonly used in childhood cancer, has also been indicated as a cytotoxic agent of the oral mucosa, which can trigger the inflammatory process and increase the vascularity of epithelial tissues during the early stages of oral mucositis. The aim of this study was to determine the production of proinflammatory cytokines IL-1beta, IL-6 y TNF-alpha in epithelial cell cultures treated with MTX. Epithelial cells of human larynx, obtained from the cell line Hep-2, were cultured with different doses of MTX during different incubation times. The drug cytotoxicity was analyzed by means of the colorimetric test, which is based on the metabolic reduction of the bromide of 3-(4, 5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol (MTT); and the proinflammatory cytokines production by the test enzyme-linked immunosorbent assay (ELISA). Cultures of HEp-2 cells showed increased production of proinflammatory cytokines at 72 hours with 0.32 microM of MTX. These results suggest that depending on the dose and exposure time, MTX alters the physiology of human epithelial cells, which may play an important role during the phases of initiation and development of oral mucositis. PMID:24758098

  4. Haemophilus haemolyticus Interaction with Host Cells Is Different to Nontypeable Haemophilus influenzae and Prevents NTHi Association with Epithelial Cells

    PubMed Central

    Pickering, Janessa L.; Prosser, Amy; Corscadden, Karli J.; de Gier, Camilla; Richmond, Peter C.; Zhang, Guicheng; Thornton, Ruth B.; Kirkham, Lea-Ann S.

    2016-01-01

    Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen that resides in the upper respiratory tract and contributes to a significant burden of respiratory related diseases in children and adults. Haemophilus haemolyticus is a respiratory tract commensal that can be misidentified as NTHi due to high levels of genetic relatedness. There are reports of invasive disease from H. haemolyticus, which further blurs the species boundary with NTHi. To investigate differences in pathogenicity between these species, we optimized an in vitro epithelial cell model to compare the interaction of 10 H. haemolyticus strains with 4 NTHi and 4 H. influenzae-like haemophili. There was inter- and intra-species variability but overall, H. haemolyticus had reduced capacity to attach to and invade nasopharyngeal and bronchoalveolar epithelial cell lines (D562 and A549) within 3 h when compared with NTHi. H. haemolyticus was cytotoxic to both cell lines at 24 h, whereas NTHi was not. Nasopharyngeal epithelium challenged with some H. haemolyticus strains released high levels of inflammatory mediators IL-6 and IL-8, whereas NTHi did not elicit an inflammatory response despite higher levels of cell association and invasion. Furthermore, peripheral blood mononuclear cells stimulated with H. haemolyticus or NTHi released similar and high levels of IL-6, IL-8, IL-10, IL-1β, and TNFα when compared with unstimulated cells but only NTHi elicited an IFNγ response. Due to the relatedness of H. haemolyticus and NTHi, we hypothesized that H. haemolyticus may compete with NTHi for colonization of the respiratory tract. We observed that in vitro pre-treatment of epithelial cells with H. haemolyticus significantly reduced NTHi attachment, suggesting interference or competition between the two species is possible and warrants further investigation. In conclusion, H. haemolyticus interacts differently with host cells compared to NTHi, with different immunostimulatory and cytotoxic

  5. Establishment of a rat nasal epithelial tumor cell line.

    PubMed

    Hood, A T; Currie, D; Garte, S J

    1987-04-01

    A new cell line designated NAS 2BL has been established from a rat nasal tumor induced by inhalation of the direct-acting carcinogen methylmethane sulfonate. The cells are epithelial in morphology, have a generation time of 34 h, require 10% fetal bovine serum for optimal growth, and exhibit keratinization at confluence. The karyotype is aneuploid, with several marker chromosomes, and the cells are transformed by the criterion of nude mouse tumorigenicity.

  6. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry.

  7. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  8. Rhinovirus Load Is High despite Preserved Interferon-β Response in Cystic Fibrosis Bronchial Epithelial Cells

    PubMed Central

    Cammisano, Maria; Chen, He; Singh, Sareen; Kooi, Cora; Leigh, Richard; Beaudoin, Trevor; Rousseau, Simon; Lands, Larry C.

    2015-01-01

    Lung disease in cystic fibrosis (CF) is often exacerbated following acute upper respiratory tract infections caused by the human rhinovirus (HRV). Pathophysiology of these exacerbations is presently unclear and may involve deficient innate antiviral or exaggerated inflammatory responses in CF airway epithelial cells. Furthermore, responses of CF cells to HRV may be adversely affected by pre-exposure to virulence factors of Pseudomonas (P.) aeruginosa, the microorganism that frequently colonizes CF airways. Here we examined production of antiviral cytokine interferon-β and inflammatory chemokine interleukin-8, expression of the interferon-responsive antiviral gene 2’-5’-oligoadenylate synthetase 1 (OAS1), and intracellular virus RNA load in primary CF (delF508 CFTR) and healthy airway epithelial cells following inoculation with HRV16. Parallel cells were exposed to virulence factors of P. aeruginosa prior to and during HRV16 inoculation. CF cells exhibited production of interferon-β and interleukin-8, and expression of OAS1 at levels comparable to those in healthy cells, yet significantly higher HRV16 RNA load during early hours post-inoculation with HRV16. In line with this, HRV16 RNA load was higher in the CFBE41o- dF cell line overexpessing delF508 CFTR, compared with the isogenic control CFBE41o- WT (wild-type CFTR). Pre-exposure to virulence factors of P. aeruginosa did not affect OAS1 expression or HRV16 RNA load, but potentiated interleukin-8 production. In conclusion, CF cells demonstrate elevated HRV RNA load despite preserved interferon-β and OAS1 responses. High HRV load in CF airway epithelial cells appears to be due to deficiencies manifesting early during HRV infection, and may not be related to interferon-β. PMID:26599098

  9. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    PubMed

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  10. In vitro differentiation of a cloned bovine mammary epithelial cell.

    PubMed

    Rose, Michael T; Aso, Hisashi; Yonekura, Shinichi; Komatsu, Tokushi; Hagino, Akihiko; Ozutsumi, Kyouhei; Obara, Yoshiaki

    2002-08-01

    The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of alpha-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced alpha-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce alpha-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

  11. Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

    PubMed Central

    Centeno, A; Davis, C P; Cohen, M S; Warren, M M

    1983-01-01

    The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Images PMID:6132878

  12. Macrophages are required for dendritic cell uptake of respiratory syncytial virus from an infected epithelium.

    PubMed

    Ugonna, Kelechi; Bingle, Colin D; Plant, Karen; Wilson, Kirsty; Everard, Mark L

    2014-01-01

    We have previously shown that the respiratory syncytial virus [RSV] can productively infect monocyte derived dendritic cells [MoDC] and remain dormant within the same cells for prolonged periods. It is therefore possible that infected dendritic cells act as a reservoir within the airways of individuals between annual epidemics. In the present study we explored the possibility that sub-epithelial DCs can be infected with RSV from differentiated bronchial epithelium and that in turn RSV from DCs can infect the epithelium. A dual co-culture model was established in which a differentiated primary airway epithelium on an Air Liquid Interface (ALI) was cultured on a transwell insert and MoDCs were subsequently added to the basolateral membrane of the insert. Further experiments were undertaken using a triple co-culture model in which in which macrophages were added to the apical surface of the differentiated epithelium. A modified RSV [rr-RSV] expressing a red fluorescent protein marker of replication was used to infect either the MoDCs or the differentiated epithelium and infection of the reciprocal cell type was assessed using confocal microscopy. Our data shows that primary epithelium became infected when rr-RSV infected MoDCs were introduced onto the basal surface of the transwell insert. MoDCs located beneath the epithelium did not become infected with virus from infected epithelial cells in the dual co-culture model. However when macrophages were present on the apical surface of the primary epithelium infection of the basal MoDCs occurred. Our data suggests that RSV infected dendritic cells readily transmit infection to epithelial cells even when they are located beneath the basal layer. However macrophages appear to be necessary for the transmission of infection from epithelial cells to basal dendritic cells.

  13. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  14. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  15. Chronic Alcohol Exposure Renders Epithelial Cells Vulnerable to Bacterial Infection

    PubMed Central

    Wood, Stephen; Pithadia, Ravi; Rehman, Tooba; Zhang, Lijuan; Plichta, Jennifer; Radek, Katherine A.; Forsyth, Christopher; Keshavarzian, Ali; Shafikhani, Sasha H.

    2013-01-01

    Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier’s capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol’s deleterious effects on cells may be reversible. PMID:23358457

  16. Chronic alcohol exposure renders epithelial cells vulnerable to bacterial infection.

    PubMed

    Wood, Stephen; Pithadia, Ravi; Rehman, Tooba; Zhang, Lijuan; Plichta, Jennifer; Radek, Katherine A; Forsyth, Christopher; Keshavarzian, Ali; Shafikhani, Sasha H

    2013-01-01

    Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier's capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol's deleterious effects on cells may be reversible. PMID:23358457

  17. Secondhand smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial cells.

    PubMed

    Savitski, Amy N; Mesaros, Clementina; Blair, Ian A; Cohen, Noam A; Kreindler, James L

    2009-01-01

    Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure. PMID:19943936

  18. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

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

    PubMed Central

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

    2013-01-01

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

  20. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

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

  1. miR-124 regulates fetal pulmonary epithelial cell maturation

    PubMed Central

    Wang, Yang; Huang, Chaoqun; Chintagari, Narendranath Reddy; Xi, Dong; Weng, Tingting

    2015-01-01

    MicroRNAs are a family of small noncoding RNAs that regulate the expression of their target proteins at the posttranscriptional level. Their functions cover almost every aspect of cell physiology. However, the roles of microRNAs in fetal lung development are not completely understood. The objective of this study is to investigate the regulation and molecular mechanisms of alveolar epithelial cell maturation during fetal lung development by miR-124. We discovered that miR-124 was downregulated during rat fetal lung development and predominantly expressed in the epithelial cells at late stage of the lung development. Overexpression of miR-124 with an adenovirus vector led to the inhibition of epithelial maturation in rat fetal lung organ cultures and fetal alveolar epithelial type II cells, as demonstrated by a decrease in the type II cell marker expression and an increase in glycogen content. We further demonstrated by luciferase reporter assays that miR-124 inhibited the NF-κB, cAMP/PKA, and MAPK/ERK pathways. In addition, nuclear factor I/B (NFIB), a critical protein in fetal lung maturation, was validated as a direct target of miR-124. Furthermore, miR-124 expression was induced by the Wnt/β-catenin signaling pathway through a direct interaction of LEF1 and the miR-124 promoter region. We concluded that miR-124 downregulation is critical to fetal lung epithelial maturation and miR-124 inhibits this maturation process at least partially through the inhibition of NFIB. PMID:26071557

  2. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells.

  3. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells. PMID:27398446

  4. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  5. Notch Signaling in Meibomian Gland Epithelial Cell Differentiation

    PubMed Central

    Gidfar, Sanaz; Afsharkhamseh, Neda; Sanjari, Sara; Djalilian, Ali R.

    2016-01-01

    Purpose Notch1 was previously shown to play a critical role in murine meibomian gland function and maintenance. In this study, we have examined the expression and activation of Notch pathway in human meibomian gland epithelial cells in vitro. Methods An immortalized human meibomian gland epithelial cell (HMGEC) line was cultured under proliferative and differentiative conditions. Expression of Notch receptors and ligands were evaluated by quantitative PCR and Western blot. The effect of Notch inhibition and induction on oil production was also assessed. Results Human meibomian gland epithelial cell expressed Notch1, Notch2, Notch3, Jagged1, Jagged2, Delta-like 1, and Delta-like 3. The level of cleaved (activated) Notch1 strongly increased with differentiation. The expression of Notch3 was inversely correlated with proliferation. Induction and inhibition of Notch1 led to an increase and decrease in the amount of oil production, respectively. Conclusions Notch signaling appears to play an important role in human meibomian gland epithelial differentiation and oil production. This may provide a potential therapeutic pathway for treating meibomian gland dysfunction. PMID:26943148

  6. Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration

    PubMed Central

    Dudakov, Jarrod A.; Jenq, Robert R.; Velardi, Enrico; Young, Lauren F.; Smith, Odette M.; Lawrence, Gillian; Ivanov, Juliet A.; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L.; O'Rourke, Kevin P.; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas; Nieuwenhuis, Edward E.; Shroyer, Noah F.; Liu, Chen; Kolesnick, Richard

    2015-01-01

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  7. Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

    PubMed

    Svirshchevskaya, E V; Poltavtseva, R A; Beletskii, I P; Selezneva, I I; Sukhikh, G T

    2016-08-01

    We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin. PMID:27590756

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

    EPA Science Inventory

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

  9. Regulation of tracheal antimicrobial peptide gene expression in airway epithelial cells of cattle.

    PubMed

    Taha-Abdelaziz, Khaled; Wyer, Leanna; Berghuis, Lesley; Bassel, Laura L; Clark, Mary Ellen; Caswell, Jeff L

    2016-01-01

    β-defensins are an important element of the mucosal innate immune response against bacterial pathogens. Tracheal antimicrobial peptide (TAP) has microbicidal activity against the bacteria that cause bovine respiratory disease, and its expression in tracheal epithelial cells is upregulated by bacterial products including lipopolysaccharide (LPS, a TLR4 agonist), Pam3CSK4 (an agonist of Toll-like receptor 2/1), and interleukin (IL)-17A. The objectives of this study were to identify the signalling pathway by which LPS, Pam3CSK4 and IL-17A induce TAP gene expression, and to determine the effect of glucocorticoid as a model of stress on this epithelial innate immune response. In primary cultures of bovine tracheal epithelial cells (bTEC), LPS, Pam3CSK4 and IL-17A each stimulated TAP gene expression. This effect was abrogated by caffeic acid phenylester (CAPE), an inhibitor of NF-κB. Similarly, western analysis showed that LPS, Pam3CSK4 and IL-17A each induced translocation of NF-κB p65 from the cytoplasm to the nucleus, but pre-treatment with CAPE inhibited this response. Finally, pre-treatment of bTEC with the glucocorticoid dexamethasone abolished the stimulatory effect of LPS, Pam3CSK4 and IL-17A on upregulation of TAP gene expression. These findings indicate that NF-κB activation is necessary for induction of TAP gene expression by LPS (a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist), or IL-17A. Furthermore, this stimulatory response is inhibited by glucocorticoid, suggesting this as one mechanism by which stress increases the risk of bacterial pneumonia. These findings have implications for understanding the pathogenesis of stress-associated bacterial pneumonia, and for developing methods to stimulate innate immune responses in the respiratory tract of cattle. PMID:26987959

  10. Midbody remnant licenses primary cilia formation in epithelial cells.

    PubMed

    Ott, Carolyn M

    2016-08-01

    Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model. PMID:27482049

  11. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    SciTech Connect

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

    1990-10-01

    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.

  12. Review: corneal epithelial stem cells, their niche and wound healing.

    PubMed

    Castro-Muñozledo, Federico

    2013-01-01

    Stem cells emerged as a concept during the second half of 19(th) century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet's membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  13. Review: Corneal epithelial stem cells, their niche and wound healing

    PubMed Central

    2013-01-01

    Stem cells emerged as a concept during the second half of 19th century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet’s membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  14. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

    PubMed

    Gomperts, Brigitte N; Belperio, John A; Rao, P Nagesh; Randell, Scott H; Fishbein, Michael C; Burdick, Marie D; Strieter, Robert M

    2006-02-01

    Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases. PMID:16424223

  15. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; hafiz, Khandakar musabbir bin; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  16. Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells.

    PubMed

    Chanoux, Rebecca A; Shubin, Calla B; Robay, Amal; Suaud, Laurence; Rubenstein, Ronald C

    2013-10-01

    The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface. PMID:23885065

  17. Pseudomonas aeruginosa pili bind to asialoGM1 which is increased on the surface of cystic fibrosis epithelial cells.

    PubMed Central

    Saiman, L; Prince, A

    1993-01-01

    The basis for the unique association of Pseudomonas aeruginosa and the cystic fibrosis (CF) lung has remained obscure despite major advances in the understanding of the molecular genetic cause of this disease. There is evidence to suggest that abnormalities in CF transmembrane conductance regulator function result in alterations in the glycosylation of epithelial components. The number of asialoGM1 residues, as representative of a class of glycolipids which contain a GalNAc beta 1-4Gal sequence for P. aeruginosa attachment, was quantified by flow cytometric studies of respiratory epithelial cells in primary culture from both CF patients and normal subjects. Superficial asialoGM1 was detected on 12% of the CF cells as compared with 2.9% of the cells from normal control subjects (P = 0.03, chi 2 = 4.73), and more asialoGM1 residues were exposed on CF cells after modification by P. aeruginosa exoproducts. AsialoGM1, but not the sialylated glycolipid GM1, was demonstrated to be a receptor for 125I-labeled P. aeruginosa pilin, a major adhesin for this organism, and exogenous asialoGM1 was found to competitively inhibit P. aeruginosa adherence to epithelial cells, thus, confirming the biological role of the asialoGM1 receptor. Quantitative and qualitative differences in the sialylation of superficial glycolipids in CF epithelial cells may directly contribute to the colonization of the CF lung by P. aeruginosa. Images PMID:8104958

  18. Alveolar epithelial type II cell: defender of the alveolus revisited

    PubMed Central

    Fehrenbach, Heinz

    2001-01-01

    In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2) cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today. PMID:11686863

  19. Host epithelial geometry regulates breast cancer cell invasiveness

    PubMed Central

    Boghaert, Eline; Gleghorn, Jason P.; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C.; Nelson, Celeste M.

    2012-01-01

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment. PMID:23150585

  20. Combustion-Derived Ultrafine Particles Transport Organic Toxicants to Target Respiratory Cells

    PubMed Central

    Penn, Arthur; Murphy, Gleeson; Barker, Steven; Henk, William; Penn, Lynn

    2005-01-01

    Epidemiologic evidence supports associations between inhalation of fine and ultrafine ambient particulate matter [aerodynamic diameter ≤ 2.5 μm (PM2.5)] and increases in cardiovascular/respiratory morbidity and mortality. Less attention has been paid to how the physical and chemical characteristics of these particles may influence their interactions with target cells. Butadiene soot (BDS), produced during combustion of the high-volume petrochemical 1,3-butadiene, is rich in polynuclear aromatic hydrocarbons (PAHs), including known carcinogens. We conducted experiments to characterize BDS with respect to particle size distribution, assembly, PAH composition, elemental content, and interaction with respiratory epithelial cells. Freshly generated, intact BDS is primarily (> 90%) PAH-rich, metals-poor (nickel, chromium, and vanadium concentrations all < 1 ppm) PM2.5, composed of uniformly sized, solid spheres (30–50 nm) in aggregated form. Cells of a human bronchial epithelial cell line (BEAS-2B) exhibit sequential fluorescent responses—a relatively rapid (~ 30 min), bright but diffuse fluorescence followed by the slower (2–4 hr) appearance of punctate cytoplasmic fluorescence—after BDS is added to medium overlying the cells. The fluorescence is associated with PAH localization in the cells. The ultrafine BDS particles move down through the medium to the cell membrane. Fluorescent PAHs are transferred from the particle surface to the cell membrane, cross the membrane into the cytosol, and appear to accumulate in lipid vesicles. There is no evidence that BDS particles pass into the cells. The results demonstrate that uptake of airborne ultrafine particles by target cells is not necessary for transfer of toxicants from the particles to the cells. PMID:16079063

  1. DNA analysis of epithelial cell suspensions

    SciTech Connect

    Wilson, J.S.; Johnson, N.F.; Holland, L.M.

    1985-01-01

    Cell suspensions of skin were obtained by animals exposed by skin painting of several crude oils. DNA analysis of these cell suspensions labeled with mithramycin provide determination of percentages of cells in the G/sub 1/, S and G/sub 2/M phases of the cell cycle. Data acquired showed differences from control animals occurring as early as 7 days after treatment and persisting through 21 days afterwards. There was histological evidence of erythema and hyperplasia in shale oil-exposed skins. Flow cytometric analysis of DNA content in shale-oil-exposed skin cells showed an increased percentage of cycling cells plus evidence of aneuploidy. Similar data from simply abraded skin showed increased percentages of cycling cells, but no aneuploidy. The shale-oil-exposed group, when compared to a standard petroleum-exposed group, had significantly increased percentages of cycling cells. This early indication of differing response to different complex mixtures was also seen in long-term skin exposures to these compounds. Similar analytical techniques were applied to tracheal cell suspensions from ozone-exposed rats. 12 refs., 4 figs., 4 tabs. (DT)

  2. To grow mouse mammary epithelial cells in culture

    PubMed Central

    1984-01-01

    Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures. PMID:6699079

  3. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  4. Helicobacter pylori increases proliferation of gastric epithelial cells.

    PubMed

    Fan, X G; Kelleher, D; Fan, X J; Xia, H X; Keeling, P W

    1996-01-01

    The direct and indirect effects of helicobacter pylori on cell kinetics of gastric epithelial cell line AGS were investigated by flow cytometric analysis of Ki-67 positive cells and by MTT assay. Flow cytometric analysis of Ki-67 positivity permits detection of cells that are in S-phase, whereas the MTT assay is a colometric measure of the number of viable cells. In the absence of added stimulants, 23.06 (4.88)% mean (SD) of AGS cells were Ki-67 positive. When cells were preincubated in the presence of H pylori, there was a significant increase in Ki-67 positivity (66.20 (7.89)%, p < 0.001). This increase was not seen in cells cultured in the presence of Campylobacter jejuni (24.63 (8.11)% or Escherichia coli (21.66 (9.78)%). Pre-incubation of AGS cells with supernatants from both H pylori and mitogen activated peripheral blood lymphocytes also increased the per cent of cells that were Ki-67 positive (72.93 (8.68) and 69.96 (12.35)%; p, 0.001) respectively. Similar results were also found in MTT assay. These data show that both H pylori directly and the immune/inflammatory response to H pylori indirectly can influence the rate of epithelial cell proliferation, suggesting this bacterium may be an initiating step in gastric carcinogenesis and an important co-carcinogenic factor in H pylori positive subjects.

  5. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  6. Clostridium difficile toxin A binding to human intestinal epithelial cells.

    PubMed

    Smith, J A; Cooke, D L; Hyde, S; Borriello, S P; Long, R G

    1997-11-01

    Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.

  7. Stiffness nanotomography of human epithelial cancer cells

    NASA Astrophysics Data System (ADS)

    Staunton, Jack R.; Doss, Bryant L.; Gilbert, C. Michael; Kasas, Sandor; Ros, Robert

    2012-02-01

    The mechanical stiffness of individual cells is important in both cancer initiation and metastasis. We present atomic force microscopy (AFM) based nanoindentation experiments on various human mammary and esophagus cell lines covering the spectrum from normal immortalized cells to highly metastatic ones. The combination of an AFM with a confocal fluorescence lifetime imaging microscope (FLIM) in conjunction with the ability to move the sample and objective independently allow for precise alignment of AFM probe and laser focus with an accuracy down to a few nanometers. This enables us to correlate the mechanical properties with the point of indentation in the FLIM image. We are using force-volume measurements as well as force indentation curves on distinct points on the cells to compare the elastic moduli of the nuclei, nucleoli, and the cytoplasm, and how they vary within and between individual cells and cell lines. Further, a detailed analysis of the force-indentation curves allows study of the cells' mechanical properties at different indentation depths and to generate 3D elasticity maps.

  8. Modulation of bronchial epithelial cell barrier function by in vitro jet propulsion fuel 8 exposure.

    PubMed

    Robledo, R F; Barber, D S; Witten, M L

    1999-09-01

    The loss of epithelial barrier integrity in bronchial and bronchiolar airways may be an initiating factor in the observed onset of toxicant-induced lung injuries. Acute 1-h inhalation exposures to aerosolized jet propulsion fuel 8 (JP-8) have been shown to induce cellular and morphological indications of pulmonary toxicity that was associated with increased respiratory permeability to 99mTc-DTPA. To address the hypothesis that JP-8 jet fuel-induced lung injury is initiated through a disruption in the airway epithelial barrier function, paracellular mannitol flux of BEAS-2B human bronchial epithelial cells was measured. Incubation of confluent cell cultures with non-cytotoxic concentrations of JP-8 or n-tetradecane (C14), a primary constituent of JP-8, for a 1-h exposure period resulted in dose-dependent increases of paracellular flux. Following exposures of 0.17, 0.33, 0.50, or 0.67 mg/ml, mannitol flux increased above vehicle controls by 10, 14, 29, and 52%, respectively, during a 2-h incubation period immediately after each JP-8 exposure. C14 caused greater mannitol flux increases of 37, 42, 63, and 78%, respectively, following identical exposure conditions. The effect on transepithelial mannitol flux reached a maximum at 12 h and spontaneously reversed to control values over a 48-h recovery period, for both JP-8 and C14 exposure. These data indicate that non-cytotoxic exposures to JP-8 or C14 exert a noxious effect on bronchial epithelial barrier function that may preclude pathological lung injury.

  9. Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

    PubMed Central

    Lee, Wing-Kee; Dittmar, Thomas

    2014-01-01

    A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin. PMID:25407650

  10. Dpp signaling directs cell motility and invasiveness during epithelial morphogenesis.

    PubMed

    Ninov, Nikolay; Menezes-Cabral, Sofia; Prat-Rojo, Carla; Manjón, Cristina; Weiss, Alexander; Pyrowolakis, George; Affolter, Markus; Martín-Blanco, Enrique

    2010-03-23

    Tissue remodeling in development and disease involves the coordinated invasion of neighboring territories and/or the replacement of entire cell populations. Cell guidance, cell matching, transitions from passive to migratory epithelia, cell growth and death, and extracellular matrix remodeling all impinge on epithelial spreading. Significantly, the extracellular signals that direct these activities and the specific cellular elements and mechanisms regulated by these signals remain in most cases to be identified. To address these issues, we performed an analysis of histoblasts (Drosophila abdominal epithelial founder cells) on their transition from a dormant state to active migration replacing obsolete larval epidermal cells (LECs). We found that during expansion, Decapentaplegic (Dpp) secreted from surrounding LECs leads to graded pathway activation in cells at the periphery of histoblast nests. Across nests, Dpp activity confers differential cellular behavior and motility by modulating cell-cell contacts, the organization and activity of the cytoskeleton, and histoblast attachment to the substrate. Furthermore, Dpp also prevents the premature death of LECs, allowing the coordination of histoblast expansion to LEC delamination. Dpp signaling activity directing histoblast spreading and invasiveness mimics transforming growth factor-beta and bone morphogenetic proteins' role in enhancing the motility and invasiveness of cancer cells, resulting in the promotion of metastasis. PMID:20226662

  11. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

  12. Vimentin contributes to human mammary epithelial cell migration.

    PubMed

    Gilles, C; Polette, M; Zahm, J M; Tournier, J M; Volders, L; Foidart, J M; Birembaut, P

    1999-12-01

    Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.

  13. Cancer stem cells from epithelial ovarian cancer patients privilege oxidative phosphorylation, and resist glucose deprivation

    PubMed Central

    Ciminale, Vincenzo; Silic-Benussi, Micol; Guzzo, Giulia; Rasola, Andrea; Frasson, Chiara; Nardo, Giorgia; Zulato, Elisabetta; Nicoletto, Maria Ornella; Manicone, Mariangela

    2014-01-01

    We investigated the metabolic profile of cancer stem cells (CSC) isolated from patients with epithelial ovarian cancer. CSC overexpressed genes associated with glucose uptake, oxidative phosphorylation (OXPHOS), and fatty acid β-oxidation, indicating higher ability to direct pyruvate towards the Krebs cycle. Consistent with a metabolic profile dominated by OXPHOS, the CSC showed higher mitochondrial reactive oxygen species (ROS) production and elevated membrane potential, and underwent apoptosis upon inhibition of the mitochondrial respiratory chain. The CSC also had a high rate of pentose phosphate pathway (PPP) activity, which is not typical of cells privileging OXPHOS over glycolysis, and may rather reflect the PPP role in recharging scavenging enzymes. Furthermore, CSC resisted in vitro and in vivo glucose deprivation, while maintaining their CSC phenotype and OXPHOS profile. These observations may explain the CSC resistance to anti-angiogenic therapies, and indicate this peculiar metabolic profile as a possible target of novel treatment strategies. PMID:24946808

  14. Cancer stem cells from epithelial ovarian cancer patients privilege oxidative phosphorylation, and resist glucose deprivation.

    PubMed

    Pastò, Anna; Bellio, Chiara; Pilotto, Giorgia; Ciminale, Vincenzo; Silic-Benussi, Micol; Guzzo, Giulia; Rasola, Andrea; Frasson, Chiara; Nardo, Giorgia; Zulato, Elisabetta; Nicoletto, Maria Ornella; Manicone, Mariangela; Indraccolo, Stefano; Amadori, Alberto

    2014-06-30

    We investigated the metabolic profile of cancer stem cells (CSC) isolated from patients with epithelial ovarian cancer. CSC overexpressed genes associated with glucose uptake, oxidative phosphorylation (OXPHOS), and fatty acid β-oxidation, indicating higher ability to direct pyruvate towards the Krebs cycle. Consistent with a metabolic profile dominated by OXPHOS, the CSC showed higher mitochondrial reactive oxygen species (ROS) production and elevated membrane potential, and underwent apoptosis upon inhibition of the mitochondrial respiratory chain. The CSC also had a high rate of pentose phosphate pathway (PPP) activity, which is not typical of cells privileging OXPHOS over glycolysis, and may rather reflect the PPP role in recharging scavenging enzymes. Furthermore, CSC resisted in vitro and in vivo glucose deprivation, while maintaining their CSC phenotype and OXPHOS profile. These observations may explain the CSC resistance to anti-angiogenic therapies, and indicate this peculiar metabolic profile as a possible target of novel treatment strategies. PMID:24946808

  15. Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells.

    PubMed

    Chen, Ying-Ting; Li, Wei; Hayashida, Yasutaka; He, Hua; Chen, Szu-Yu; Tseng, David Y; Kheirkhah, Ahmad; Tseng, Scheffer C G

    2007-08-01

    Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.

  16. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  17. Vitamin C inhibit the proliferation, migration and epithelial-mesenchymal-transition of lens epithelial cells by destabilizing HIF-1α

    PubMed Central

    Zhao, Lin; Quan, Yanlong; Wang, Jianming; Wang, Feng; Zheng, Yuping; Zhou, Aiyi

    2015-01-01

    Posterior capsular opacification (PCO), the main complication of cataract surgery, is mainly caused by the proliferation, migration, and epithelial-mesenchymal transition (EMT) of the residual lens epithelial cells (LECs).Vitamin C was reported to reduce the risk of forming a cataract. However, there has been no study showing the association between vitamin C and PCO. In this study, we found that vitamin C could inhibit the migration and proliferation of human lens epithelial cells. We also found that vitamin C could increase the proline hydroxylation of HIF-1α and reduce the activity of HIF-1α. Moreover, vitamin C could not inhibit the activity of proline-mutant HIF-1α (402/564). Overexpression of wild-type HIF-1α or proline-mutant HIF-1α was found to increase the proliferation and migration of human lens epithelial cells. Differently, vitamin C could inhibit the proliferation and migration in wild-type HIF-1α-overexpressing lens epithelial cells but not the proline-mutant HIF-1α-overexpressing cells. Additionally, vitamin C was also found to inhibit the expression of EMT transcription factors TWIST. We then found that vitamin C could repress the EMT phenotypes induced by the overexpression of wild-type HIF-1α but not the proline-mutant HIF-1α. These results provide evidence that vitamin C plays a role in the repression of proliferation, migration, and EMT of human lens epithelial cells by destabilizing HIF-1α. PMID:26628999

  18. Proteomic profiling of rat lung epithelial cells induced by acrolein

    PubMed Central

    Sarkar, Poonam; Hayes, Barbara E.

    2009-01-01

    Aims Acrolein is a highly toxic unsaturated aldehyde and is also an endogenous byproduct produced from lipid peroxidation. It can be formed from the breakdown of certain pollutants in outdoor air or from burning tobacco or gasoline. Inhalation and dermal exposure to acrolein are extremely toxic to human tissue. Although it is known that acrolein is toxic to lung tissue, no studies have attempted to address the changes induced by acrolein on a global scale. Main methods In the present study we have attempted to address the changes in global protein expression induced by acrolein using proteomics analysis in rat lung epithelial cells. Key findings Our analysis reveals a comprehensive profiling of the proteins that includes a heterogeneous class of proteins and this compels one to consider that the toxic response to acrolein is very complex. There were 34 proteins that showed changes between the control cells and after acrolein treatment. The expression of 18 proteins was increased and the expression of 16 proteins was decreased following exposure to acrolein. We have further validated two differentially expressed proteins namely annexin II (ANXII) and prohibitin (PHB) in lung epithelial cells treated with acrolein. Significance Based on the results of the overall proteomic analysis, acrolein appears to induce changes in a diverse range of proteins suggesting a complex mechanism of acrolein-induced toxicity in lung epithelial cells. PMID:19490921

  19. Serratia marcescens is injurious to intestinal epithelial cells.

    PubMed

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  20. TLR2 Activation Limits Rhinovirus-Stimulated CXCL-10 by Attenuating IRAK-1-Dependent IL-33 Receptor Signaling in Human Bronchial Epithelial Cells.

    PubMed

    Ganesan, Shyamala; Pham, Duc; Jing, Yaxun; Farazuddin, Mohammad; Hudy, Magdalena H; Unger, Benjamin; Comstock, Adam T; Proud, David; Lauring, Adam S; Sajjan, Uma S

    2016-09-15

    Airway epithelial cells are the major target for rhinovirus (RV) infection and express proinflammatory chemokines and antiviral cytokines that play a role in innate immunity. Previously, we demonstrated that RV interaction with TLR2 causes ILR-associated kinase-1 (IRAK-1) depletion in both airway epithelial cells and macrophages. Further, IRAK-1 degradation caused by TLR2 activation was shown to inhibit ssRNA-induced IFN expression in dendritic cells. Therefore, in this study, we examined the role of TLR2 and IRAK-1 in RV-induced IFN-β, IFN-λ1, and CXCL-10, which require signaling by viral RNA. In airway epithelial cells, blocking TLR2 enhanced RV-induced expression of IFNs and CXCL-10. By contrast, IRAK-1 inhibition abrogated RV-induced expression of CXCL-10, but not IFNs in these cells. Neutralization of IL-33 or its receptor, ST2, which requires IRAK-1 for signaling, inhibited RV-stimulated CXCL-10 expression. In addition, RV induced expression of both ST2 and IL-33 in airway epithelial cells. In macrophages, however, RV-stimulated CXCL-10 expression was primarily dependent on TLR2/IL-1R. Interestingly, in a mouse model of RV infection, blocking ST2 not only attenuated RV-induced CXCL-10, but also lung inflammation. Finally, influenza- and respiratory syncytial virus-induced CXCL-10 was also found to be partially dependent on IL-33/ST2/IRAK-1 signaling in airway epithelial cells. Together, our results indicate that RV stimulates CXCL-10 expression via the IL-33/ST2 signaling axis, and that TLR2 signaling limits RV-induced CXCL-10 via IRAK-1 depletion at least in airway epithelial cells. To our knowledge, this is the first report to demonstrate the role of respiratory virus-induced IL-33 in the induction of CXCL-10 in airway epithelial cells. PMID:27503209

  1. A novel cholinergic epithelial cell with chemosensory traits in the murine conjunctiva.

    PubMed

    Wiederhold, Stephanie; Papadakis, Tamara; Chubanov, Vladimir; Gudermann, Thomas; Krasteva-Christ, Gabriela; Kummer, Wolfgang

    2015-11-01

    We recently identified a specialized cholinergic cell type in tracheal and urethral epithelium that utilizes molecules of the canonical taste transduction signaling cascade to sense potentially harmful substances in the luminal content. Upon stimulation, this cell initiates protective reflexes. Assuming a sentinel role of such cells at mucosal surfaces exposed to bacteria, we hypothesized their occurrence also in ocular mucosal surfaces. Utilizing a mouse strain expressing eGFP under the promoter of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT-eGFP), we observed a cholinergic cell in the murine conjunctiva. Singular cholinergic cells reaching the epithelial surface with slender processes were detected in fornical, but neither in bulbar nor palpebral epithelia. These cells were found neither in the lacrimal canaliculi, nor in the lacrimal sac and the nasolacrimal duct. Cholinergic conjunctival epithelial cells were immunoreactive for components of the canonical taste transduction signaling cascade, i.e. α-gustducin, phospholipase Cβ2 and the monovalent cation channel TRPM5. Calcitonin gene-related peptide- and substance P-immunoreactive sensory nerve fibers were observed extending into the conjunctival epithelium approaching slender ChAT-eGFP-positive cells. In addition, we noted both ChAT-eGFP expression and α-gustducin-immunoreactivity, albeit in different cell populations, in occasionally occurring lymphoid follicles of the nictitating membrane. The data show a previously unidentified cholinergic cell in murine conjunctiva with chemosensory traits that presumably utilizes acetylcholine for signaling. In analogy to similar cells described in the respiratory and urethral epithelium, it might serve to detect bacterial products and to initiate protective reflexes. PMID:26119492

  2. In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

    2015-01-01

    Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue.

  3. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left–right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left–right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left–right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  4. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis.

    PubMed

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  5. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  6. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  7. Bone marrow stem cells do not repopulate the healthy upper respiratory tract.

    PubMed

    Davies, Jane C; Potter, Mike; Bush, Andrew; Rosenthal, Mark; Geddes, Duncan M; Alton, Eric W F W

    2002-10-01

    Recent studies reported differentiation of both bone marrow and tissue-specific stem cells into cells of other organs. The demonstration that bone marrow stem cells differentiate into human hepatocytes in vivo has raised the possibility of new therapeutic approaches for liver disease. For diseases such as cystic fibrosis (CF), correction of the respiratory epithelium is being attempted by gene therapy. Differentiation of bone marrow stem cells into epithelium of the lung and airway was recently reported in an animal model, and would provide an alternative approach. We examined the nasal epithelium of female patients up to 15 years after gender-mismatched bone marrow transplantation. Donor-derived epithelial cells were sought with a combination of Y-chromosome fluorescence in situ hybridization and anti-cytokeratin antibody. In nasal brushing samples from 6 transplant-recipients, a median of 2.5% (range, 0.7-18.1%) of nuclei was male and identified as being of donor-origin. However, a complete absence of staining with anti-cytokeratin antibodies confirmed that these were not epithelial cells, but were likely to be either intraepithelial lymphocytes or mesenchymal cells. Following whole bone marrow transplantation, bone marrow progenitor cells do not differentiate into respiratory epithelium of the healthy upper airway. The differences between this and other studies could relate to the cells transplanted, to differential rates of turnover, or to the requirement for specific triggers to stimulate migration and differentiation. In the absence of such conditions, whole bone marrow transplantation is unlikely to provide a route for correction of the CF airway. PMID:12205565

  8. Oral epithelial cell responses to multispecies microbial biofilms.

    PubMed

    Peyyala, R; Kirakodu, S S; Novak, K F; Ebersole, J L

    2013-03-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms.

  9. LOXL2 in epithelial cell plasticity and tumor progression.

    PubMed

    Cano, Amparo; Santamaría, Patricia G; Moreno-Bueno, Gema

    2012-09-01

    Several members of the lysyl oxidase family have recently emerged as important regulators of tumor progression. Among them, LOXL2 has been shown to be involved in tumor progression and metastasis of several tumor types, including breast carcinomas. Secreted LOXL2 participates in the remodeling of the extracellular matrix of the tumor microenvironment, in a similar fashion to prototypical lysyl oxidase. In addition, new intracellular functions of LOXL2 have been described, such as its involvement in the regulation of the epithelial-to-mesenchymal transition, epithelial cell polarity and differentiation mediated by transcriptional repression mechanisms. Importantly, intracellular (perinuclear) expression of LOXL2 is associated with poor prognosis and distant metastasis of specific tumor types, such as larynx squamous cell carcinoma and basal breast carcinomas. These recent findings open new avenues for the therapeutic utility of LOXL2.

  10. Epithelial Cell Polarity Determinant CRB3 in Cancer Development

    PubMed Central

    Li, Pingping; Mao, Xiaona; Ren, Yu; Liu, Peijun

    2015-01-01

    Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development. PMID:25552927

  11. Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell

    PubMed Central

    Kikuchi, Hiroyuki; Prasad, Ankush; Matsuoka, Ryo; Aoyagi, Shigeo; Matsue, Tomokazu; Kasai, Shigenobu

    2016-01-01

    Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials. PMID:26903876

  12. Scanning Electrochemical Microscopy Imaging during Respiratory Burst in Human Cell.

    PubMed

    Kikuchi, Hiroyuki; Prasad, Ankush; Matsuoka, Ryo; Aoyagi, Shigeo; Matsue, Tomokazu; Kasai, Shigenobu

    2016-01-01

    Phagocytic cells, such as neutrophils and monocytes, consume oxygen and generate reactive oxygen species (ROS) in response to external stimuli. Among the various ROS, the superoxide anion radical is known to be primarily produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. In the current study, we attempt to evaluate the respiratory burst by monitoring the rapid consumption of oxygen by using scanning electrochemical microscopy (SECM) imaging. The respiratory burst was measured in a human monocytic cell line (THP-1 cells) derived from an acute monocytic leukemia patient under the effect of the exogenous addition of phorbol 12-myristate 13-acetate, which acts as a differentiation inducer. SECM imaging composed of a microelectrode was used to compare oxygen consumption between normal cellular respiration and during respiratory burst in THP-1 cells. Two-dimensional respiratory activity imaging was performed using XY-scan. In addition, the quantitative evaluation of oxygen consumption in THP-1 cells was performed using a Z-scan. The results obtained show higher consumption of oxygen in cells undergoing respiratory burst. SECM imaging is thus claimed to be a highly sensitive and appropriate technique compared to other existing techniques available for evaluating oxidative stress in human cells, making it potentially useful for widespread applications in biomedical research and clinical trials.

  13. Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

    PubMed Central

    Davis, A. Sally; Chertow, Daniel S.; Moyer, Jenna E.; Suzich, Jon; Sandouk, Aline; Dorward, David W.; Logun, Carolea; Shelhamer, James H.

    2015-01-01

    Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic. PMID:25604814

  14. Ivermectin inhibits growth of Chlamydia trachomatis in epithelial cells.

    PubMed

    Pettengill, Matthew A; Lam, Verissa W; Ollawa, Ikechukwu; Marques-da-Silva, Camila; Ojcius, David M

    2012-01-01

    Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia. PMID:23119027

  15. Characterization of foamy epithelial surface cells in the canine endometrium.

    PubMed

    Bartel, C; Tichy, A; Walter, I

    2014-06-01

    In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, β-catenin and E-cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E-cadherin and β-catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid-accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.

  16. Role of medullary progenitor cells in epithelial cell migration and proliferation

    PubMed Central

    Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

    2014-01-01

    This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

  17. Hyperoxia alters the mechanical properties of alveolar epithelial cells.

    PubMed

    Roan, Esra; Wilhelm, Kristina; Bada, Alex; Makena, Patrudu S; Gorantla, Vijay K; Sinclair, Scott E; Waters, Christopher M

    2012-06-15

    Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability. PMID:22467640

  18. TCDD alters medial epithelial cell differentiation during palatogenesis

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1989-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of (3H)TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation.

  19. Cytotoxicity of Different Excipients on RPMI 2650 Human Nasal Epithelial Cells.

    PubMed

    Horváth, Tamás; Bartos, Csilla; Bocsik, Alexandra; Kiss, Lóránd; Veszelka, Szilvia; Deli, Mária A; Újhelyi, Gabriella; Szabó-Révész, Piroska; Ambrus, Rita

    2016-01-01

    The nasal route receives a great deal of attention as a non-invasive method for the systemic administration of drugs. For nasal delivery, specific formulations containing excipients are used. Because of the sensitive respiratory mucosa, not only the active ingredients, but also additives need to be tested in appropriate models for toxicity. The aim of the study was to measure the cytotoxicity of six pharmaceutical excipients, which could help to reach larger residence time, better permeability, and increased solubility dissolution rate. The following excipients were investigated on RPMI 2650 human nasal septum tumor epithelial cells: β-d-mannitol, sodium hyaluronate, α and β-cyclodextrin, polyvinyl alcohol and methylcellulose. 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye conversion assay and real-time impedance analysis were used to investigate cytotoxicity. No excipient showed toxicity at 0.3% (w/v) concentration or below while 1% concentration a significantly reduced metabolic activity was measured by MTT assay for methylcellulose and cyclodextrins. Using impedance measurements, only β-cyclodextrin (1%) was toxic to cells. Mannitol at 1% concentration had a barrier opening effect on epithelial cells, but caused no cellular damage. Based on the results, all additives at 0.3%, sodium hyaluronate and polyvinyl alcohol at 1% concentrations can be safely used for nasal formulations. PMID:27213303

  20. Chlorobenzene induces oxidative stress in human lung epithelial cells in vitro

    SciTech Connect

    Feltens, Ralph; Moegel, Iljana; Roeder-Stolinski, Carmen; Simon, Jan-Christoph; Herberth, Gunda; Lehmann, Irina

    2010-01-01

    Chlorobenzene is a volatile organic compound (VOC) that is widely used as a solvent, degreasing agent and chemical intermediate in many industrial settings. Occupational studies have shown that acute and chronic exposure to chlorobenzene can cause irritation of the mucosa of the upper respiratory tract and eyes. Using in vitro assays, we have shown in a previous study that human bronchial epithelial cells release inflammatory mediators such as the cytokine monocyte chemoattractant protein-1 (MCP-1) in response to chlorobenzene. This response is mediated through the NF-kappaB signaling pathway. Here, we investigated the effects of monochlorobenzene on human lung cells, with emphasis on potential alterations of the redox equilibrium to clarify whether the chlorobenzene-induced inflammatory response in lung epithelial cells is caused via an oxidative stress-dependent mechanism. We found that expression of cellular markers for oxidative stress, such as heme oxygenase 1 (HO-1), glutathione S-transferase pi1 (GSTP1), superoxide dismutase 1 (SOD1), prostaglandin-endoperoxide synthase 2 (PTGS2) and dual specificity phosphatase 1 (DUSP1), were elevated in the presence of monochlorobenzene. Likewise, intracellular reactive oxygen species (ROS) were increased in response to exposure. However, in the presence of the antioxidants N-(2-mercaptopropionyl)-glycine (MPG) or bucillamine, chlorobenzene-induced upregulation of marker proteins and release of the inflammatory mediator MCP-1 are suppressed. These results complement our previous findings and point to an oxidative stress-mediated inflammatory response following chlorobenzene exposure.

  1. Interleukin-13 Inhibits Lipopolysaccharide-Induced BPIFA1 Expression in Nasal Epithelial Cells.

    PubMed

    Tsou, Yung-An; Lin, Chia-Der; Chen, Hui-Chen; Hsu, Hui-Ying; Wu, Lii-Tzu; Chiang-Ni, Chuan; Chen, Chih-Jung; Wu, Tsu-Fang; Kao, Min-Chuan; Chen, Yu-An; Peng, Ming-Te; Tsai, Ming-Hsui; Chen, Chuan-Mu; Lai, Chih-Ho

    2015-01-01

    Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is expressed in human nasopharyngeal and respiratory epithelium and has demonstrated antimicrobial activity. SPLUNC1 is now referred to as bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1). Reduced BPIFA1 expression is associated with bacterial colonization in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Interleukin 13 (IL-13), predominately secreted by T helper 2 (TH2) cells, has been found to contribute to airway allergies and suppress BPIFA1 expression in nasal epithelial cells. However, the molecular mechanism of IL-13 perturbation of bacterial infection and BPIFA1 expression in host airways remains unclear. In this study, we found that lipopolysaccharide (LPS)-induced BPIFA1 expression in nasal epithelial cells was mediated through the JNK/c-Jun signaling pathway and AP-1 activation. We further demonstrated that IL-13 downregulated the LPS-induced activation of phosphorylated JNK and c-Jun, followed by attenuation of BPIFA1 expression. Moreover, the immunohistochemical analysis showed that IL-13 prominently suppressed BPIFA1 expression in eosinophilic CRSwNP patients with bacterial infection. Taken together, these results suggest that IL-13 plays a critical role in attenuation of bacteria-induced BPIFA1 expression that may result in eosinophilic CRSwNP. PMID:26646664

  2. Interleukin-13 Inhibits Lipopolysaccharide-Induced BPIFA1 Expression in Nasal Epithelial Cells

    PubMed Central

    Chen, Hui-Chen; Hsu, Hui-Ying; Wu, Lii-Tzu; Chiang-Ni, Chuan; Chen, Chih-Jung; Wu, Tsu-Fang; Kao, Min-Chuan; Chen, Yu-An; Peng, Ming-Te; Tsai, Ming-Hsui; Chen, Chuan-Mu; Lai, Chih-Ho

    2015-01-01

    Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is expressed in human nasopharyngeal and respiratory epithelium and has demonstrated antimicrobial activity. SPLUNC1 is now referred to as bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1). Reduced BPIFA1 expression is associated with bacterial colonization in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Interleukin 13 (IL-13), predominately secreted by T helper 2 (TH2) cells, has been found to contribute to airway allergies and suppress BPIFA1 expression in nasal epithelial cells. However, the molecular mechanism of IL-13 perturbation of bacterial infection and BPIFA1 expression in host airways remains unclear. In this study, we found that lipopolysaccharide (LPS)-induced BPIFA1 expression in nasal epithelial cells was mediated through the JNK/c-Jun signaling pathway and AP-1 activation. We further demonstrated that IL-13 downregulated the LPS-induced activation of phosphorylated JNK and c-Jun, followed by attenuation of BPIFA1 expression. Moreover, the immunohistochemical analysis showed that IL-13 prominently suppressed BPIFA1 expression in eosinophilic CRSwNP patients with bacterial infection. Taken together, these results suggest that IL-13 plays a critical role in attenuation of bacteria-induced BPIFA1 expression that may result in eosinophilic CRSwNP. PMID:26646664

  3. Acrolein induced both pulmonary inflammation and the death of lung epithelial cells.

    PubMed

    Sun, Yang; Ito, Sachiko; Nishio, Naomi; Tanaka, Yuriko; Chen, Nana; Isobe, Ken-Ichi

    2014-09-01

    Acrolein, a compound found in cigarette smoke, is a major risk factor for respiratory diseases. Previous research determined that both acrolein and cigarette smoke produced reactive oxygen species (ROS). As many types of pulmonary injuries are associated with inflammation, this study sought to ascertain the extent to which exposure to acrolein advanced inflammatory state in the lungs. Our results showed that intranasal exposure of mice to acrolein increased CD11c(+)F4/80(high) macrophages in the lungs and increased ROS formation via induction of NF-κB signaling. Treatment with acrolein activated macrophages and led to their increased production of ROS and expression of several key pro-inflammatory cytokines. In in vitro studies, acrolein treatment of bone marrow-derived GM-CSF-dependent immature macrophages (GM-IMs), activated the cells and led to their increased production of ROS and expression of several key pro-inflammatory cytokines. Acrolein treatment of macrophages induced apoptosis of lung epithelial cells. Inclusion of an inhibitor of ROS formation markedly decreased acrolein-mediated macrophage activation and reduced the extent of epithelial cell death. These results indicate that acrolein can cause lung damage, in great part by mediating the increased release of pro-inflammatory cytokines/factors by macrophages.

  4. N-acetylcysteine inhibits Na+ absorption across human nasal epithelial cells.

    PubMed

    Rochat, Thierry; Lacroix, Jean-Silvain; Jornot, Lan

    2004-10-01

    N-acetylcysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders. The mechanism of action is based on rupture of the disulfide bridges of the high molecular glycoproteins present in the mucus, resulting in smaller subunits of the glycoproteins and reduced viscosity of the mucus. Because Na(+) absorption regulates airway surface liquid volume and thus the efficiency of mucociliary clearance, we asked whether NAC affects the bioelectric properties of human nasal epithelial cells. A 24-h basolateral treatment with 10 mM of NAC decreased the transepithelial potential difference and short-circuit current (I(SC)) by 40%, and reduced the amiloride-sensitive current by 50%, without affecting the transepithelial resistance. After permeabilization of the basolateral membranes of cells with amphotericin B in the presence of a mucosal-to-serosal Na(+) gradient (135:25 mM), NAC inhibited 45% of the amiloride-sensitive current. The Na(+)-K(+)-ATPase pump activity and the basolateral K(+) conductance were not affected by NAC treatment. NAC did not alter total cell mRNA and protein levels of alpha-epithelial Na(+) channel (EnaC) subunit, but reduced abundance of alpha-ENaC subunits in the apical cell membrane as quantified by biotinylation. This effect can be ascribed to the sulphydryl (SH) group of NAC, since N-acetylserine and S-carboxymethyl-l-cysteine were ineffective. Given the importance of epithelial Na(+) channels in controlling the thin layer of fluid that covers the surface of the airways, the increase in the fluidity of the airway mucus following NAC treatment in vivo might be in part related to downregulation of Na(+) absorption and consequently water transport. PMID:15281093

  5. N-acetylcysteine inhibits Na+ absorption across human nasal epithelial cells.

    PubMed

    Rochat, Thierry; Lacroix, Jean-Silvain; Jornot, Lan

    2004-10-01

    N-acetylcysteine (NAC) is a widely used mucolytic drug in patients with a variety of respiratory disorders. The mechanism of action is based on rupture of the disulfide bridges of the high molecular glycoproteins present in the mucus, resulting in smaller subunits of the glycoproteins and reduced viscosity of the mucus. Because Na(+) absorption regulates airway surface liquid volume and thus the efficiency of mucociliary clearance, we asked whether NAC affects the bioelectric properties of human nasal epithelial cells. A 24-h basolateral treatment with 10 mM of NAC decreased the transepithelial potential difference and short-circuit current (I(SC)) by 40%, and reduced the amiloride-sensitive current by 50%, without affecting the transepithelial resistance. After permeabilization of the basolateral membranes of cells with amphotericin B in the presence of a mucosal-to-serosal Na(+) gradient (135:25 mM), NAC inhibited 45% of the amiloride-sensitive current. The Na(+)-K(+)-ATPase pump activity and the basolateral K(+) conductance were not affected by NAC treatment. NAC did not alter total cell mRNA and protein levels of alpha-epithelial Na(+) channel (EnaC) subunit, but reduced abundance of alpha-ENaC subunits in the apical cell membrane as quantified by biotinylation. This effect can be ascribed to the sulphydryl (SH) group of NAC, since N-acetylserine and S-carboxymethyl-l-cysteine were ineffective. Given the importance of epithelial Na(+) channels in controlling the thin layer of fluid that covers the surface of the airways, the increase in the fluidity of the airway mucus following NAC treatment in vivo might be in part related to downregulation of Na(+) absorption and consequently water transport.

  6. Particles induce apical plasma membrane enlargement in epithelial lung cell line depending on particle surface area dose

    PubMed Central

    Brandenberger, Christina; Rothen-Rutishauser, Barbara; Blank, Fabian; Gehr, Peter; Mühlfeld, Christian

    2009-01-01

    Background Airborne particles entering the respiratory tract may interact with the apical plasma membrane (APM) of epithelial cells and enter them. Differences in the entering mechanisms of fine (between 0.1 μm and 2.5 μm) and ultrafine ( ≤ 0.1 μm) particles may be associated with different effects on the APM. Therefore, we studied particle-induced changes in APM surface area in relation to applied and intracellular particle size, surface and number. Methods Human pulmonary epithelial cells (A549 cell line) were incubated with various concentrations of different sized fluorescent polystyrene spheres without surface charge (∅ fine – 1.062 μm, ultrafine – 0.041 μm) by submersed exposure for 24 h. APM surface area of A549 cells was estimated by design-based stereology and transmission electron microscopy. Intracellular particles were visualized and quantified by confocal laser scanning microscopy. Results Particle exposure induced an increase in APM surface area compared to negative control (p < 0.01) at the same surface area concentration of fine and ultrafine particles a finding not observed at low particle concentrations. Ultrafine particle entering was less pronounced than fine particle entering into epithelial cells, however, at the same particle surface area dose, the number of intracellular ultrafine particles was higher than that of fine particles. The number of intracellular particles showed a stronger increase for fine than for ultrafine particles at rising particle concentrations. Conclusion This study demonstrates a particle-induced enlargement of the APM surface area of a pulmonary epithelial cell line, depending on particle surface area dose. Particle uptake by epithelial cells does not seem to be responsible for this effect. We propose that direct interactions between particle surface area and cell membrane cause the enlargement of the APM. PMID:19284624

  7. Elastic properties of epithelial cells probed by atomic force microscopy.

    PubMed

    Brückner, Bastian R; Janshoff, Andreas

    2015-11-01

    Cellular mechanics plays a crucial role in many biological processes such as cell migration, cell growth, embryogenesis, and oncogenesis. Epithelia respond to environmental cues comprising biochemical and physical stimuli through defined changes in cell elasticity. For instance, cells can differentiate between certain properties such as viscoelasticity or topography of substrates by adapting their own elasticity and shape. A living cell is a complex viscoelastic body that not only exhibits a shell architecture composed of a membrane attached to a cytoskeleton cortex but also generates contractile forces through its actomyosin network. Here we review cellular mechanics of single cells in the context of epithelial cell layers responding to chemical and physical stimuli. This article is part of a Special Issue entitled: Mechanobiology. PMID:26193077

  8. Characterization of goldfish fin cells in culture: some evidence of an epithelial cell profile.

    PubMed

    Mauger, P-E; Labbé, C; Bobe, J; Cauty, C; Leguen, I; Baffet, G; Le Bail, P-Y

    2009-03-01

    Comprehensive characterization of cultured cells in fish was little explored and cell origin is often deduced from morphological analogies with either epithelial of fibroblastic cells. This study aims to characterize cell origin in goldfish fin culture using morphological, immunochemical, and molecular approaches. Time lapse analysis revealed that cultured cell morphology changed within minutes. Therefore, cell morphology cannot predict whether cells are from fibroblastic or epithelial origin. The labeling pattern of heterologous anti-cytokeratin and anti-vimentin antibodies against goldfish epithelial cells and fibroblasts was first tested on skin sections and the corresponding labeling of the cultured cells was analyzed. No cell origin specificity could be obtained with the chosen antibodies. In the molecular approach, detection levels of three cytokeratin (CauK8-IIS, CauK49-IE and CauK50-Ie) and one vimentin transcripts were assessed on skin and fin samples. Specificity for epithelial cells of the most abundant mRNA, CauK49-Ie, was thereafter validated on skin sections by in situ hybridization. The selected markers were used afterwards to characterize fin cultures. CauK49-IE riboprobe labeled every cell in young cultures whereas no labeling was observed in older cultures. Accordingly, CauK49-IE transcript levels decreased after 15 days culture while CauK8-IIS ones increased. The use of homologous marker gave evidence that young cultured cells from goldfish fin are homogeneously of epithelial type and that cell characteristics may change over culture time.

  9. Comparison of functional limbal epithelial stem cell isolation methods.

    PubMed

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Dziasko, Marc; Galindo, Sara; Rey, Esther; Herreras, José M; Corrales, Rosa M; Daniels, Julie T; Calonge, Margarita

    2016-05-01

    The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either

  10. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  11. Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture.

    PubMed

    Leguen; Carlsson; Perdu-Durand; Prunet; Pärt; Cravedi

    2000-03-01

    The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10(-6) to 10(-12) M. After 24 h, TCDD showed a maximal induction at a concentration of 10(-9) M while BNF showed a maximal induction at a concentration of 10(-7) M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 µM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6beta and 16beta and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate.

  12. Unexpected Role for Adaptive αβTH17 Cells in Acute Respiratory Distress Syndrome1

    PubMed Central

    Li, John T.; Melton, Andrew C.; Su, George; Hamm, David E.; LaFemina, Michael; Howard, James; Fang, Xiaohui; Bhat, Sudarshan; Huynh, Kieu-My; O’Kane, Cecilia M.; Ingram, Rebecca J.; Muir, Roshell R.; McAuley, Daniel F.; Matthay, Michael A.; Sheppard, Dean

    2015-01-01

    Acute respiratory distress syndrome (ARDS) is a devastating disorder characterized by increased alveolar permeability with no effective treatment beyond supportive care. Current mechanisms underlying ARDS focus on alveolar endothelial and epithelial injury caused by products of innate immune cells and platelets. However, the role of adaptive immune cells in ARDS remains largely unknown. Here we report that expansion of antigen-specific αβT helper 17 (αβTH17) cells contribute to ARDS by local secretion of IL-17A, which in turn directly increases alveolar epithelial permeability. Mice with a highly restrictive defect in antigen-specific αβTH17 cells were protected from experimental ARDS induced by a single dose of endotracheal lipopolysaccharide (LPS). Loss of IL-17 receptor C or antibody blockade of IL-17A was similarly protective, further suggesting that IL-17A released by these cells was responsible for this effect. LPS induced a rapid and specific clonal expansion of αβTH17 cells in the lung, as determined by deep sequencing of the hypervariable CD3RβVJ region of the T cell receptor. Our findings could be relevant to ARDS in humans, since we found significant elevation of IL-17A in bronchoalveolar lavage (BAL) fluid from patients with ARDS and recombinant IL-17A directly increased permeability across cultured human alveolar epithelial monolayers. These results reveal a previously unexpected role for adaptive immune responses that increase alveolar permeability in ARDS and suggest that αβTH17 cells and IL-17A could be novel therapeutic targets for this currently untreatable disease. PMID:26002979

  13. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation.

  14. Mammary Epithelial Cell Hierarchy in the Dairy Cow Throughout Lactation.

    PubMed

    Perruchot, Marie-Hélène; Arévalo-Turrubiarte, Magdalena; Dufreneix, Florence; Finot, Laurence; Lollivier, Vanessa; Chanat, Eric; Mayeur, Frédérique; Dessauge, Frédéric

    2016-10-01

    The plasticity of the mammary gland relies on adult mammary stem cells (MaSCs) and their progenitors, which give rise to various populations of mammary epithelial cells (MECs). To face global challenges, an in-depth characterization of milk-producing animal mammary gland plasticity is required, to select more sustainable and robust dairy cows. The identification and characterization of MaSC and their progenitors will also provide innovative tools in veterinary/human medicine regarding mammary tissue damage (carcinogenesis, bacterial infections). This study aimed to determine the dynamics of mammary cell populations throughout a lactation cycle. Using mammary biopsies from primiparous lactating dairy cows at 30, 90, 150, and 250 days of lactation, we phenotyped cell populations by flow cytometry. To investigate cell lineages, we used specific cell-surface markers, including CD49f, CD24, EpCAM (epithelial cell adhesion molecule), and CD10. Two cell populations linked to milk production were identified: CD49f(+)/EpCAM(-) (y = 0.88x + 4.42, R(2) = 0.36, P < 0.05) and CD49f(-)/EpCAM(-) (y = -1.15x + 92.44, R(2) = 0.51, P < 0.05) cells. Combining immunostaining analysis, flow cytometry, daily milk production data, and statistical approaches, we defined a stem cell population (CD24(+)/CD49f(+)) and four progenitor cell populations that include bipotent luminal progenitors (CD24(-)/CD49f(+)), lumino-alveolar progenitors (CD24(-)/EpCAM(+)), myoepithelial progenitors (CD24(+)/CD10(-)), and lumino-ductal progenitors (CD49f(-)/EpCAM(+)). Interestingly, we found that the bipotent luminal progenitors (CD24(-)/CD49f(+)) decreased significantly (P < 0.05) during lactation. This study provides the first results of mammary cell lineage, allowing insight into mammary cell plasticity during lactation. PMID:27520504

  15. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

    PubMed Central

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs. PMID:27409796

  16. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells.

    PubMed

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  17. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells

    PubMed Central

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  18. Lung epithelial cell-derived extracellular vesicles activate macrophage-mediated inflammatory responses via ROCK1 pathway.

    PubMed

    Moon, H-G; Cao, Y; Yang, J; Lee, J H; Choi, H S; Jin, Y

    2015-12-10

    Despite decades of research, the pathogenesis of acute respiratory distress syndrome (ARDS) remains poorly understood, thus impeding the development of effective treatment. Diffuse alveolar damage (DAD) and lung epithelial cell death are prominent features of ARDS. Lung epithelial cells are the first line of defense after inhaled stimuli, such as in the case of hyperoxia. We hypothesized that lung epithelial cells release 'messenger' or signaling molecules to adjacent or distant macrophages, thereby initiating or propagating inflammatory responses after noxious insult. We found that, after hyperoxia, a large amount of extracellular vesicles (EVs) were generated and released into bronchoalveolar lavage fluid (BALF). These hyperoxia-induced EVs were mainly derived from live lung epithelial cells as the result of hyperoxia-associated endoplasmic reticulum (ER) stress. These EVs were remarkably different from epithelial 'apoptotic bodies', as reflected by the significantly smaller size and differentially expressed protein markers. These EVs fall mainly in the size range of the exosomes and smaller microvesicles (MVs) (50-120 nm). The commonly featured protein markers of apoptotic bodies were not found in these EVs. Treating alveolar macrophages with hyperoxia-induced, epithelial cell-derived EVs led to an increased secretion of pro-inflammatory cytokines and macrophage inflammatory protein 2 (MIP-2). Robustly increased macrophage and neutrophil influx was found in the lung tissue of the mice intranasally treated with hyperoxia-induced EVs. It was determined that EV-encapsulated caspase-3 was largely responsible for the alveolar macrophage activation via the ROCK1 pathway. Caspase-3-deficient EVs induced less cytokine/MIP-2 release, reduced cell counts in BALF, less neutrophil infiltration and less inflammation in lung parenchyma, both in vitro and in vivo. Furthermore, the serum circulating EVs were increased and mainly derived from lung epithelial cells after

  19. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.

    PubMed

    Luissint, Anny-Claude; Parkos, Charles A; Nusrat, Asma

    2016-10-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  20. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

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

    2010-03-15

    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{sup -/-} 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{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  1. Studies in human skin epithelial cell carcinogenesis

    SciTech Connect

    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 /sup 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 /sup 32/P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts.

  2. Hertwig's epithelial root sheath cells do not transform into cementoblasts in rat molar cementogenesis.

    PubMed

    Yamamoto, Tsuneyuki; Takahashi, Shigeru

    2009-12-01

    It is generally accepted that cementoblasts originate in the process of differentiation of the mesenchymal cells of the dental follicle. Recently, a different hypothesis for the origin of cementoblasts has been proposed. Hertwig's epithelial root sheath cells undergo the epithelial-mesenchymal transformation to differentiate into cementoblasts. To elucidate whether the epithelial-mesenchymal transformation occurs in the epithelial sheath, developing rat molars were examined by keratin-vimentin and Runx2 (runt-related transcription factor 2)-keratin double immunostaining. In both acellular and cellular cementogenesis, epithelial sheath and epithelial cells derived from the epithelial sheath expressed keratin, but did not express vimentin or Runx2. Dental follicle cells and cementoblasts, however, expressed vimentin and Runx2, but did not express keratin. No cells showed coexisting keratin-vimentin or Runx2-keratin staining. These findings suggest that there is no intermediate phenotype transforming epithelial to mesenchymal cells, and that epithelial sheath cells do not generate mineralized tissue. This study concludes that the epithelial-mesenchymal transformation does not occur in Hertwig's epithelial root sheath in rat acellular or cellular cementogenesis and that the dental follicle is the origin of cementoblasts, as has been proposed in the original hypothesis. PMID:19716687

  3. Thymic epithelial cells: working class heroes for T cell development and repertoire selection.

    PubMed

    Anderson, Graham; Takahama, Yousuke

    2012-06-01

    The thymus represents an epithelial-mesenchymal tissue, anatomically structured into discrete cortical and medullary regions that contain phenotypically and functionally distinct stromal cells, as well as thymocytes at defined stages of maturation. The stepwise progression of thymocyte development seems to require serial migration through these distinct thymic regions, where interactions with cortical thymic epithelial cell (cTEC) and medullary thymic epithelial cell (mTEC) subsets take place. Recent work on TEC subsets provides insight into T cell development and selection, such as the importance of tumour necrosis factor (TNF) receptor superfamily members in thymus medulla development, and the specialised antigen processing/presentation capacity of the thymic cortex for positive selection. Here, we summarise current knowledge on the development and function of the thymic microenvironment, paying particular attention to the cortical and medullary epithelial compartments.

  4. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  5. Incremental responses to light recorded from pigment epithelial cells and horizontal cells of the cat retina

    PubMed Central

    Steinberg, Roy H.

    1971-01-01

    1. Rod-dependent incremental responses were recorded intracellularly in both pigment epithelial cells and horizontal cells of the cat retina. They were elicited by test flashes which were superimposed on background flashes after a delay. 2. In pigment epithelial cells smaller test responses were produced as background intensity was raised. The incremental sensitivity function was linear for about 1·4 log units, with a slope of 0·86, and the approach of saturation occurred at about 2·5 log td scotopic. 3. The amplitude of pigment epithelial test responses could be estimated from the dark-adapted amplitude—log intensity function obtained with single flashes. Test flashes produced the voltage increment predicted by the slope of this function just above the point on the curve equal to the background intensity. The pigment epithelial response to a test flash, therefore, is the response expected if the background were presented alone and made more intense by the amount of the test flash. 4. Rod-dependent incremental sensitivity functions of horizontal cells closely resembled the ones obtained from pigment epithelial cells. 5. It was concluded that the adaptive effects observed in pigment epithelial cells originated in individual rods. These effects arose from the compressive nature of the dark-adapted amplitude—intensity function. In horizontal cell responses these effects may be modified by the failure of the background response to maintain its initial voltage. PMID:5571955

  6. Continuous mucociliary transport by primary human airway epithelial cells in vitro

    PubMed Central

    Sears, Patrick R.; Yin, Wei-Ning

    2015-01-01

    Mucociliary clearance (MCC) is an important innate defense mechanism that continuously removes inhaled pathogens and particulates from the airways. Normal MCC is essential for maintaining a healthy respiratory system, and impaired MCC is a feature of many airway diseases, including both genetic (cystic fibrosis, primary ciliary dyskinesia) and acquired (chronic obstructive pulmonary disease, bronchiectasis) disorders. Research into the fundamental processes controlling MCC, therefore, has direct clinical application, but has been limited in part due to the difficulty of studying this complex multicomponent system in vitro. In this study, we have characterized a novel method that allows human airway epithelial cells to differentiate into a mucociliary epithelium that transports mucus in a continuous circular track. The mucociliary transport device allows the measurement and manipulation of all features of mucociliary transport in a controlled in vitro system. In this initial study, the effect of ciliary beat frequency and mucus concentration on the speed of mucociliary transport was investigated. PMID:25979076

  7. Oral microbial biofilm stimulation of epithelial cell responses.

    PubMed

    Peyyala, Rebecca; Kirakodu, Sreenatha S; Novak, Karen F; Ebersole, Jeffrey L

    2012-04-01

    Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria.

  8. Human epithelial cell cultures from superficial limbal explants

    PubMed Central

    Basli, E.; Goldschmidt, P.; Pecha, F.; Chaumeil, C.; Laroche, L.; Borderie, V.

    2011-01-01

    Purpose To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer. Methods Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR). Results The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm2 and 429 µm2, and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human

  9. Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

    PubMed

    Martínez-Martínez, Eduardo; Uribe-Querol, Eileen; Galván-Hernández, Claudio I; Gutiérrez-Ospina, Gabriel

    2016-01-01

    We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology. PMID:27431250

  10. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  11. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  12. Expression of bone morphogenetic proteins of human neoplastic epithelial cells.

    PubMed

    Hatakeyama, S; Gao, Y H; Ohara-Nemoto, Y; Kataoka, H; Satoh, M

    1997-07-01

    Bone morphogenetic proteins (BMPs) are crucial factors of osteogenesis. We investigated the expressions of BMP subtypes in human salivary adenocarcinoma cell line (HSG-S8), tongue squamous cell (HSC-4) and gingival squamous cell (Ca9-22) carcinoma cell lines, gastric poorly differentiated adenocarcinoma cell (MNK45) and signet ring cell (KATOIII) carcinoma cell lines, rectal adenocarcinoma (RCM-1, RCM-2, and RCM-3), and thyroid (8505C) and bladder (T24) carcinoma cell lines by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR disclosed that BMP-1 was expressed in all cell lines examined, and BMP-2 was amplified in almost all cells except MKN45. Two squamous cell carcinomas, HSC-4 and Ca9-22, and KATOIII expressed only BMP-1 and BMP-2. MKN45 did not express BMP-2, but expressed BMP-7 and weakly BMP-4 and BMP-5. In addition to the expression BMP-7, and HSG-S8 expressed BMP-6. These findings indicated that the neoplastic epithelial cells possessed a rather great potency to express BMP mRNAs. On the other hand, among these carcinoma cells, HSG-S8 solely induced bone in nude mouse tumors, and HSC-4 and KATOIII contained many calcified masses in tumors while the rest did not induce either. PMID:9247707

  13. Interleukin-6 induces epithelial-mesenchymal transition in human intrahepatic biliary epithelial cells

    PubMed Central

    JIANG, GUI-XING; CAO, LI-PING; KANG, PENG-CHENG; ZHONG, XIANG-YU; LIN, TIAN-YU; CUI, YUN-FU

    2016-01-01

    The aim of the present study was to determine the role of interleukin-6 (IL-6) in the epithelial-mesenchymal transition (EMT) of human intrahepatic biliary epithelial cell (HIBEC) lines in vitro. HIBECs were stimulated with IL-6 at concentrations of 0, 10, 20, 50 and 100 µg/l for 24 h. A wound healing and Transwell assay were performed to determine the migratory and invasive capacity of HIBECs, respectively. Following 24 h of incubation, IL-6 at 10 and 20 µg/l significantly increased the number of migrated and invaded cells (P<0.05), while stimulation with 50 and 100 µg/l IL-6 resulted in a further increase of the migratory and invasive capacity compared to that in all other groups (P<0.05). Furthermore, reverse-transcription quantitative polymerase chain reaction and western blot analyses were used to detect the mRNA and protein expression of EMT markers E-cadherin and vimentin in HIBECs. Decreased mRNA levels of E-cadherin accompanied by higher mRNA levels of vimentin were observed in the 10, 20, 50, 100 µg/l IL-6 groups compared to those in the 0 µg/l group (all P<0.05). Furthermore, the protein expression of E-cadherin was decreased, while that of vimentin was increased in the 50 and 100 µg/l IL-6 groups compared to those in the 0, 10 and 20 µg/l IL-6 groups (all P<0.05). The present study therefore indicated that IL-6 promoted the process of EMT in HIBECs as characterized by increased migration and invasion of HIBECs and the typical changes in mRNA and protein expression of the EMT markers E-cadherin and vimentin. PMID:26708270

  14. Differential response of human nasal and bronchial epithelial cells upon exposure to size-fractionated dairy dust.

    PubMed

    Hawley, Brie; Schaeffer, Joshua; Poole, Jill A; Dooley, Gregory P; Reynolds, Stephen; Volckens, John

    2015-01-01

    Exposure to organic dusts is associated with increased respiratory morbidity and mortality in agricultural workers. Organic dusts in dairy farm environments are complex, polydisperse mixtures of toxic and immunogenic compounds. Previous toxicological studies focused primarily on exposures to the respirable size fraction; however, organic dusts in dairy farm environments are known to contain larger particles. Given the size distribution of dusts from dairy farm environments, the nasal and bronchial epithelia represent targets of agricultural dust exposures. In this study, well-differentiated normal human bronchial epithelial cells and human nasal epithelial cells were exposed to two different size fractions (PM10 and PM>10) of dairy parlor dust using a novel aerosol-to-cell exposure system. Levels of proinflammatory transcripts (interleukin [IL]-8, IL-6, and tumor necrosis factor [TNF]-α) were measured 2 h after exposure. Lactate dehydrogenase (LDH) release was also measured as an indicator of cytotoxicity. Cell exposure to dust was measured in each size fraction as a function of mass, endotoxin, and muramic acid levels. To our knowledge, this is the first study to evaluate the effects of distinct size fractions of agricultural dust on human airway epithelial cells. Our results suggest that both PM10 and PM>10 size fractions elicit a proinflammatory response in airway epithelial cells and that the entire inhalable size fraction needs to be considered when assessing potential risks from exposure to agricultural dusts. Further, data suggest that human bronchial cells respond differently to these dusts than human nasal cells, and therefore that the two cell types need to be considered separately in airway cell models of agricultural dust toxicity. PMID:25965193

  15. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    PubMed

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  16. Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair

    PubMed Central

    Quantius, Jennifer; Schmoldt, Carole; Vazquez-Armendariz, Ana I.; Becker, Christin; El Agha, Elie; Wilhelm, Jochen; Morty, Rory E.; Vadász, István; Mayer, Konstantin; Gattenloehner, Stefan; Fink, Ludger; Matrosovich, Mikhail; Li, Xiaokun; Seeger, Werner; Lohmeyer, Juergen; Bellusci, Saverio; Herold, Susanne

    2016-01-01

    Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high. This cell fraction expressed the stem cell antigen-1, highly enriched lung stem/progenitor cells previously characterized by the signature integrin(β4)+CD200+, and upregulated the p63/krt5 regeneration program after IV-induced injury. Using 3-dimensional organoid cultures derived from these epithelial stem/progenitor cells (EpiSPC), and in vivo infection models including transgenic mice, we reveal that their expansion, barrier renewal and outcome after IV-induced injury critically depended on Fgfr2b signaling. Importantly, IV infected EpiSPC exhibited severely impaired renewal capacity due to IV-induced blockade of β-catenin-dependent Fgfr2b signaling, evidenced by loss of alveolar tissue repair capacity after intrapulmonary EpiSPC transplantation in vivo. Intratracheal application of exogenous Fgf10, however, resulted in increased engagement of non-infected EpiSPC for tissue regeneration, demonstrated by improved proliferative potential, restoration of alveolar barrier function and increased survival following IV pneumonia. Together, these data suggest that tropism of IV to distal lung stem cell niches represents an important factor of pathogenicity and highlight impaired Fgfr2b signaling as underlying mechanism. Furthermore, increase of alveolar Fgf10

  17. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells.

    PubMed

    Saroj, Sunil D; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics. PMID:27524981

  18. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells.

    PubMed

    Saroj, Sunil D; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics.

  19. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells

    PubMed Central

    Saroj, Sunil D.; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics. PMID:27524981

  20. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.

  1. Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

    NASA Astrophysics Data System (ADS)

    Chen, Kun; Qin, Yejun; Zheng, Feng; Sun, Menghong; Shi, Daren

    2006-07-01

    A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis.

  2. Direct contact between dendritic cells and bronchial epithelial cells inhibits T cell recall responses towards mite and pollen allergen extracts in vitro

    PubMed Central

    Papazian, D; Wagtmann, V R; Hansen, S; Würtzen, P A

    2015-01-01

    Airway epithelial cells (AECs) form a polarized barrier along the respiratory tract. They are the first point of contact with airborne antigens and are able to instruct resident immune cells to mount appropriate immune responses by either soluble or contact-dependent mechanisms. We hypothesize that a healthy, polarized epithelial cell layer inhibits inflammatory responses towards allergens to uphold homeostasis. Using an in-vitro co-culture model of the airway epithelium, where a polarized cell layer of bronchial epithelial cells can interact with dendritic cells (DCs), we have investigated recall T cell responses in allergic patients sensitized to house dust mite, grass and birch pollen. Using allergen extract-loaded DCs to stimulate autologous allergen-specific T cell lines, we show that AEC-imprinted DCs inhibit T cell proliferation significantly of Bet v 1-specific T cell lines as well as decrease interleukin (IL)-5 and IL-13 production, whereas inhibition of Phl p 5-specific T cells varied between different donors. Stimulating autologous CD4+ T cells from allergic patients with AEC-imprinted DCs also inhibited proliferation significantly and decreased production of both T helper type 1 (Th1) and Th2 cytokines upon rechallenge. The inhibitory effects of AECs’ contact with DCs were absent when allergen extract-loaded DCs had been exposed only to AECs supernatants, but present after direct contact with AECs. We conclude that direct contact between DCs and AECs inhibits T cell recall responses towards birch, grass and house dust mite allergens in vitro, suggesting that AECs-DC contact in vivo constitute a key element in mucosal homeostasis in relation to allergic sensitisation. PMID:25707463

  3. Sensing, signaling and sorting events in kidney epithelial cell physiology.

    PubMed

    Brown, Dennis; Breton, Sylvie; Ausiello, Dennis A; Marshansky, Vladimir

    2009-03-01

    The kidney regulates body fluid, ion and acid/base homeostasis through the interaction of a host of channels, transporters and pumps within specific tubule segments, specific cell types and specific plasma membrane domains. Furthermore, renal epithelial cells have adapted to function in an often unique and challenging environment that includes high medullary osmolality, acidic pHs, variable blood flow and constantly changing apical and basolateral 'bathing' solutions. In this review, we focus on selected protein trafficking events by which kidney epithelial cells regulate body fluid, ion and acid-base homeostasis in response to changes in physiological conditions. We discuss aquaporin 2 and G-protein-coupled receptors in fluid and ion balance, the vacuolar H(+)-adenosine triphosphatase (V-ATPase) and intercalated cells in acid/base regulation and acidification events in the proximal tubule degradation pathway. Finally, in view of its direct role in vesicle trafficking that we outline in this study, we propose that the V-ATPase itself should, under some circumstances, be considered a fourth category of vesicle 'coat' protein (COP), alongside clathrin, caveolin and COPs. PMID:19170982

  4. Sensing, signaling and sorting events in kidney epithelial cell physiology

    PubMed Central

    Brown, Dennis; Breton, Sylvie; Ausiello, Dennis A.; Marshansky, Vladimir

    2010-01-01

    The kidney regulates body fluid, ion, and acid/base homeostasis via the interaction of a host of channels, transporters, and pumps within specific tubule segments, specific cell types, and specific plasma membrane domains. Furthermore, renal epithelial cells have adapted to function in an often unique and challenging environment that includes high medullary osmolality, acidic pHs, variable blood flow and constantly changing apical and basolateral “bathing” solutions. In this review, we focus on selected protein trafficking events by which kidney epithelial cells regulate body fluid, ion and acid-base homeostasis in response to changes in physiological conditions. We discuss aquaporin 2 and G-protein coupled receptors (GPCRs) in fluid and ion balance, the V-ATPase and intercalated cells (IC) in acid/base regulation, and acidification events in the proximal tubule degradation pathway. Finally, in view of its direct role in vesicle trafficking that we outline here, we propose that the V-ATPase itself should, under some circumstances, be considered as a fourth category of vesicle “coat” protein, alongside clathrin, caveolin, and COPs. PMID:19170982

  5. Organization of the cytokeratin network in an epithelial cell.

    PubMed

    Portet, Stéphanie; Arino, Ovide; Vassy, Jany; Schoëvaërt, Damien

    2003-08-01

    The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.

  6. CsrRS and Environmental pH Regulate Group B Streptococcus Adherence to Human Epithelial Cells and Extracellular Matrix

    PubMed Central

    Park, Su Eun; Jiang, Shengmei

    2012-01-01

    Streptococcus agalactiae (group B Streptococcus or GBS) is a common colonizer of the gastrointestinal and genital tracts and an important cause of invasive infections in newborn infants and in adults with predisposing chronic conditions or advanced age. Attachment to epithelial surfaces at mucosal sites is a critical step in the successful colonization of a human host, and regulation of this process is likely to play an important role in both commensalism and dissemination to cause invasive disease. We found that inactivation of the CsrRS (or CovRS) two-component system increased GBS adherence to epithelial cells derived from human vaginal, cervical, and respiratory epithelium, as well as increasing adherence to extracellular matrix proteins and increasing biofilm formation on polystyrene. Neutral (as opposed to acidic) pH enhanced GBS binding to vaginal epithelial cells and to fibrinogen and fibronectin, effects that were partially dependent on CsrRS. The regulatory effects of CsrRS and environmental pH on bacterial adherence correlated with their effects on the expression of multiple surface adhesins, as assessed by quantitative reverse transcription-PCR. We conclude that GBS adherence to epithelial and abiotic surfaces is regulated by the CsrRS two-component system and by environmental pH through their regulatory effects on the expression of bacterial surface adhesins. Dynamic regulation of GBS adherence enhances the organism's adaptability to survival in multiple niches in the human host. PMID:22949550

  7. Comparison of innate immune agonists for induction of tracheal antimicrobial peptide gene expression in tracheal epithelial cells of cattle.

    PubMed

    Berghuis, Lesley; Abdelaziz, Khaled Taha; Bierworth, Jodi; Wyer, Leanna; Jacob, Gabriella; Karrow, Niel A; Sharif, Shayan; Clark, Mary Ellen; Caswell, Jeff L

    2014-10-12

    Bovine respiratory disease is a complex of bacterial and viral infections of economic and welfare importance to the beef industry. Although tracheal antimicrobial peptide (TAP) has microbicidal activity against bacterial pathogens causing bovine respiratory disease, risk factors for bovine respiratory disease including BVDV and stress (glucocorticoids) have been shown to inhibit the induced expression of this gene. Lipopolysaccharide is known to stimulate TAP gene expression, but the maximum effect is only observed after 16 h of stimulation. The present study investigated other agonists of TAP gene expression in primary cultures of bovine tracheal epithelial cells. PCR analysis of unstimulated tracheal epithelial cells, tracheal tissue and lung tissue each showed mRNA expression for Toll-like receptors (TLRs) 1-10. Quantitative RT-PCR analysis showed that Pam3CSK4 (an agonist of TLR1/2) and interleukin (IL)-17A significantly induced TAP gene expression in tracheal epithelial cells after only 4-8 h of stimulation. Flagellin (a TLR5 agonist), lipopolysaccharide and interferon-α also had stimulatory effects, but little or no response was found with class B CpG ODN 2007 (TLR9 agonist) or lipoteichoic acid (TLR2 agonist). The use of combined agonists had little or no enhancing effect above that of single agonists. Thus, Pam3CSK4, IL-17A and lipopolysaccharide rapidly and significantly induce TAP gene expression, suggesting that these stimulatory pathways may be of value for enhancing innate immunity in feedlot cattle at times of susceptibility to disease.

  8. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  9. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  10. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  11. Plasticity of epithelial stem cells in tissue regeneration

    PubMed Central

    Blanpain, Cédric; Fuchs, Elaine

    2015-01-01

    Tissues rely upon stem cells for homeostasis and repair. Recent studies show that the fate and multilineage potential of epithelial stem cells can change depending on whether a stem cell exists within its resident niche and responds to normal tissue homeostasis, whether it is mobilized to repair a wound, or whether it is taken from its niche and challenged to de novo tissue morphogenesis after transplantation. In this Review, we discuss how different populations of naturally lineage-restricted stem cells and committed progenitors can display remarkable plasticity and reversibility and reacquire long-term self-renewing capacities and multilineage differentiation potential during physiological and regenerative conditions. We also discuss the implications of cellular plasticity for regenerative medicine and for cancer. PMID:24926024

  12. Comparative effects of cocaine and cocaethylene on alveolar epithelial type II cells.

    PubMed

    Bazuaye-Ekwuyasi, Eseosa A; Ogunbileje, John O; Kaphalia, Bhupendra S; Eltorky, Mahmoud A; Okorodudu, Anthony O

    2015-01-01

    Abuse of cocaine (COC) and alcohol have been among the leading causes of non-prescription drug-related deaths in the USA and are known to cause acute and chronic lung diseases. The co-abuse of COC and alcohol results in the production of an active metabolite, cocaethylene (CE). The effects of COC and its metabolites on the respiratory system have been scarcely studied. This study was aimed at comparing the toxic effects of eqimolar concentration (1 mM) of COC and CE on alveolar epithelial type II cells. This was performed by measuring cell growth, viability, clonogenic activity, cell cycle and reactive oxygen species (ROS) generation. The treatment of CE and COC resulted in a significant inhibition of cell proliferation with the formation of an average of three colonies which measured about 1.74×10(-15) m each and 25 colonies each of about 5.73×10(-15) m, respectively, while untreated cells yielded 31 colonies of 8.75×10(-15) m (p<0.05). The treatments of CE and COC resulted in the reduction of the growth fraction of alveolar epithelial type II cells without significant decrease in viability. In addition, there was an approximately twofold increase in ROS generation as compared to the controls (p<0.05). Therefore, CE-induced inhibition of cellular proliferation may contribute to the pathogenesis of diffuse alveolar damage in co-abusers of COC and alcohol. PMID:26364649

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

    SciTech Connect

    Chinet, T.; Dusser, D.; Labrune, S.; Collignon, M.A.; Chretien, J.; Huchon, G.J. )

    1990-02-01

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

  14. Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells

    PubMed Central

    1994-01-01

    Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double- immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed. PMID:7798314

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

    SciTech Connect

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

    2008-04-01

    The water-soluble, hydroxylated fullerene [fullerol, nano-C{sub 60}(OH){sub 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{sub 60}(OH){sub 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 {mu}M. Exposure to either UVA or visible light in the presence of > 5 {mu}M fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 {mu}M 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 {alpha}-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{sub 60}(OH){sub 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.

  16. In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

    PubMed Central

    Hu, Han; Wang, Jiaqi; Bu, Dengpan; Wei, Hongyang; Zhou, Linyun; Li, Fadi; Loor, Juan J.

    2009-01-01

    Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs). PMID:19888476

  17. Cell crawling mediates collective cell migration to close undamaged epithelial gaps

    PubMed Central

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C.; Sheetz, Michael P.; Trepat, Xavier; Ladoux, Benoît

    2012-01-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  18. Cell crawling mediates collective cell migration to close undamaged epithelial gaps.

    PubMed

    Anon, Ester; Serra-Picamal, Xavier; Hersen, Pascal; Gauthier, Nils C; Sheetz, Michael P; Trepat, Xavier; Ladoux, Benoît

    2012-07-01

    Fundamental biological processes such as morphogenesis and wound healing involve the closure of epithelial gaps. Epithelial gap closure is commonly attributed either to the purse-string contraction of an intercellular actomyosin cable or to active cell migration, but the relative contribution of these two mechanisms remains unknown. Here we present a model experiment to systematically study epithelial closure in the absence of cell injury. We developed a pillar stencil approach to create well-defined gaps in terms of size and shape within an epithelial cell monolayer. Upon pillar removal, cells actively respond to the newly accessible free space by extending lamellipodia and migrating into the gap. The decrease of gap area over time is strikingly linear and shows two different regimes depending on the size of the gap. In large gaps, closure is dominated by lamellipodium-mediated cell migration. By contrast, closure of gaps smaller than 20 μm was affected by cell density and progressed independently of Rac, myosin light chain kinase, and Rho kinase, suggesting a passive physical mechanism. By changing the shape of the gap, we observed that low-curvature areas favored the appearance of lamellipodia, promoting faster closure. Altogether, our results reveal that the closure of epithelial gaps in the absence of cell injury is governed by the collective migration of cells through the activation of lamellipodium protrusion. PMID:22711834

  19. Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

    PubMed Central

    Pantenburg, Birte; Castellanos-Gonzalez, Alejandro; Dann, Sara M.; Connelly, Rhykka L.; Lewis, Dorothy E.; Ward, Honorine D.; Clinton White, A.

    2010-01-01

    Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4+ T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8+ T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8+ T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8+ T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite. PMID:20348507

  20. The 2009 pandemic A/Wenshan/01/2009 H1N1 induces apoptotic cell death in human airway epithelial cells.

    PubMed

    Yang, Ning; Hong, Xiaoxu; Yang, Penghui; Ju, Xiangwu; Wang, Yuguo; Tang, Jun; Li, Chenggang; Fan, Quanshui; Zhang, Fuqiang; Chen, Zhongwei; Xing, Li; Zhao, Zhongpeng; Gao, Xiao; Liao, Guoyang; Li, Qihan; Wang, Xiliang; Li, Dangsheng; Jiang, Chengyu

    2011-08-01

    In 2009, a novel swine-origin H1N1 influenza virus emerged in Mexico and quickly spread to other countries, including China. This 2009 pandemic H1N1 can cause human respiratory disease, but its pathogenesis remains poorly understood. Here, we studied the infection and pathogenesis of a new 2009 pandemic strain, A/Wenshan/01/2009 H1N1, in China in human airway epithelial cell lines compared with contemporary seasonal H1N1 influenza virus. Our results showed that viral infection by the A/Wenshan H1N1 induced significant apoptotic cell death in both the human nasopharyngeal carcinoma cell line CNE-2Z and the human lung adenocarcinoma cell line A549. The A/Wenshan H1N1 virus enters both of these cell types more efficiently than the seasonal influenza virus. Viral entry in both cell lines was shown to be mediated by clathrin- and dynamin-dependent endocytosis. Therefore, we discovered that the 2009 pandemic H1N1 strain, A/Wenshan/01/2009, can induce apoptotic cell death in epithelial cells of the human respiratory tract, suggesting a molecular pathogenesis for the 2009 pandemic H1N1. PMID:21816972

  1. AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

    PubMed

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis. PMID:27263094

  2. Contacts between pigmented retina epithelial cells in culture.

    PubMed

    Middleton, C A; Pegrum, S M

    1976-11-01

    The behaviour of primary cultures of dissociated embryonic chick pigmented retina epithelial (PRE) cells has been investigated. Isolated PRE cells have a mean speed of locomotion of 7-16 mum/h. Collisions between the cells normally result in the development of stable contacts between the cells involved. This leads to a gradual reduction in the number of isolated cells and an increase in the number of cells incorporated into islands. Ultrastructural observations of islands of cells after 24 h in culture show that junctional complexes are present between the cells. These complexes consist of 2 components: (a) an apically situated region of focal tight junctions and/or gap junctions, and (b) a more ventrally located zonula adhaerens with associated cytoplasmic filaments forming a band running completely around the periphery of each cell. The intermembrane gap in the region of the zonula is 6-0-12-0 nm. The junctional complexes become more differentiated with time and after 48 h in culture consist of an extensive region of tight junctions and/or gap junctions and a more specialized zonula adhaerens. It is suggested that the development of junctional complexes may be responsible for the stable contacts that the cells display in culture.

  3. Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

    PubMed

    Chen, Xiuping; Guo, Feiye; LeBlanc, Michelle E; Ding, Ying; Zhang, Chenming; Shakya, Akhalesh; Li, Wei

    2016-08-01

    Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance.

  4. Barrier Epithelial Cells and the Control of Type 2 Immunity.

    PubMed

    Hammad, Hamida; Lambrecht, Bart N

    2015-07-21

    Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease.

  5. Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

    PubMed

    Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

    2016-05-01

    Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

  6. Expression of tracheal antimicrobial peptide in bovine mammary epithelial cells.

    PubMed

    López-Meza, Joel E; Gutiérrez-Barroso, Angelina; Ochoa-Zarzosa, Alejandra

    2009-08-01

    The production of antimicrobial peptides is an important key of innate immunity. Tracheal antimicrobial peptide (TAP) expression has been reported in bovine tracheal epithelial cells and it can be modulated by bacterial infection or bacterial components. In mammary gland TAP expression has been reported, but the cell type that produces it is unknown. The objective of this work was to evaluate if bovine mammary epithelial cells (bMEC) express TAP mRNA, and evaluate the regulation of its expression in response to Staphylococcus aureus infection, bovine prolactin (bPRL) or acetyl salicylic acid (ASA). By retrotranscription and PCR, we demonstrated that bMEC express TAP mRNA. bMEC infected with live S. aureus down-regulates TAP expression, whereas the challenge with gentamicin-killed S. aureus up-regulates it. Also, bPRL do not significantly modify TAP expression, but in the presence of 5 mM ASA it was down-regulated, suggesting that nuclear factor kappaB (NF-kappaB) pathway can be involved in its regulation. PMID:19181355

  7. Proteomic analysis of nasal epithelial cells from cystic fibrosis patients.

    PubMed

    Jeanson, Ludovic; Guerrera, Ida Chiara; Papon, Jean-François; Chhuon, Cerina; Zadigue, Patricia; Prulière-Escabasse, Virginie; Amselem, Serge; Escudier, Estelle; Coste, André; Edelman, Aleksander

    2014-01-01

    The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

  8. Adiponectin stimulates proliferation and cytokine secretion in colonic epithelial cells.

    PubMed

    Ogunwobi, Olorunseun Olatunji; Beales, Ian L P

    2006-05-15

    Adiponectin is a recently described mediator secreted by adipose tissue. Here we report the growth promoting and pro-inflammatory actions of adiponectin on colonic epithelial cancer cells. Full-length and globular adiponectin produced an identical stimulation of HT-29 cell growth that was blocked by inhibition of adenylate cyclase and protein kinase A and partially inhibited by a pan-specific protein kinase C inhibitor, but was unaffected by specific inhibition of extracellular signal-related kinase (ERK) or p38 MAP kinase. Globular adiponectin but not full-length adiponectin significantly increased the secretion and mRNA levels of IL-8, GM-CSF and MCP-1. Globular adiponectin doubled IL-1beta-stimulated IL-8 and GM-CSF secretion. Adiponectin-stimulated cytokine secretion was blocked by pharmacological inhibitors of NF-kappaB, ERK and p38 MAP kinase. Globular adiponectin increased phosphorylation of both ERK and p38 MAP kinase and increased the nuclear translocation of active NF-kappaB. Adiponectin has pro-proliferative and pro-inflammatory actions on colonic epithelial cells; these appear to be differentially activated by the adiponectin isoforms. Adiponectin may have a role in the regulation of gastrointestinal mucosal function, inflammation and colon carcinogenesis.

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

    PubMed Central

    2009-01-01

    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. PMID:19650888

  10. Specific parasitism of purified vaginal epithelial cells by Trichomonas vaginalis.

    PubMed Central

    Alderete, J F; Demeś, P; Gombosova, A; Valent, M; Fabusová, M; Jánoska, A; Stefanovic, J; Arroyo, R

    1988-01-01

    Human vaginal epithelial cells<