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Sample records for alveolar epithelial apoptosis

  1. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

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    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Deng, Yubin, E-mail: dengyub@mail.sysu.edu.cn [Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Zeng, Mian, E-mail: zengmian2004@163.com [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China)

    2016-05-20

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

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

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

    2015-01-01

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

  3. Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells.

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    Li, Xiaopeng; Rayford, Heather; Shu, Ruijie; Zhuang, Jiaju; Uhal, Bruce D

    2004-07-01

    Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.

  4. Dexmedetomidine Attenuates Oxidative Stress Induced Lung Alveolar Epithelial Cell Apoptosis In Vitro

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    Jian Cui

    2015-01-01

    Full Text Available Background. Oxidative stress plays a pivotal role in the lung injuries of critical ill patients. This study investigates the protection conferred by α2 adrenoceptor agonist dexmedetomidine (Dex from lung alveolar epithelial cell injury induced by hydrogen peroxide (H2O2 and the underlying mechanisms. Methods. The lung alveolar epithelial cell line, A549, was cultured and then treated with 500 μM H2O2 with or without Dex (1 nM or Dex in combination with atipamezole (10 nM, an antagonist of α2 receptors. Their effect on mitochondrial membrane potential (Δψm, reactive oxygen species (ROS, and the cell cycle was assessed by flow cytometry. Cleaved-caspases 3 and 9, BAX, Bcl-2, phospho-mTOR (p-mTOR, ERK1/2, and E-cadherin expression were also determined with immunocytochemistry. Results. Upregulation of cleaved-caspases 3 and 9 and BAX and downregulation of Bcl-2, p-mTOR, and E-cadherin were found following H2O2 treatment, and all of these were reversed by Dex. Dex also prevented the ROS generation, cytochrome C release, and cell cycle arrest induced by H2O2. The effects of Dex were partially reversed by atipamezole. Conclusion. Our study demonstrated that Dex protected lung alveolar epithelial cells from apoptotic injury, cell cycle arrest, and loss of cell adhesion induced by H2O2 through enhancing the cell survival and proliferation.

  5. Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing.

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    Crosby, Lynn M; Luellen, Charlean; Zhang, Zhihong; Tague, Larry L; Sinclair, Scott E; Waters, Christopher M

    2011-10-01

    After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.

  6. Alveolar epithelial and endothelial cell apoptosis in emphysema: What we know and what we need to know

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    Mathieu C Morissette

    2008-12-01

    Full Text Available Mathieu C Morissette, Julie Parent, Julie MilotCentre de Recherche de l’Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l’Université Laval, Québec, CanadaAbstract: Emphysema is mainly caused by cigarette smoking and is characterized by the loss of alveolar integrity and an enlargement of the alveolar space. However, mechanisms involved in its development are not fully understood. Alveolar cell apoptosis has been previously investigated in the lung of emphysematous subjects as a potential contributor to the loss of alveolar cell and has been found abnormally elevated. Though, mechanisms involved in the increased alveolar apoptosis that occurs in emphysema have now become a prolific field of research. Those mechanisms are reviewed here with special focus on how they affect cell viability and how they may be implicated in emphysema. Moreover, we suggest a model that integrates all those mechanisms to explain the increased alveolar apoptosis observed in emphysema. This review also includes some reflections and suggestions on the research to come.Keywords: emphysema, apoptosis, proteases, VEGF, oxidative stress, TRAIL, autoimmunity

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

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

    2017-09-16

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

  8. Hydrogen protects against hyperoxia-induced apoptosis in type II alveolar epithelial cells via activation of PI3K/Akt/Foxo3a signaling pathway.

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    Wu, Dan; Liang, Mulin; Dang, Hongxing; Fang, Fang; Xu, Feng; Liu, Chengjun

    2018-01-08

    Oxidative stress is regarded as a key regulator in the pathogenesis of prolonged hyperoxia-induced lung injury, which causes injury to alveolar epithelial cells and eventually leads to development of bronchopulmonary dysplasia (BPD). Many studies have shown that hydrogen has a protective effect in a variety of cells. However, the mechanisms by which hydrogen rescues cells from damage due to oxidative stress in BPD remains to be fully elucidated. This study sought to evaluate the effects of hydrogen on hyperoxia-induced lung injury and to investigate the underlying mechanism. Primary type II alveolar epithelial cells (AECIIs) were divided into four groups: control (21% oxygen), hyperoxia (95% oxygen), hyperoxia + hydrogen, and hyperoxia + hydrogen + LY294002 (a PI3K/Akt inhibitor). Proliferation and apoptosis of AECIIs were assessed using MTS assay and flow cytometry (FCM), respectively. Gene and protein expression were detected by quantitative polymerase chain reaction (q-PCR) and western blot analysis. Stimulation with hyperoxia decreased the expression of P-Akt, P- FoxO3a, cyclinD1 and Bcl-2. Hyperoxic conditions increased levels of Bim, Bax, and Foxo3a, which induced proliferation restriction and apoptosis of AECIIs. These effects of hyperoxia were reversed with hydrogen pretreatment. Furthermore, the protective effects of hydrogen were abrogated by PI3K/Akt inhibitor LY294002. The results indicate that hydrogen protects AECIIs from hyperoxia-induced apoptosis by inhibiting apoptosis factors and promoting the expression of anti-apoptosis factors. These effects were associated with activation of the PI3K/Akt/FoxO3a pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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    Nagai A

    2003-09-01

    Full Text Available Abstract 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.

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

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng, Li-Wei Pan, Sheng-Rong Zhang, Bin-Yu Ying, Ben-Ji Wang, Guo-Liang Lin, Shi-Fang Ding ...

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

    African Journals Online (AJOL)

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

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

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    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

  13. Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK

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    Elena A. Goncharova

    2014-04-01

    Full Text Available Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD syndrome, which is caused by mutations of the tumor suppressor gene folliculin (FLCN. The underlying mechanism of the lung manifestations in BHD is unclear. We show that BHD lungs exhibit increased alveolar epithelial cell apoptosis and that Flcn deletion in mouse lung epithelium leads to cell apoptosis, alveolar enlargement, and an impairment of both epithelial barrier and overall lung function. We find that Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Correspondingly, Flcn-null cell survival is rescued by the AMPK activator AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cre mice. Our data suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival, attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.

  14. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

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    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

  15. Induction of Programmed Cell Death in Human Alveolar Epithelial Cells Infected with Influenza Virus

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    Sh Shahsavandi

    2015-11-01

    Full Text Available Introduction: Avian influenza viruses are considered as a serious threat to human and animal health. An increase in expression of proinflammatory cytokines and type I IFN genes, as well as host cell death responses contribute to the pathogenesis of influenza infection. Hence, this study aimed to evaluate the growth dynamics of subacute avian influenza virus in human respiratory alveolar epithelium cells (A549. Methods: The A549 cell cultures were infected at MOIs 0.1 and 2.0 viral doses in the presence and absence of trypsin. The virus growth kinetics were elucidated by the plaque assay and the cell viability was determined by MTT at various times after the infection. The induction quality of programmed cell death as well as the signal transduction pathway of death were assessed by genomic DNA fragmentation and western blotting respectively. Results: The study findings indicated that although the H9N2 virus replication did produce a marked cytopathic effect on the alveolar cells, which led to a reduction in the cell viability, the viral titers were increased in the infected cells. The virus replication of in these cells indicated repression of host defense mechanism as well as activation of cell death. The induction of apoptosis in A549 cells was correlated with the increased virus titers as well as virus replication (p< 0.05. Conclusion: H9N2 avian influenza virus were demonstrated to induce apoptosis in human alveolar epithelial cells via the intrinsic pathway in a dose-dependent manner.

  16. Ontogeny of pulmonary alveolar epithelial markers of differentiation.

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    Joyce-Brady, M F; Brody, J S

    1990-02-01

    We studied differentiation of the pulmonary epithelium in the periphery of fetal rat lung in vivo and in vitro by comparing the ontogeny of cell-surface glycoconjugates with that of surfactant phospholipids. Apical surface binding of the lectin Maclura pomifera agglutinin (MPA) and expression of a 200-kDa MPA-binding glycoprotein (MPA-gp200) was evident at 20 days gestation in type 2 cells, but did not correlate with ultrastructural features of type 2 cell differentiation. Epithelial cells isolated from peripheral lung of 18-day gestation fetal rats displayed hormone-sensitive surfactant synthesis prior to the hormone-insensitive expression of MPA-gp200. Expression of MPA-gp200 occurred in association with the appearance of many new apical surface proteins suggesting a hormone-independent process of polar membrane differentiation. Thus membrane and secretory differentiation are discordant and can be dissociated. In vivo binding of Ricinus communis 1 agglutinin (RCA1), an apical marker of the differentiated alveolar type 1 cell occurred in undifferentiated peripheral lung epithelial cells as early as 18 days gestation, disappeared from differentiating type 2 cells and appeared in differentiated type 1 cells. Both undifferentiated fetal epithelial cells at 18 days gestation and fully differentiated type 1 cells express multiple glycoproteins with terminal beta-linked galactose residues which bind RCA1. Some of these RCA1-binding glycoproteins appear to be similar. These observations suggest that alveolar epithelial type 1 cells may derive directly from undifferentiated peripheral lung epithelial cells as well as from fully differentiated type 2 cells. In addition, terminal differentiation of fetal lung peripheral epithelium into type 1 and type 2 cells may involve repression as well as induction of differentiation-related genes.

  17. Effect of cigarette smoke and dexamethasone on Hsp72 system of alveolar epithelial cells.

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    Gál, Krisztina; Cseh, Aron; Szalay, Balázs; Rusai, Krisztina; Vannay, Adám; Lukácsovits, József; Heemann, Uwe; Szabó, Attila J; Losonczy, György; Tamási, Lilla; Müller, Veronika

    2011-07-01

    Smoking is the leading risk factor of chronic obstructive pulmonary disease (COPD) and lung cancer. Corticosteroids are abundantly used in these patients; however, the interaction of smoking and steroid treatment is not fully understood. Heat shock proteins (Hsps) play a central role in the maintenance of cell integrity, apoptosis and cellular steroid action. To better understand cigarette smoke-steroid interaction, we examined the effect of cigarette smoke extract (CSE) and/or dexamethasone (DEX) on changes of intracellular heat shock protein-72 (Hsp72) in lung cells. Alveolar epithelial cells (A549) were exposed to increasing doses (0; 0.1; 1; and 10 μM/μl) of DEX in the medium in the absence(C) and presence of CSE. Apoptosis, necrosis, Hsp72 messenger-ribonucleic acid (mRNA) and protein expression of cells were measured, and the role of Hsp72 on steroid effect examined. CSE reduced the number of viable cells by significantly increasing the number of apoptotic and necrotic cells. DEX dose-dependently decreased the ratio of apoptosis when CSE was administered, without change in necrosis. CSE - DEX co-treatment dose-dependently increased Hsp72 mRNA and protein expression, with the highest level measured in CSE + DEX (10) cells, while significantly lower levels were noted in all respective C groups. Pretreatment with Hsp72 silencing RNA confirmed that increased survival observed following DEX administration in CSE-treated cells was mainly mediated via the Hsp72 system. CSE significantly decreases cell survival by inducing apoptosis and necrosis. DEX significantly increases Hsp72 mRNA and protein expression only in the presence of CSE resulting in increased cellular protection and survival. DEX exerts its cell protective effects by decreasing apoptotic cell death via the Hsp72 system in CSE-treated alveolar epithelial cells.

  18. Simulation of lung alveolar epithelial wound healing in vitro.

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    Kim, Sean H J; Matthay, Michael A; Mostov, Keith; Hunt, C Anthony

    2010-08-06

    The mechanisms that enable and regulate alveolar type II (AT II) epithelial cell wound healing in vitro and in vivo remain largely unknown and need further elucidation. We used an in silico AT II cell-mimetic analogue to explore and better understand plausible wound healing mechanisms for two conditions: cyst repair in three-dimensional cultures and monolayer wound healing. Starting with the analogue that validated for key features of AT II cystogenesis in vitro, we devised an additional cell rearrangement action enabling cyst repair. Monolayer repair was enabled by providing 'cells' a control mechanism to switch automatically to a repair mode in the presence of a distress signal. In cyst wound simulations, the revised analogue closed wounds by adhering to essentially the same axioms available for alveolar-like cystogenesis. In silico cell proliferation was not needed. The analogue recovered within a few simulation cycles but required a longer recovery time for larger or multiple wounds. In simulated monolayer wound repair, diffusive factor-mediated 'cell' migration led to repair patterns comparable to those of in vitro cultures exposed to different growth factors. Simulations predicted directional cell locomotion to be critical for successful in vitro wound repair. We anticipate that with further use and refinement, the methods used will develop as a rigorous, extensible means of unravelling mechanisms of lung alveolar repair and regeneration.

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

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    Mohammad Reza Etemadi

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

  20. Alveolocapillary model system to study alveolar re-epithelialization

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    Willems, Coen H.M.P.; Zimmermann, Luc J.I.; Sanders, Patricia J.L.T.; Wagendorp, Margot; Kloosterboer, Nico [Department of Paediatrics, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht (Netherlands); Cohen Tervaert, Jan Willem [Division of Clinical and Experimental Immunology, Department of Internal Medicine, Maastricht University Medical Centre, Maastricht (Netherlands); Duimel, Hans J.Q.; Verheyen, Fons K.C.P. [Electron Microscopy Unit, Department of Molecular Cell Biology, Maastricht University Medical Centre, Maastricht (Netherlands); Iwaarden, J. Freek van, E-mail: f.vaniwaarden@maastrichtuniversity.nl [Department of Paediatrics, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht (Netherlands)

    2013-01-01

    In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process. -- Highlights: ► Model system for vital imaging and high throughput screening. ► Microvascular endothelium influences re-epithelialization. ► A549 cells form protrusions through membrane to contact HPMEC. ► A549 cells and HPMECs form heterocellular tight-, gap- and adherens-junctions.

  1. Epithelial apoptosis: cause or consequence of ulcerative colitis?

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    Seidelin, Jakob Benedict; Nielsen, Ole Haagen

    2009-01-01

    OBJECTIVE: Epithelial apoptosis rates are increased in ulcerative colitis (UC). The increased apoptosis rate could expose mucosal cells to luminal pathogens and thereby be regarded as a primary pathogenic factor in UC. On the other hand, the local inflammatory reaction could cause epithelial...... apoptosis secondary to the release of cytotoxic mediators. If apoptosis is a primary defect, apoptosis rates could influence the degree of spreading of inflammation and the clinical course of UC. If apoptosis is a side effect of local inflammation, apoptosis rates would be expected only to correlate...

  2. A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing.

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    Ghosh, Manik C; Makena, Patrudu S; Kennedy, Joseph; Teng, Bin; Luellen, Charlean; Sinclair, Scott E; Waters, Christopher M

    2017-05-19

    Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

  3. An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

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    Birch, Nigel P.; Suresh, Vinod

    2016-01-01

    Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI

  4. Differential effects of hypoxic stress in alveolar epithelial cells and microvascular endothelial cells

    NARCIS (Netherlands)

    Signorelli, Sara; Jennings, Paul; Leonard, Martin O; Pfaller, Walter

    2010-01-01

    Under hypoxic conditions eukaryotic cells and tissues undergo adaptive responses involving glycolysis, angiogenesis, vasoconstriction and inflammation. The underlying molecular mechanisms are not yet fully elucidated and are most likely cell and tissue specific. In the lung, alveolar epithelial

  5. HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Jacob Barbara A

    2009-02-01

    Full Text Available Abstract Background HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. Results HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. Conclusion Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

  6. Mitochondrial quality control in alveolar epithelial cells damaged byS. aureuspneumonia in mice.

    Science.gov (United States)

    Suliman, Hagir B; Kraft, Bryan; Bartz, Raquel; Chen, Lingye; Welty-Wolf, Karen E; Piantadosi, Claude A

    2017-10-01

    Mitochondrial damage is often overlooked in acute lung injury (ALI), yet most of the lung's physiological processes, such as airway tone, mucociliary clearance, ventilation-perfusion (Va/Q) matching, and immune surveillance require aerobic energy provision. Because the cell's mitochondrial quality control (QC) process regulates the elimination and replacement of damaged mitochondria to maintain cell survival, we serially evaluated mitochondrial biogenesis and mitophagy in the alveolar regions of mice in a validated Staphylococcus aureus pneumonia model. We report that apart from cell lysis by direct contact with microbes, modest epithelial cell death was detected despite significant mitochondrial damage. Cell death by TdT-mediated dUTP nick-end labeling staining occurred on days 1 and 2 postinoculation: apoptosis shown by caspase-3 cleavage was present on days 1 and 2, while necroptosis shown by increased levels of phospho- mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was present on day 1 Cell death in alveolar type I (AT 1 ) cells assessed by bronchoalveolar lavage fluid receptor for advanced glycation end points (RAGE) levels was high, yet AT 2 cell death was limited while both mitochondrial biogenesis and mitophagy were induced. These mitochondrial QC mechanisms were evaluated mainly in AT 2 cells by localizing increases in citrate synthase content, increases in nuclear mitochondrial biogenesis regulators nuclear respiratory factor-1 (NRF-1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and increases in light chain 3B protein (LC3-I)/LC3II ratios. Concomitant changes in p62, Pink 1, and Parkin protein levels indicated activation of mitophagy. By confocal microscopy, mitochondrial biogenesis and mitophagy were often observed on day 1 within the same AT 2 cells. These findings imply that mitochondrial QC activation in pneumonia-damaged AT 2 cells promotes cell

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Inositol-trisphosphate reduces alveolar apoptosis and pulmonary edema in neonatal lung injury.

    Science.gov (United States)

    Preuss, Stefanie; Stadelmann, Sabrina; Omam, Friede D; Scheiermann, Julia; Winoto-Morbach, Supandi; von Bismarck, Philipp; Knerlich-Lukoschus, Friederike; Lex, Dennis; Adam-Klages, Sabine; Wesch, Daniela; Held-Feindt, Janka; Uhlig, Stefan; Schütze, Stefan; Krause, Martin F

    2012-08-01

    D-myo-inositol-1,2,6-trisphosphate (IP3) is an isomer of the naturally occurring second messenger D-myo-inositol-1,4,5-trisphosphate, and exerts anti-inflammatory and antiedematous effects in the lung. Myo-inositol (Inos) is a component of IP3, and is thought to play an important role in the prevention of neonatal pulmonary diseases such as bronchopulmonary dysplasia and neonatal acute lung injury (nALI). Inflammatory lung diseases are characterized by augmented acid sphingomyelinase (aSMase) activity leading to ceramide production, a pathway that promotes increased vascular permeability, apoptosis, and surfactant alterations. A novel, clinically relevant triple-hit model of nALI was developed, consisting of repeated airway lavage, injurious ventilation, and lipopolysaccharide instillation into the airways, every 24 hours. Thirty-five piglets were randomized to one of four treatment protocols: control (no intervention), surfactant alone, surfactant + Inos, and surfactant + IP3. After 72 hours of mechanical ventilation, lungs were excised from the thorax for subsequent analyses. Clinically, oxygenation and ventilation improved, and extravascular lung water decreased significantly with the S + IP3 intervention. In pulmonary tissue, we observed decreased aSMase activity and ceramide concentrations, decreased caspase-8 concentrations, reduced alveolar epithelial apoptosis, the reduced expression of interleukin-6, transforming growth factor-β1, and amphiregulin (an epithelial growth factor), reduced migration of blood-borne cells and particularly of CD14(+)/18(+) cells (macrophages) into the airspaces, and lower surfactant surface tensions in S + IP3-treated but not in S + Inos-treated piglets. We conclude that the admixture of IP3 to surfactant, but not of Inos, improves gas exchange and edema in our nALI model by the suppression of the governing enzyme aSMase, and that this treatment deserves clinical evaluation.

  9. The Milieu of Damaged Alveolar Epithelial Type 2 Cells Stimulates Alveolar Wound Repair by Endogenous and Exogenous Progenitors

    Science.gov (United States)

    Buckley, Susan; Shi, Wei; Carraro, Gianni; Sedrakyan, Sargis; Da Sacco, Stefano; Driscoll, Barbara A.; Perin, Laura; De Filippo, Roger E.

    2011-01-01

    Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation. PMID:21700959

  10. Cultured alveolar epithelial cells from septic rats mimic in vivo septic lung.

    Directory of Open Access Journals (Sweden)

    Taylor S Cohen

    2010-06-01

    Full Text Available Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200-300 g were sacrificed 24 hours after cecal ligation and double puncture (2CLP or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A and MAPk (JNK, ERK, an p38 signaling activation, or barrier function was examined by measuring transepithelial resistance (TER or the flux of two molecular tracers (5 and 20 A. Inhibitors of JNK (SP600125, 20 microM and ERK (U0126, 10 microM were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm(2, respectively, however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique

  11. Barrier-protective effects of activated protein C in human alveolar epithelial cells.

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    Ferranda Puig

    Full Text Available Acute lung injury (ALI is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC on mechanical tension and barrier integrity in human alveolar epithelial cells (A549 exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml or vehicle (control. Subsequently, thrombin (50 nM or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

  12. Alveolar epithelial type II cells induce T cell tolerance to specific antigen

    DEFF Research Database (Denmark)

    Lo, Bernice; Hansen, Søren; Evans, Kathy

    2008-01-01

    The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Ty...

  13. Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor.

    Science.gov (United States)

    Yang, Jibing; Velikoff, Miranda; Canalis, Ernesto; Horowitz, Jeffrey C; Kim, Kevin K

    2014-04-15

    Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

  14. Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Marco Checa

    Full Text Available Idiopathic pulmonary fibrosis (IPF is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549, was exposed to cigarette smoke extract (CSE for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-β and Wnt. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-12 and rat (RLE-6TN epithelial cells. The secretion of activated TGF-β1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-β1, which may explain at least partially, the increased risk of developing IPF in smokers.

  15. Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

    Science.gov (United States)

    Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

    2015-09-04

    Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

  16. RAGE-induced changes in the proteome of alveolar epithelial cells.

    Science.gov (United States)

    Downs, Charles A; Johnson, Nicholle M; Tsaprailis, George; Helms, My N

    2018-02-12

    The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1β and phosphorylated p65. We also assessed RAGE's effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. In the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have

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

    Science.gov (United States)

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

    2015-09-15

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

  18. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

    Science.gov (United States)

    Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

    2014-01-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

  19. Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells.

    Science.gov (United States)

    Van Overbeke, Ingrid; Chiers, Koen; Charlier, Gerard; Vandenberghe, Isabel; Van Beeumen, Jozef; Ducatelle, Richard; Haesebrouck, Freddy

    2002-08-02

    Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture. For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD). This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae. For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions. The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein. Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat. These findings suggest that proteins are involved in adhesion. The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A. pleuropneumoniae to alveolar epithelial cells. Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes. This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A. pleuropneumoniae to alveolar epithelial cells. The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

  20. In vivo metabolism of pulmonary alveolar epithelial type II pneumonocytes and macrophages from Syrian hamsters

    International Nuclear Information System (INIS)

    Pfleger, R.C.; Waide, J.J.

    1976-01-01

    Young adult Syrian hamsters were injected intraperitoneally with 14 C-glycerol and 3 H-palmitate 17 hr before they were sacrificed and pulmonary alveolar epithelial type II cells and pulmonary alveolar macrophages (PAM) were isolated. Incorporation of the two labeled components into the cellular lipids showed that the 3 H-specific activity of the phospholipids from the type II cells was three times that of the PAM and the utilization of 14 C-glycerol into phosphatidyl choline (PC) was 50% greater than incorporation into the PC from PAMs. The PC from type II cells showed that 30% was disaturated and from PAMs 21% was disaturated. Another phosphatide, phosphatidyl glycerol contained about one-third of the molecules in disaturated form. These data are consistent with the view that both type II cells and PAMs can synthesize surface-active phospholipids but it is generally accepted that only the pulmonary alveolar epithelial type II cells excrete the disaturated phospholipids which comprise the surface-active components of pulmonary surfactant

  1. Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage.

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P; Morales-Nebreda, Luisa; Cheng, Yuan; Hogan, Erin; Yeldandi, Anjana; Chi, Monica; Piseaux, Raul; Ridge, Karen; Michael Hart, C; Chandel, Navdeep; Scott Budinger, G R; Kamp, David W

    2016-12-01

    Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. Crocidolite asbestos (100µg/50µL), TiO 2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role

  2. Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation.

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    Nissim Arish

    Full Text Available High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT. Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP. Telomerase in mouse (MLE and human (A549 lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may

  3. Establishment and evaluation of a stable cattle type II alveolar epithelial cell line.

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    Feng Su

    Full Text Available Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-α and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.

  4. Carbon black nanoparticles induce type II epithelial cells to release chemotaxins for alveolar macrophages

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    Donaldson Ken

    2005-12-01

    Full Text Available Abstract Background Alveolar macrophages are a key cell in dealing with particles deposited in the lungs and in determining the subsequent response to that particle exposure. Nanoparticles are considered a potential threat to the lungs and the mechanism of pulmonary response to nanoparticles is currently under intense scrutiny. The type II alveolar epithelial cell has previously been shown to release chemoattractants which can recruit alveolar macrophages to sites of particle deposition. The aim of this study was to assess the responses of a type II epithelial cell line (L-2 to both fine and nanoparticle exposure in terms of secretion of chemotactic substances capable of inducing macrophage migration. Results Exposure of type II cells to carbon black nanoparticles resulted in significant release of macrophage chemoattractant compared to the negative control and to other dusts tested (fine carbon black and TiO2 and nanoparticle TiO2 as measured by macrophage migration towards type II cell conditioned medium. SDS-PAGE analysis of the conditioned medium from particle treated type II cells revealed that a higher number of protein bands were present in the conditioned medium obtained from type II cells treated with nanoparticle carbon black compared to other dusts tested. Size-fractionation of the chemotaxin-rich supernatant determined that the chemoattractants released from the epithelial cells were between 5 and 30 kDa in size. Conclusion The highly toxic nature and reactive surface chemistry of the carbon black nanoparticles has very likely induced the type II cell line to release pro-inflammatory mediators that can potentially induce migration of macrophages. This could aid in the rapid recruitment of inflammatory cells to sites of particle deposition and the subsequent removal of the particles by phagocytic cells such as macrophages and neutrophils. Future studies in this area could focus on the exact identity of the substance(s released by the

  5. Decreased CXCL12 is associated with impaired alveolar epithelial cell migration and poor lung healing after lung resection.

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    Kanter, Jacob A; Sun, Haiying; Chiu, Stephen; DeCamp, Malcolm M; Sporn, Peter H S; Sznajder, Jacob I; Bharat, Ankit

    2015-10-01

    Prolonged air leak (PAL) is an important cause of morbidity and mortality after lung resection, but its pathogenesis has not been elucidated. Migration of alveolar type II epithelial cells is essential for lung wound repair. Here we determined the role of C-X-C motif chemokine 12 (CXCL12) on alveolar epithelial cell migration and lung wound healing. CXCL12 in the pleural fluid of patients was analyzed using enzyme-linked immunosorbent assay. Human A549 and murine MLE12 alveolar epithelial cell lines were used for wound closure, cell migration, and proliferation assays. Western blot was used to analyze Rac1 and cofilin. Pleural CXCL12 was decreased in patients with PAL (1,389 ± 192 vs 3,270 ± 247 pg/mL; P alveolar epithelial cell migration by binding to its receptor CXCR4 and may have a role in lung healing. CXCL12-mediated alveolar epithelial cell migration is associated with Rac1 and cofilin activation. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Alveolar epithelial type II cells induce T cell tolerance to specific antigen

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    Lo, Bernice; Hansen, Søren; Evans, Kathy

    2008-01-01

    The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Type...... II) constitutively express the class II MHC led us to hypothesize that Type II cells play a role in the adaptive immune response. Because Type II cells do not express detectable levels of the costimulatory molecules CD80 and CD86, we propose that Type II cells suppress activation of naive T cells...

  7. Cell stress induces upregulation of osteopontin via the ERK pathway in type II alveolar epithelial cells.

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    Aki Kato

    Full Text Available Osteopontin (OPN is a multifunctional protein that plays important roles in cell growth, differentiation, migration and tissue fibrosis. In human idiopathic pulmonary fibrosis and murine bleomycin-induced lung fibrosis, OPN is upregulated in type II alveolar epithelial cells (AEC II. However, the mechanism of OPN induction in AEC II is not fully understood. In this study, we demonstrate the molecular mechanism of OPN induction in AEC II and elucidate the functions of OPN in AEC II and lung fibroblasts. Human lung adenocarcinoma cells (A549 and mouse alveolar epithelial cells (MLE12, used as type II alveolar epithelial cell lines for in vitro assays, and human pulmonary alveolar epithelial cells (HPAEpiC were treated with either bleomycin, doxorubicin or tunicamycin. The mechanism of OPN induction in these cells and its function as a pro-fibrotic cytokine on A549 and lung fibroblasts were analyzed. The DNA damaging reagents bleomycin and doxorubicin were found to induce OPN expression in A549, MLE12 and HPAEpiC. OPN expression was induced via activation of the extracellular signal-regulated protein kinase (ERK-dependent signaling pathway in A549 and MLE12. The endoplasmic reticulum (ER stress-inducing reagent tunicamycin induced OPN mRNA expression in A549, MLE12 and HPAEpiC, and OPN mRNA expression was induced via activation of the ERK-dependent signaling pathway in A549 and MLE12. Another ER stress-inducing reagent thapsigargin induced the expression of OPN mRNA as well as the subsequent production of OPN in A549 and MLE12. Furthermore, OPN promoted the proliferation of A549 and the migration of normal human lung fibroblasts. Inhibition of OPN by small interference RNA or neutralizing antibody suppressed both of these responses. The results of this study suggest that cell stress induces the upregulation of OPN in AEC II by signaling through the ERK pathway, and that upregulated OPN may play a role in fibrogenesis of the lung.

  8. Silver nanowire interactions with primary human alveolar type-II epithelial cell secretions: contrasting bioreactivity with human alveolar type-I and type-II epithelial cells

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    Sweeney, Sinbad; Theodorou, Ioannis G.; Zambianchi, Marta; Chen, Shu; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng (Jim); Chung, Kian Fan; Shaffer, Milo S. P.; Ryan, Mary P.; Porter, Alexandra E.; Tetley, Teresa D.

    2015-06-01

    Inhaled nanoparticles have a high deposition rate in the alveolar units of the deep lung. The alveolar epithelium is composed of type-I and type-II epithelial cells (ATI and ATII respectively) and is bathed in pulmonary surfactant. The effect of native human ATII cell secretions on nanoparticle toxicity is not known. We investigated the cellular uptake and toxicity of silver nanowires (AgNWs; 70 nm diameter, 1.5 μm length) with human ATI-like cells (TT1), in the absence or presence of Curosurf® (a natural porcine pulmonary surfactant with a low amount of protein) or harvested primary human ATII cell secretions (HAS; containing both the complete lipid as well as the full protein complement of human pulmonary surfactant i.e. SP-A, SP-B, SP-C and SP-D). We hypothesised that Curosurf® or HAS would confer improved protection for TT1 cells, limiting the toxicity of AgNWs. In agreement with our hypothesis, HAS reduced the inflammatory and reactive oxygen species (ROS)-generating potential of AgNWs with exposed TT1 cells. For example, IL-8 release and ROS generation was reduced by 38% and 29%, respectively, resulting in similar levels to that of the non-treated controls. However in contrast to our hypothesis, Curosurf® had no effect. We found a significant reduction in AgNW uptake by TT1 cells in the presence of HAS but not Curosurf. Furthermore, we show that the SP-A and SP-D are likely to be involved in this process as they were found to be specifically bound to the AgNWs. While ATI cells appear to be protected by HAS, evidence suggested that ATII cells, despite no uptake, were vulnerable to AgNW exposure (indicated by increased IL-8 release and ROS generation and decreased intracellular SP-A levels one day post-exposure). This study provides unique findings that may be important for the study of lung epithelial-endothelial translocation of nanoparticles in general and associated toxicity within the alveolar unit.Inhaled nanoparticles have a high deposition rate in

  9. Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells.

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    Armstrong, Lynne; Medford, Andrew R L; Uppington, Kay M; Robertson, John; Witherden, Ian R; Tetley, Teresa D; Millar, Ann B

    2004-08-01

    The recognition of potentially harmful microorganisms involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. TLR-4 is the major recognition receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, whereas TLR-2 responds to bacterial products from gram-positive organisms. Although resident alveolar macrophages are the first line of defense against microbial attack, it is now understood that the alveolar epithelium also plays a pivotal role in the innate immunity of the lung. The purpose of the current study was to determine whether human primary type II alveolar epithelial cells (ATII) express functional TLR-2 and TLR-4 and how they may be regulated by inflammatory mediators. We have used reverse transcriptase-polymerase chain reaction and flow cytometry to determine basal and inducible expression on ATII. We have used highly purified preparations of the gram-positive bacterial product lipoteichoic acid (LTA) and LPS to look at the functional consequences of TLR-2 and TLR-4 ligation, respectively, in terms of interleukin-8 release. We have shown that human primary ATII cells express mRNA and protein for both TLR-2 and TLR-4, which can be modulated by incubation with LPS and tumor necrosis factor. Furthermore, we have demonstrated that these receptors are functional. This suggests that ATII have the potential to contribute significantly to the host defense of the human alveolus against bacteria.

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

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

    2015-08-13

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

  11. Ultrastructural Study of Alveolar Epithelial Type II Cells by High-Frequency Oscillatory Ventilation

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    Xiaofei Qin

    2013-01-01

    Full Text Available Alveolar epithelial type II cells (AECIIs containing lamellar bodies (LBs are alveolar epithelial stem cells that have important functions in the repair of lung structure and function after lung injury. The ultrastructural changes in AECIIs after high-frequency oscillatory ventilation (HFOV with a high lung volume strategy or conventional ventilation were evaluated in a newborn piglet model with acute lung injury (ALI. After ALI with saline lavage, newborn piglets were randomly assigned into five study groups (three piglets in each group, namely, control (no mechanical ventilation, conventional ventilation for 24 h, conventional ventilation for 48 h, HFOV for 24 h, and HFOV for 48 h. The lower tissues of the right lung were obtained to observe the AECII ultrastructure. AECIIs with reduced numbers of microvilli, decreased LBs electron density, and vacuole-like LBs deformity were commonly observed in all five groups. Compared with conventional ventilation groups, the decrease in numbers of microvilli and LBs electron density, as well as LBs with vacuole-like appearance and polymorphic deformity, was less severe in HFOV with high lung volume strategy groups. AECIIs were injured during mechanical ventilation. HFOV with a high lung volume strategy resulted in less AECII damage than conventional ventilation.

  12. Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation.

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    Loscertales, Maria; Nicolaou, Fotini; Jeanne, Marion; Longoni, Mauro; Gould, Douglas B; Sun, Yunwei; Maalouf, Faouzi I; Nagy, Nandor; Donahoe, Patricia K

    2016-07-13

    Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown. We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture. Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.

  13. Genomic signature and toxicogenomics comparison of polycationic gene delivery nanosystems in human alveolar epithelial A549 cells

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    J Barar

    2009-10-01

    Full Text Available "nBackground and the purpose of the study: Of the gene delivery systems, non-viral polycationic gene delivery nanosystems have been alternatively exploited as a relatively safe delivery reagents compared to viral vectors. However, little is known about the genomic impacts of these delivery systems in target cells/tissues. In this study, the toxicogenomics and genotoxicity potential of some selected polycationic lipid/polymer based nanostructures (i.e., Oligofectamine® (OF, starburst polyamidoamine Polyfect® (PF and diaminobutane (DAB dendrimers were investigated in human alveolar epithelial A549 cells. "nMethods: To study the nature and the ontology of the gene expression changes in A549 cells upon treatment with polycationic nanostructures, MTT assay and microarray gene expression profiling methodology were employed. For microarray analysis, cyanine (Cy3/Cy5 labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. "nResults and major conclusions: The polycationic nanosystems induced significant gene expression changes belonging to different genomic ontologies such as cell defence and apoptosis pathways. These data suggest that polycationic nanosystems can elicit multiple gene expression changes in A549 cells upon their chemical structures and interactions with cellular/subcellular components. Such impacts may interfere with the main goals of the desired genemedicine.

  14. P2X7R: independent modulation of aquaporin 5 expression in CdCl2-injured alveolar epithelial cells.

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    Heupel, Julia; Bläsche, Robert; Wesslau, Karl-Philipp; Kasper, Michael; Barth, Kathrin

    2018-03-01

    The expression of aquaporin 5 in alveolar epithelial type I cells under conditions of cadmium-induced injury has not yet been discovered. We investigated the effect of the P2X7R agonist BzATP under this condition, since P2X7R is involved in altered regulation of aquaporin 5 in pulmonary fibrosis. CdCl 2 /TGF-β1 treatment of lung epithelial MLE-12 cells was leading to increasing P2X7R, and aquaporin 5 protein levels. The aquaporin 5 expression was P2X7R-independent in MLE-12 cells under cadmium, as was shown in blocking experiments with oxATP. Further, the expression of both proteins increased after 24 h CdCl 2 /TGF-β1 treatment of precision-cut lung slices, but decreased after 72 h. Using immunohistochemistry, the activation of the P2X7R with the agonist BzATP modulated the aquaporin 5 immunoreactivity in the alveolar epithelium of precision-cut lung slices from wild-type but not from P2X7R knockout mice. Similarly, aquaporin 5 protein was reduced in BzATP-treated immortal lung epithelial E10 cells. Surprisingly, untreated alveolar epithelial type II cells of P2X7R knockouts exhibited a pronounced apical immunoreactivity in addition to the remaining alveolar epithelial type I cells. BzATP exposure did not alter this distribution pattern, but increased the number of apoptotic alveolar epithelial type II cells in wild-type lung slices.

  15. Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages.

    Science.gov (United States)

    Granado, María H; Gangoiti, Patricia; Ouro, Alberto; Arana, Lide; Gómez-Muñoz, Antonio

    2009-04-01

    We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99-105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.

  16. Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling.

    Science.gov (United States)

    Ito, Yoko; Correll, Kelly; Schiel, John A; Finigan, Jay H; Prekeris, Rytis; Mason, Robert J

    2014-07-01

    There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS. Copyright © 2014 the American Physiological Society.

  17. Sustained distribution of aerosolized PEGylated liposomes in epithelial lining fluids on alveolar surfaces.

    Science.gov (United States)

    Kaneko, Keita; Togami, Kohei; Yamamoto, Eri; Wang, Shujun; Morimoto, Kazuhiro; Itagaki, Shirou; Chono, Sumio

    2016-10-01

    The distribution characteristics of aerosolized PEGylated liposomes in alveolar epithelial lining fluid (ELF) were examined in rats, and the ensuing mechanisms were investigated in the in vitro uptake and protein adsorption experiments. Nonmodified or PEGylated liposomes (particle size 100 nm) were aerosolized into rat lungs. PEGylated liposomes were distributed more sustainably in ELFs than nonmodified liposomes. Furthermore, the uptake of PEGylated liposomes by alveolar macrophages (AMs) was less than that of nonmodified liposomes. In further in vitro uptake experiments, nonmodified and PEGylated liposomes were opsonized with rat ELF components and then added to NR8383 cells as cultured rat AMs. The uptake of opsonized PEGylated liposomes by NR8383 cells was lower than that of opsonized nonmodified liposomes. Moreover, the protein absorption levels in opsonized PEGylated liposomes were lower than those in opsonized nonmodified liposomes. These findings suggest that sustained distributions of aerosolized PEGylated liposomes in ELFs reflect evasion of liposomal opsonization with surfactant proteins and consequent reductions in uptake by AMs. These data indicate the potential of PEGylated liposomes as aerosol-based drug delivery system that target ELF for the treatment of respiratory diseases.

  18. Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat.

    Science.gov (United States)

    Mitsopoulos, Panagiotis; Suntres, Zacharias E

    2010-12-05

    Paraquat (PQ), a commonly used herbicide, is highly toxic to humans and animals. The primary injury occurs in the lung, where PQ is actively taken up by alveolar epithelial cells and consequently produces damaging reactive oxygen species (ROS) via redox cycling. ROS have also been shown to induce expression of several early response genes and to activate transcription factors, which may contribute to the inflammatory response associated with PQ injury. In order to further elucidate the mechanism(s) of PQ injury, we investigated its effects on the cellular status and gene expression profile of immortalized human alveolar epithelial A549 cells in vitro. Incubation of cells with PQ resulted in concentration- and time-dependent PQ uptake, which correlated with increases in intracellular ROS levels and decreases in intracellular glutathione content, mitochondrial membrane potential, and cell viability. Gene array analysis showed differential expression in response to PQ exposure over time, particularly increases in: (i) the expression of growth arrest and cell cycle-related genes (e.g. CDKN1A, DDIT3 GADD45A, GDF15, MDM2, EGR1, CASP10, CASP8) and (ii) the expression of pro-inflammatory genes (e.g. IL1A, IL6, IL18, NFKB1, SERPINE1), which correlated with increases in the secretion of pro-inflammatory cytokines (e.g. IL-8, IL-6). These data suggest that uptake of PQ by A549 cells altered the cellular redox status and the expression of several early response genes, including the inflammatory response, all of which might contribute to the overall cytotoxicity of PQ. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  19. Suppression of ICE and Apoptosis in Mammary Epithelial Cells by Extracellular Matrix

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    Boudreau, Nancy; Sympson, C. J.; Werb, Zena; Bissell, Mina J.

    1994-12-01

    Apoptosis (programmed cell death) plays a major role in development and tissue regeneration. Basement membrane extracellular matrix (ECM), but not fibronectin or collagen, was shown to suppress apoptosis of mammary epithelial cells in tissue culture and in vivo. Apoptosis was induced by antibodies to beta 1 integrins or by overexpression of stromelysin-1, which degrades ECM. Expression of interleukin-1 beta converting enzyme (ICE) correlated with the loss of ECM, and inhibitors of ICE activity prevented apoptosis. These results suggest that ECM regulates apoptosis in mammary epithelial cells through an integrin-dependent negative regulation of ICE expression.

  20. Different particle determinants induce apoptosis and cytokine release in primary alveolar macrophage cultures

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    Schwarze Per E

    2006-06-01

    Full Text Available Abstract Background Particles are known to induce both cytokine release (MIP-2, TNF-α, a reduction in cell viability and an increased apoptosis in alveolar macrophages. To examine whether these responses are triggered by the same particle determinants, alveolar macrophages were exposed in vitro to mineral particles of different physical-chemical properties. Results The crystalline particles of the different stone types mylonite, gabbro, basalt, feldspar, quartz, hornfels and fine grain syenite porphyr (porphyr, with a relatively equal size distribution (≤ 10 μm, but different chemical/mineral composition, all induced low and relatively similar levels of apoptosis. In contrast, mylonite and gabbro induced a marked MIP-2 response compared to the other particles. For particles of smaller size, quartz (≤ 2 μm seemed to induce a somewhat stronger apoptotic response than even smaller quartz (≤ 0.5 μm and larger quartz (≤ 10 μm in relation to surface area, and was more potent than hornfels and porphyr (≤ 2 μm. The reduction in cell viability induced by quartz of the different sizes was roughly similar when adjusted to surface area. With respect to cytokines, the release was more marked after exposure to quartz ≤ 0.5 μm than to quartz ≤ 2 μm and ≤ 10 μm. Furthermore, hornfels (≤ 2 μm was more potent than the corresponding hornfels (≤ 10 μm and quartz (≤ 2 μm to induce cytokine responses. Pre-treatment of hornfels and quartz particles ≤ 2 μm with aluminium lactate, to diminish the surface reactivity, did significantly reduce the MIP-2 response to hornfels. In contrast, the apoptotic responses to the particles were not affected. Conclusion These results indicate that different determinants of mineral/stone particles are critical for inducing cytokine responses, reduction in cell viability and apoptosis in alveolar macrophages. The data suggest that the particle surface reactivity was critical for cytokine responses

  1. Apoptosis and Vocal Fold Disease: Clinically Relevant Implications of Epithelial Cell Death

    Science.gov (United States)

    Novaleski, Carolyn K.; Carter, Bruce D.; Sivasankar, M. Preeti; Ridner, Sheila H.; Dietrich, Mary S.; Rousseau, Bernard

    2017-01-01

    Purpose: Vocal fold diseases affecting the epithelium have a detrimental impact on vocal function. This review article provides an overview of apoptosis, the most commonly studied type of programmed cell death. Because apoptosis can damage epithelial cells, this article examines the implications of apoptosis on diseases affecting the vocal fold…

  2. Helicobacter pylori enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in human gastric epithelial cells

    Science.gov (United States)

    Wu, Yi-Ying; Tsai, Hwei-Fang; Lin, We-Cheng; Chou, Ai-Hsiang; Chen, Hui-Ting; Yang, Jyh-Chin; Hsu, Ping-I; Hsu, Ping-Ning

    2004-01-01

    AIM: To investigate the relations between tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Helicobacter pylori (H pylori) infection in apoptosis of gastric epithelial cells and to assess the expression of TRAIL on the surface of infiltrating T-cells in H pylori-infected gastric mucosa. METHODS: Human gastric epithelial cell lines and primary gastric epithelial cells were co-cultured with H pylori in vitro, then recombinant TRAIL proteins were added to the culture. Apoptosis of gastric epithelial cells was determined by a specific ELISA for cell death. Infiltrating lymphocytes were isolated from H pylori-infected gastric mucosa, and expression of TRAIL in T cells was analyzed by flow cytometry. RESULTS: The apoptosis of gastric epithelial cell lines and primary human gastric epithelial cells was mildly increased by interaction with either TRAIL or H pylori alone. Interestingly, the apoptotic indices were markedly elevated when gastric epithelial cells were incubated with both TRAIL and H pylori (Control vs TRAIL and H pylori: 0.51 ± 0.06 vs 2.29 ± 0.27, P = 0.018). A soluble TRAIL receptor (DR4-Fc) could specifically block the TRAIL-mediated apoptosis. Further studies demonstrated that infiltrating T-cells in gastric mucosa expressed TRAIL on their surfaces, and the induction of TRAIL sensitivity by H pylori was dependent upon direct cell contact of viable bacteria, but not CagA and VacA of H pylori. CONCLUSION: H pylori can sensitize human gastric epithelial cells and enhance susceptibility to TRAIL-mediated apoptosis. Modulation of host cell sensitivity to apoptosis by bacterial interaction adds a new dimension to the immunopathogenesis of H pylori infection. PMID:15285015

  3. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    block alloactivation, had no inhibitory effect on RPE-mediated T-cell apoptotic responses in MHC class II-specific CD4+ T-cell lines. CONCLUSIONS: Retinal pigment epithelial cells express FasL and induce TCR-independent apoptosis in activated human T cells through Fas-FasL interaction. Retinal pigment...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  4. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  5. P2X7R-dependent regulation of glycogen synthase kinase 3β and claudin-18 in alveolar epithelial type I cells of mice lung.

    Science.gov (United States)

    Barth, K; Bläsche, R; Neißer, A; Bramke, S; Frank, J A; Kasper, M

    2016-12-01

    The purinergic receptor P2X7 represents an ATP-gated ionotropic receptor with a selective localization in alveolar epithelial type I cells of the lung. Despite the involvement of the receptor in inflammatory processes of the lung, it is not established whether this receptor plays a specific role in the alveolar epithelial cell biology. There is evidence that P2X7 receptor influences Wnt/β-catenin signalling pathways in alveolar epithelial cells under conditions of injury. Here, we investigated the expression of GSK-3β, a potent protein kinase involved in alveolar epithelial barrier functions, and of tight junction molecules occludin, claudin-4 and claudin-18 in wild-type and P2X7 -/- mice. Western blot analysis, immunohistochemistry and quantitative real-time RT-PCR revealed a remarkable increase in claudin-18 mRNA and protein in lungs of P2X7 -/- mice animals. Furthermore, alveolar epithelial cells from P2X7 -/- animals showed decreased levels of GSK-3β protein and its inactive form GSK-3β (pS9). Conversely, claudin-18 knockout mice exhibited decreased P2X7 mRNA transcript abundance as measured by mRNA expression microarray and quantitative PCR. Our data are consistent with the hypothesis that P2X7R contributes to alveolar epithelial barrier function through effects on GSK-3β. Furthermore, these data suggest a potential reciprocal regulation of claudin-18 and P2X7R in the alveolar epithelium.

  6. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  7. TNF-α increases Staphylococcus aureus-induced death of human alveolar epithelial cell line A549 associated with RIP3-mediated necroptosis.

    Science.gov (United States)

    Wen, Shun-Hang; Lin, Luo-Na; Wu, Hu-Jun; Yu, Lu; Lin, Li; Zhu, Li-Li; Li, Hai-Yan; Zhang, Hai-Lin; Li, Chang-Chong

    2018-02-15

    To explore the role of tumor necrosis factor-alpha (TNF-α) on Staphylococcus aureus-induced necroptosis in alveolar epithelial cells. The A549 alveolar epithelial cell line was pretreated with small interfering RNA (siRNA) against receptor interacting protein-3 (RIP3) and then stimulated by S. aureus, where some cells were pretreated with TNF-α or TNF-α with anti-TNF-α antibody simultaneously. A549 cell death was assessed using lactate dehydrogenase (LDH) leakage and flow cytometry analyses. The protein expressions of RIP1, RIP3, cleaved caspase-1, and cleaved caspase-8 were analyzed by western blot. S. aureus-induced LDH release was increased significantly by TNF-α. In addition, flow cytometry showed that TNF-α increased A549 cell apoptosis and necrosis in S. aureus-infected cell cultures. Levels of RIP3 and cleaved caspase-1 protein in A549 cells infected with S. aureus increased at 12 h post-infection, as shown by western blot. Significant additional increases in RIP3 expression were observed following the addition of TNF-α. Decreasing RIP3 levels by siRNA significantly suppressed the release of LDH induced by TNF-α and S. aureus. RIP3 siRNA also significantly suppressed A549 cell necrosis induced by S. aureus and TNF-α at 6 and 12 h post-infection as shown by flow cytometry analysis. TNF-α enhances the damage of S. aureus on lung epithelial cells, and its mechanism is associated with RIP3 mediated necroptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing.

    Science.gov (United States)

    Godin, Lindsay M; Vergen, Jorge; Prakash, Y S; Pagano, Richard E; Hubmayr, Rolf D

    2011-04-01

    Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of "barotrauma" and "biotrauma." In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ∼10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process.

  9. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    Science.gov (United States)

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

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

    Directory of Open Access Journals (Sweden)

    Shigehisa Yanagi

    2015-01-01

    Full Text Available 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.

  11. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    Directory of Open Access Journals (Sweden)

    Hiroshi Kondo

    2015-01-01

    Full Text Available Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12 were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC, an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5, an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1 expression levels were enhanced. After treatment with dexamethasone (DEX, 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX, and keratinocyte growth factor (KGF, surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

  12. Graphene-induced apoptosis in lung epithelial cells through EGFR

    Science.gov (United States)

    Tsai, Shih-Ming; Bangalore, Preeti; Chen, Eric Y.; Lu, David; Chiu, Meng-Hsuen; Suh, Andrew; Gehring, Matthew; Cangco, John P.; Garcia, Santiago G.; Chin, Wei-Chun

    2017-07-01

    Expanding interest in nanotechnology applied to electronic and biomedical fields has led to fast-growing development of various nanomaterials. Graphene is a single-atom thick, two-dimensional sheet of hexagonally arranged carbon atoms with unique physical and chemical properties. Recently, graphene has been used in many studies on electronics, photonics, composite materials, energy generation and storage, sensors, and biomedicine. However, the current health risk assessment for graphene has been relatively limited and inconclusive. This study evaluated the toxicity effects of graphene on the airway epithelial cell line BEAS-2B, which represents the first barrier of the human body to interact with airborne graphene particles. Our result showed that graphene can induce the cellular Ca2+ by phospholipase C (PLC) associated pathway by activating epidermal growth factor receptor (EGFR). Subsequently, inositol 1,4,5-triphosphate (IP3) receptors activate the release of Ca2+ from the endoplasmic reticulum (ER) Ca2+ stores. Those Ca2+ signals further trigger the calcium-regulated apoptosis in the cell. Furthermore, the stimulation can cause EGFR upregulation, which have been demonstrated to associate with diseases such as lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular diseases. This study highlights the additional health risk considering that it can function as a contributing factor for other respiratory diseases.

  13. Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

    Directory of Open Access Journals (Sweden)

    Danyi Peng

    2017-12-01

    Full Text Available Alveolar epithelial cells (AECs injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF. Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ model, as well as a new SMARCA4-deleted alveolar type II (ATII-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.

  14. Effect of SLC34A2 gene mutation on extracellular phosphorus transport in PAM alveolar epithelial cells.

    Science.gov (United States)

    Ma, Tiangang; Qu, Danhua; Yan, Bingdi; Zhang, Qinghua; Ren, Jin; Hu, Yanbing

    2018-01-01

    A mutation in the IIb sodium phosphate transporter SLC34A2 gene has recently been described in pulmonary alveolar microlithiasis (PAM) patients. Experiments in this study were aimed at confirming the role of the gene product in PAM by comparing phosphorylated products in extracellular fluid of alveolar epithelial cells overexpressing the SLC34A2 gene or its mutated version. Eukaryotic expression vectors were constructed and transfected into A549 human alveolar epithelial cells. There were three groups of cells including those transfected with empty vector plasmid pcDNA3.1(+) (plasmid control group), those transfected with normal SLC34A2 gene expressed from pcDNA3.1 (normal control group), and those transfected with a version of the PAM SLC34A2 gene linked to the pcDNA3.1(+) (PAM group). Transfection efficiencies were detected by reverse transcription-polymerase chain reaction (RT-PCR). At 48 h after transfection, the concentration of inorganic phosphorus in the culture medium was detected using an automatic biochemical analyzer. Our results showed the concentration of inorganic phosphorus in the supernatant of the normal control group was significantly lower than that in the plasmid control and PAM groups (PPAM group was significantly lower than that in the plasmid control group (PPAM patients, given that the function of the phosphate transporter seems to be affected and it is conceivable that it would lead to extracellular fluid alterations in vivo .

  15. Effect of mechanical vs dilute ethanol epithelial removal on keratocyte apoptosis and polymorphonuclear leukocyte migration.

    Science.gov (United States)

    Gurelik, G; Bilgihan, K; Sezer, C; Akyol, G; Hasanreisoglu, B

    2002-03-01

    To investigate keratocyte apoptosis and polymorphonuclear (PMN) cell infiltration to the corneal stroma after mechanical epithelial scraping and chemical de-epithelialization with 18% ethanol solution. Twelve New Zealand Albino rabbits (24 eyes) were randomly divided into three groups. Group A was the control group with no epithelial removal. Group B underwent a 7.5-mm mechanical epithelial removal with a blunt spatula. Group C underwent 7.5-mm chemical de-epithelialization with 18% ethanol-balanced salt solution. Corneas were stained with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay after 24 h. Only nuclear staining in keratocytes was counted. Polymorphonuclear (PMN) leukocyte densities were also assessed by light microscopy. Mechanical de-epithelialization (group B) and chemical de-epithelialization with 18% ethanol (group C) showed no difference in keratocyte apoptosis compared with the control group. There was also no difference between groups B and C. Group B showed no difference in PMN leukocyte counts compared with the control group. But the number of PMN leukocytes observed in group C was significantly higher than those encountered in the corneas of the control group (P < 0.05) and group B (P < 0.05). Dilute alcohol induces more PMN cell infiltration when compared with mechanical de-epithelialization although there is no difference in the apoptosis rates.

  16. Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

    LENUS (Irish Health Repository)

    O'Connell, J

    2012-02-03

    Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell\\'s sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95\\/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.

  17. [Role of oxidative stress in endoplasmic reticulum stress? induced apoptosis of alveolar macrophages triggered by quartz dust].

    Science.gov (United States)

    Song, Jing; Lu, Xiaoting; Li, Qiuying; Liu, Chengyun; Liu, Ying

    2014-07-01

    To investigate the role of oxidative stress in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust. Seventy-two healthy adult Wistar rats were randomly divided into control group, quartz dust group, quartz dust plus N-acetyl cysteine (NAC) group, and NAC group, with 18 rats in each group. One milliliter of sterile saline (for the control and NAC groups) or 1 ml of saline with 5%ultrafine quartz dust (for dust group and dust plus NAC group) was given to each rat by non-exposed endotracheal infusion. From the second day after dust infusion, rats in dust plus NAC group and NAC group received intragastric administration of NAC (100 mg/kg). In each week, the treatment with NAC lasted for 5 consecutive days, followed by 2 days' interval. For each group, 6 rats were randomly selected on the 14th, 28th, or 56th day after dust exposure; they were sacrificed by bloodletting from the femoral artery, and the lungs were collected. Bronchoalveolar lavage fluid was collected to separate macrophages. The protein expression of caspase-12 in alveolar macrophages, the apoptosis rate and reactive oxygen species (ROS) content of alveolar macrophages, and the protein carbonyl content of alveolar macrophages were determined by Western blot, flow cytometry, and colorimetry, respectively. Increased protein expression of caspase-12, apoptosis rate, and content of ROS and protein carbonyl were discovered on the 14th day in the dust group, in comparison with the control group (P quartz dust. Oxidative damage of protein in the endoplasmic reticulum may play an important role in the process.

  18. Quercetogetin protects against cigarette smoke extract-induced apoptosis in epithelial cells by inhibiting mitophagy.

    Science.gov (United States)

    Son, Eun Suk; Kim, Se-Hee; Ryter, Stefan W; Yeo, Eui-Ju; Kyung, Sun Young; Kim, Yu Jin; Jeong, Sung Hwan; Lee, Chang Soo; Park, Jeong-Woong

    2018-04-01

    Recent studies demonstrate that the autophagy-dependent turnover of mitochondria (mitophagy) mediates pulmonary epithelial cell death in response to cigarette smoke extract (CSE) exposure, and contributes to emphysema development in vivo during chronic cigarette smoke (CS)-exposure, although the underlying mechanisms remain unclear. Here, we investigated the role of mitophagy in regulating apoptosis in CSE-exposed human lung bronchial epithelial cells. Furthermore, we investigated the potential of the polymethoxylated flavone antioxidant quercetogetin (QUE) to inhibit CSE-induced mitophagy-dependent apoptosis. Our results demonstrate that CSE induces mitophagy in epithelial cells via mitochondrial dysfunction, and causes increased expression levels of the mitophagy-regulator protein PTEN-induced putative kinase-1 (PINK1) and the mitochondrial fission protein dynamin-1-like protein (DRP-1). CSE induced epithelial cell death and increased the expression of the apoptosis-related proteins cleaved caspase-3, -8 and -9. Caspase-3 activity was significantly increased in Beas-2B cells exposed to CSE, and decreased by siRNA-dependent knockdown of DRP-1. Treatment of epithelial cells with QUE inhibited CSE-induced mitochondrial dysfunction and mitophagy by inhibiting phospho (p)-DRP-1 and PINK1 expression. QUE suppressed mitophagy-dependent apoptosis by inhibiting the expression of cleaved caspase-3, -8 and -9 and downregulating caspase activity in human bronchial epithelial cells. These findings suggest that QUE may serve as a potential therapeutic in CS-induced pulmonary diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. The protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Xue - Fang Chen

    2013-06-01

    Full Text Available AIM: To investigate the protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis. METHODS:Subcultured human lens epithelial cell line, ultraviolet induced cell apoptosis, 20μmol/L resveratrol pretreated cell, the indicators change was observed: rate of apoptosis was detected by flow cytometry and apoptosis-related factors of caspses-3 and caspase-9 were detected by colorimetric detection, ultrastructure changes were observed under transmission electron microscope. RESULTS: Flow cytometry instrument testing found that resveratrol can suppress the apoptosis induced by ultraviolet irradiation, caspses-3 and caspase-9 content in positive control group were significantly higher than that of the negative control group at the same time period, the difference was statistically significant(P<0.05; caspses-3 and caspase-9 content in experimental group were lower than that in the positive control group at the same time, the difference was statistically significant(P<0.05. In addition, the damage of human lens epithelial cells was alleviated with the incubation time of resveratrol elongated. CONCLUSION:Resveratrol may inhibit ultraviolet-induced apoptosis of human lens epithelial cells, it has preventive function against radioactive cataract, and it can provide reliable evidence for pursuing effective medicine to prevent and treat cataract.

  20. Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Rahman Irfan

    2006-10-01

    Full Text Available Abstract Background Cigarette smoke mediated oxidative stress and inflammatory events in the airway and alveolar epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Previously, individual cell lines were used to assess the oxidative and proinflammatory effects of cigarette smoke with confounding results. In this study, a panel of human and rodent transformed epithelial cell lines were used to determine the effects of cigarette smoke extract (CSE on oxidative stress markers, cell toxicity and proinflammatory cytokine release and compared the effects with that of primary human small airway epithelial cells (SAEC. Methods Primary human SAEC, transformed human (A549, H1299, H441, and rodent (murine MLE-15, rat L2 alveolar epithelial cells were treated with different concentrations of CSE (0.2–10% ranging from 20 min to 24 hr. Cytotoxicity was assessed by lactate dehydrogenase release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide. Glutathione concentration was measured by enzymatic recycling assay and 4-hydroxy-2-nonenal levels by using lipid peroxidation assay kit. The levels of proinflammatory cytokines (e.g. IL-8 and IL-6 were measured by ELISA. Nuclear translocation of the transcription factor, NF-κB was assessed by immunocytochemistry and immunoblotting. Results Cigarette smoke extract dose-dependently depleted glutathione concentration, increased 4-hydroxy-2-nonenal (4-HNE levels, and caused necrosis in the transformed cell lines as well as in SAEC. None of the transformed cell lines showed any significant release of cytokines in response to CSE. CSE, however, induced IL-8 and IL-6 release in primary cell lines in a dose-dependent manner, which was associated with the nuclear translocation of NF-κB in SAEC. Conclusion This study suggests that primary, but not transformed, lung epithelial cells are an appropriate model to study the inflammatory

  1. The influence of volatile anesthetics on alveolar epithelial permeability measured by noninvasive radionuclide lung scan

    International Nuclear Information System (INIS)

    Hung, Chih-Jen; Wu, Rick Sai-Chuen; Lin, Cheng-Chieh; Kao, Albert; Tsai, Jeffrey J.P.

    2003-01-01

    Many volatile anesthetics have long been thought to affect pulmonary functions including lung ventilation (LV) and alveolar epithelial permeability (AEP). The purpose of this study is to examine the influence of volatile anesthetics on LV and AEP by noninvasive radionuclide lung imaging of technetium-99m labeled diethylene triamine pentaacetic acid radioaerosol inhalation lung scan (DTPA lung scan). Twenty patients undergoing surgery and receiving volatile anesthesia with 1% halothane were enrolled as the study group 1. The other 20 patients undergoing surgery and receiving volatile anesthesia with 1.5% isoflurane were enrolled as the study group 2. At the same time, 20 patients undergoing surgery with intravenous anesthesia drugs were included as a control group. Before surgery, 1 hour after surgery, and 1 week after surgery, we investigated the 3 groups of patients with DTPA lung scan to evaluate LV and AEP by 99m Tc DTPA clearance halftime (T1/2). No significant change or abnormality of LV before surgery, 1 hour after surgery, or 1 week after surgery was found among the 3 groups of patients. In the control group, the 99m Tc DTPA clearance T1/2 was 63.5±16.4, 63.1±18.4, and 62.8±17.0 minutes, before surgery, 1 hour after surgery, and 1 week after surgery, respectively. In group 1, it was 65.9±9.3, 62.5±9.1, and 65.8±10.3 minutes, respectively. No significant change in AEP before surgery, 1 hour after surgery, or 1 week after surgery was found. However, in group 2, the 99m Tc DTPA clearance T1/2 was 65.5±13.2, 44.9±10.5, and 66.1±14.0 minutes, respectively. A significant transient change in AEP was found 1 hour after surgery, but it recovered 1 week after surgery. We conclude that volatile anesthesia is safe for LV and AEP, and only isoflurane can induce transient change of AEP. (author)

  2. The effects of benzene exposure on apoptosis in epithelial lung cells: localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene products.

    Science.gov (United States)

    Weaver, C V; Liu, S-P; Lu, J-F; Lin, B-S

    2007-05-01

    Although benzene, a well-known human carcinogen, has been shown to induce apoptosis in vitro, no studies have been carried out to confirm and characterize its role in activating apoptosis in vivo. The present study investigated the effects of benzene inhalation on the epithelial cells lining the respiratory tract including bronchioles, terminal bronchioles, respiratory bronchioles and alveoli of male Sprague-Dawley rats. Inhalation of benzene 300 ppm for 7 days induced apoptotic changes in the parenchymal components in the lung that significantly exceeded the events of programmed cell death in normal control tissues. Apoptosis was confirmed by the electrophoretic analysis of internucleosomal DNA fragmentation of benzene-exposed lung tissues, which exhibited 180-200 bp laddering subunits indicative of genomic DNA degradation. Furthermore, semi-quantitative analysis of intracellular localization of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling TUNEL) showed a significant (p respiratory bronchiolar 60.8% segmental epithelial components as well as alveolar (55%) epithelia. Analysis of immunohistochemical expression of apoptosis-related gene products also supported the hypothesis that benzene can induce apoptosis in chemosensitive target cells in the lung parenchyma. Quantitative immunhistochemistry showed a statistically significant increase p respiratory system that demonstrates that benzene inhalation induces lung cell apoptosis as confirmed by DNA electrophoresis, in situ nick end labeling, and the upregulation of apoptosis-related gene products that facilitate caspase-cleaved enzymes which lead to cell degradation via programmed cell death. These responses may represent an important defense mechanism within the parenchymal cells of the respiratory system that reduce mutational hazard and the potential carcinogenic effects of benzene-initiated pathogenesis.

  3. Apoptosis in oral epithelial dysplastic lesions and oral squamous cell carcinoma: A prognostic marker

    OpenAIRE

    Shwetha Nambiar; Veda Hegde

    2016-01-01

    Background: Apoptotic index (AI) using light microscopy as an indirect measure to assess the significance of apoptosis as a proliferative marker in dysplastic lesions and malignant epithelial lesions of the oral cavity. Aims: (1) To quantify the apoptotic bodies/cells in oral epithelial dysplastic (OED) lesions and oral squamous cell carcinoma (OSCC). (2) To measure AI in OED and OSCC. (3) To compare AI in OED and OSCC. Settings and Design: The proposed laboratory-based retrospective study in...

  4. CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis.

    Science.gov (United States)

    Stromberg, Paul E; Woolsey, Cheryl A; Clark, Andrew T; Clark, Jessica A; Turnbull, Isaiah R; McConnell, Kevin W; Chang, Katherine C; Chung, Chun-Shiang; Ayala, Alfred; Buchman, Timothy G; Hotchkiss, Richard S; Coopersmith, Craig M

    2009-06-01

    Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1(-/-) and wild-type (WT) mice. However, Rag-1(-/-) animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1(-/-) mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4(+) but not CD8(+), gammadelta, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1(-/-) mice. Further, adoptively transferring lymphocytes to Rag-1(-/-) mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4(+) lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.

  5. High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells.

    Science.gov (United States)

    Zhang, Xiao-Qian; Dong, Jian-Jun; Cai, Tian; Shen, Xue; Zhou, Xiao-Jun; Liao, Lin

    2017-04-11

    Diabetic nephropathy is the primary cause of end-stage renal disease. Apoptosis of tubule epithelial cells is a major feature of diabetic nephropathy. The mechanisms of high glucose (HG) induced apoptosis are not fully understood. Here we demonstrated that, HG induced apoptosis via upregulating the expression of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim protein, but not bring a significant change in the baseline level of autophagy in HK2 cells. The increase of Bim expression was caused by the ugregulation of transcription factors, FOXO1 and FOXO3a. Bim expression initiates BAX/BAK-mediated mitochondria-dependent apoptosis. Silence of Bim by siRNA in HK2 cells prevented HG-induced apoptosis and also sensitized HK2 cells to autophagy during HG treatment. The autophagy inhibitor 3-MA increased the injury in Bim knockdown HK2 cells by retriggering apoptosis. The above results suggest a Bim-independent apoptosis pathway in HK2 cells, which normally could be inhibited by autophagy. Overall, our results indicate that HG induces apoptosis via up-regulation of Bim expression in proximal tubule epithelial cells.

  6. Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

    Directory of Open Access Journals (Sweden)

    Peiris Malik

    2010-03-01

    Full Text Available Abstract Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1, which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97 virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280 and A/Chicken/Hong Kong/G9/97 (H9N2/G9, replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells.

  7. Mechanism research of miR-181 regulating human lens epithelial cell apoptosis

    Directory of Open Access Journals (Sweden)

    Yu Qin

    2015-05-01

    Full Text Available AIM: To investigate the expression of miR-181 in the lens tissue of cataract and the regulating mechanism of miR-181 on apoptosis of human lens epithelial cell.METHODS:Real time q-PCR was used to measure the expression of miR-181 in the anterior lens capsules of age-related cataract and human lens epithelial cell apoptosis model. miR-181 mimic and inhibitor were transfected using Lipofectamine 2 000 to regulate the expression of miR-181, and then Real time q-PCR was used to verify transfection efficiency. Flow cytometry was used to detect the change of cell apoptosis rate. RESULTS: Compared with control group, the expression of miR-181 was significantly higher in both the anterior lens capsules of age-related cataract and human lens epithelial cell apoptosis model; the relative expression of miR-181 in lens epithelial cells transfected with miR-181 mimic was increased, whereas decreased in cells transfected with miR-181 inhibitor; the apoptosis rate of cells transfected with miR-181 mimic was increased, while reduced in miR-181 inhibitor group. Each result was statistically significant(PCONCLUSION: High expression of miR-181 is detected in anterior lens capsule of age-related cataract. miR-181 might play a certain role in the pathogenesis of cataract via promoting human lens epithelial cell apoptosis. miR-181 probably becomes a new approach for the nonoperative treatment of cataract, but the concrete mechanism still needs to be further studied.

  8. Mechanisms of decreased intestinal epithelial proliferation and increased apoptosis in murine acute lung injury.

    Science.gov (United States)

    Husain, Kareem D; Stromberg, Paul E; Woolsey, Cheryl A; Turnbull, Isaiah R; Dunne, W Michael; Javadi, Pardis; Buchman, Timothy G; Karl, Irene E; Hotchkiss, Richard S; Coopersmith, Craig M

    2005-10-01

    The aim of this study was to determine the effects of acute lung injury on the gut epithelium and examine mechanisms underlying changes in crypt proliferation and apoptosis. The relationship between severity and timing of lung injury to intestinal pathology was also examined. Randomized, controlled study. University research laboratory. Genetically inbred mice. Following induction of acute lung injury, gut epithelial proliferation and apoptosis were assessed in a) C3H/HeN wild-type and C3H/HeJ mice, which lack functional Toll-like receptor 4 (n = 17); b) C57Bl/6 mice that received monoclonal anti-tumor necrosis factor-alpha or control antibody (n = 22); and c) C57Bl/6 wild-type and transgenic mice that overexpress Bcl-2 in their gut epithelium (n = 21). Intestinal epithelial proliferation and death were also examined in animals with differing degrees of lung inflammation (n = 24) as well as in a time course analysis following a fixed injury (n = 18). Acute lung injury caused decreased proliferation and increased apoptosis in crypt epithelial cells in all animals studied. C3H/HeJ mice had higher levels of proliferation than C3H/HeN animals without additional changes in apoptosis. Anti-tumor necrosis factor-alpha antibody had no effect on gut epithelial proliferation or death. Overexpression of Bcl-2 did not change proliferation despite decreasing gut apoptosis. Proliferation and apoptosis were not correlated to severity of lung injury, as gut alterations were lost in mice with more severe acute lung injury. Changes in both gut epithelial proliferation and death were apparent within 12 hrs, but proliferation was decreased 36 hrs following acute lung injury while apoptosis returned to normal. Acute lung injury causes disparate effects on crypt proliferation and apoptosis, which occur, at least in part, through differing mechanisms involving Toll-like receptor 4 and Bcl-2. Severity of lung injury does not correlate with perturbations in proliferation or death in the

  9. Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell-cell junctions.

    Science.gov (United States)

    Gagat, Maciej; Grzanka, Dariusz; Izdebska, Magdalena; Sroka, Wiktor Dariusz; Hałas-Wiśniewska, Marta; Grzanka, Alina

    2016-04-01

    The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract. Copyright © 2016 Elsevier GmbH. All rights reserved.

  10. Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis.

    Science.gov (United States)

    Jilling, Tamas; Lu, Jing; Jackson, Michele; Caplan, Michael S

    2004-04-01

    The histopathology of necrotizing enterocolitis (NEC) is characterized by destruction of the mucosal layer in initial stages and by transmural necrosis of the intestinal wall in advanced stages of the disease. To test the hypothesis that enhanced epithelial apoptosis is an initial event underlying the gross histologic changes, we analyzed epithelial apoptosis and tissue morphology in an animal model of NEC and evaluated the effect of caspase inhibition on the incidence of experimental NEC in this model. Apoptosis was analyzed with terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end labeling (TUNEL) staining in intestinal sections and by measuring caspase 3 activity from intestinal lysates of neonatal rats subjected to formula feeding and cold/asphyxia stress (FFCAS) and from mother-fed (MF) controls. Morphologic evaluation was based on hematoxylin and eosin staining of intestinal sections. FFCAS resulted in histologic changes consistent with NEC, which were absent from MF animals. FFCAS was also associated with a significantly increased rate of nuclear DNA fragmentation in the small intestinal epithelium compared with MF. Elevated tissue caspase 3 activity confirmed the presence of apoptosis in samples with increased DNA fragmentation. Analysis of the coincidence of morphologic damage and apoptosis in corresponding tissue sections indicated that apoptosis precedes gross morphologic changes in this model. Furthermore, supplementation of formula with 8 boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, significantly reduced the incidences of apoptosis and experimental NEC. These findings indicate that in neonatal rats FFCAS induces epithelial apoptosis that serves as an underlying cause for subsequent gross tissue necrosis.

  11. Taurine Protects Lens Epithelial Cells Against Ultraviolet B-Induced Apoptosis.

    Science.gov (United States)

    Dayang, Wu; Dongbo, Pang

    2017-10-01

    The massive uptake of compatible osmolytes is a self-protective response shared by lens exposed to hypertonic stress and ultraviolet stress. This study aimed to investigate the protective effects of taurine against ultraviolet B-induced cytotoxicity in the lens epithelial cells. Real-time PCR was used to measure osmolytes transport. Radioimmunoassay was used to measure osmolytes uptake. Cell counting kit-8 assays were used to measure cellular viability. Flow cytometry analysis was used to measure apoptosis level. Compared with normotonic stress, hypertonic stress-induced osmolytes uptake into the lens epithelial cells such as betaine, myoinositol and taurine. UVB exposure increased osmolytes transporter mRNA expression together with osmolytes uptake. Moreover, taurine suppressed UVB-induced cell apoptosis in the lens epithelial cells significantly. The effect of compatible osmolyte taurine on cell survival rate may play an important role in cell resistance and adaption to UVB exposure.

  12. Mammary alveolar epithelial cells convert to brown adipocytes in post-lactating mice

    DEFF Research Database (Denmark)

    Giordano, Antonio; Perugini, Jessica; Kristensen, David Møbjerg

    2017-01-01

    found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X...

  13. Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing.

    Science.gov (United States)

    Maquerlot, François; Galiacy, Stephane; Malo, Michel; Guignabert, Christophe; Lawrence, Daniel A; d'Ortho, Maria-Pia; Barlovatz-Meimon, Georgia

    2006-11-01

    Epithelium repair, crucial for restoration of alveolo-capillary barrier integrity, is orchestrated by various cytokines and growth factors. Among them keratinocyte growth factor plays a pivotal role in both cell proliferation and migration. The urokinase plasminogen activator (uPA) system also influences cell migration through proteolysis during epithelial repair. In addition, the complex formed by uPAR-uPA and matrix-bound plasminogen activator inhibitor type-1 (PAI-1) exerts nonproteolytic roles in various cell types. Here we present new evidence about the dual role of PAI-1 under keratinocyte growth factor stimulation using an in vitro repair model of rat alveolar epithelial cells. Besides proteolytic involvement of the uPA system, the availability of matrix-bound-PAI-1 is also required for an efficient healing. An unexpected decrease of healing was shown when PAI-1 activity was blocked. However, the proteolytic action of uPA and plasmin were still required. Moreover, immediately after wounding, PAI-1 was dramatically increased in the newly deposited matrix at the leading edge of wounds. We thus propose a dual role for PAI-1 in epithelial cell wound healing, both as a soluble inhibitor of proteolysis and also as a matrix-bound regulator of cell migration. Matrix-bound PAI-1 could thus be considered as a new member of the matricellular protein family.

  14. Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells.

    Science.gov (United States)

    Ribeiro, Giovanni Monteiro; Matsumoto, Cristianne Kayoko; Real, Fernando; Teixeira, Daniela; Duarte, Rafael Silva; Mortara, Renato Arruda; Leão, Sylvia Cardoso; de Souza Carvalho-Wodarz, Cristiane

    2017-09-13

    Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection. CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h. M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by

  15. Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine

    Science.gov (United States)

    Vyas, Dinesh; Robertson, Charles M; Stromberg, Paul E; Martin, James R.; Dunne, W. Michael; Houchen, Courtney W; Barrett, Terrence A; Ayala, Alfred; Perl, Mario; Buchman, Timothy G; Coopersmith, Craig M

    2007-01-01

    Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death. PMID:17357092

  16. Hydrogen Sulfide Inhibits Cigarette Smoke-Induced Endoplasmic Reticulum Stress and Apoptosis in Bronchial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Fan Lin

    2017-09-01

    Full Text Available Background: Apoptosis of lung structural cells contributes to the process of lung damage and remodeling in chronic obstructive pulmonary disease (COPD. Our previous studies demonstrated that exogenous hydrogen sulfide (H2S can reduce the lung tissue pathology score, anti-inflammation and anti-oxidation effects in COPD, but the effect of H2S in regulating cigarette smoke (CS induced bronchial epithelial cell apoptosis and the underlying mechanisms are not clear.Objectives: To investigate the effect of H2S on CS induced endoplasmic reticulum stress (ERS and bronchial epithelial cell apoptosis.Methods: Male Sprague–Dawley rats randomly divided into four groups for treatment: control, CS, NaHS + CS, and propargylglycine (PPG + CS. The rats in the CS group were exposed to CS generated from 20 commercial unfiltered cigarettes for 4 h/day, 7 days/week for 4 months. Since the beginning of the third month, freshly prepared NaHS (14 μmol/kg and PPG (37.5 mg/kg were intraperitoneally administered 30 min before CS-exposure in the NaHS and PPG groups. 16HBE cells were pretreated with Taurine (10 mM, 5 mmol/L 4-phenylbutyric acid (4-PBA or NaHS (100, 200, and 400 μM for 30 min, and then cells were exposed to 40 μmol/L nicotine for 72 h. ERS markers (GRP94, GRP78 and ERS-mediated apoptosis markers 4-C/EBP homologous protein (CHOP, caspase-3 and caspase-12 were assessed in rat lung tissues and human bronchial epithelial cells. The apoptotic bronchial epithelial cells were detected by Hoechst staining in vitro and TUNEL staining in vivo.Results: In CS exposed rats, peritoneal injection of NaHS significantly inhibited CS induced overexpression ERS-mediated apoptosis markers and upregulation of apoptotic rate in rat lungs, and inhibiting the endogenous H2S production by peritoneal injection of PPG exacerbated these effects. In the nicotine-exposed bronchial epithelial cells, appropriate concentration of NaHS and ERS inhibitors taurine and 4-PBA inhibited

  17. PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation

    Science.gov (United States)

    Qi, Y; Liu, J; Saadat, S; Tian, X; Han, Y; Fong, G-H; Pandolfi, P P; Lee, L Y; Li, S

    2015-01-01

    The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3. PMID:25394489

  18. Water-pipe smoke condensate increases the internalization of Mycobacterium Bovis of type II alveolar epithelial cells (A549).

    Science.gov (United States)

    Mortaz, Esmaeil; Alipoor, Shamila D; Movassaghi, Masoud; Varahram, Mohammad; Ghorbani, Jahangir; Folkerts, Gert; Garssen, Johan; Adcock, Ian M

    2017-04-21

    Tuberculosis (TB) is a major global health problem, and there is an association between tobacco smoke and TB. Water pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally because users consider it as safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be a predisposing factor that enhances the incidence of pulmonary disorders. For example, uncontrolled macropinocytosis in alveolar epithelial cells following exposure to water-pipe smoke may predispose subjects to pulmonary infection. Here, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium Bovis BCG by macropinocytosis in the alveolar epithelial cell line A549. A549 cells were exposed to WPC (4 mg/ml) for 24, 48, 72 and 96 h. Cell viability was studied using the methyl thiazolyldipenyl-tetrazolium bromide (MTT) reduction assay and proliferation by bromodeoxyUridine (BrdU) incorporation. Cells were exposed to FITC-Dextran (1 mg/ml) (as a control) and FITC-BCG (MOI = 10) for 20 min at 37 °C before cells were collected and the uptake of BCG-FITC determined by flow cytometry. Similar experiments were performed at 4 °C as a control. The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (1 μM) was used to assess the mechanism by which WPC enhanced BCG uptake. WPC (4 mg/ml) increased the uptake of BCG-FITC after 72 (1.3 ± 0.1 fold, p WPC also significantly increased the uptake of FITC-Dextran (2.9 ± 0.3 fold, p WPC significantly decreased cell viability after 24 (84 ± 2%, p WPC. Cell proliferation showed a decreasing trend in a time-dependent manner with WPC exposure. WPC exposure increased epithelial cell endocytosis activity and death as well as enhancing their capacity for macropinocytosis. Our in vitro data indicates possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacterium.

  19. Wnt-inducible protein (WISP-1 is a key regulator of alveolar epithelial cell hyperplasia in pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Melanie Königshoff

    2006-12-01

    Full Text Available Fibrotic lung disease is characterized by distorted lung architecture and severe loss of respiratory function secondary to alveolar epithelial cell (AEC hyperplasia, enhanced extracellular matrix (ECM deposition and fibroblast proliferation. Repetitive epithelial injuries with impaired alveolar wound healing and altered AEC gene expression represent a trigger mechanism for development of fibrosis. To reveal gene regulatory networks in lung fibrosis, we compared gene expression profiles of freshly isolated AEC obtained from mice 14 days after saline or bleomycin (BM instillation using whole genome microarray analysis. Several genes of the Wnt signaling pathway, in particular WISP-1, a member of the CCN family, were highly regulated. WISP-1 protein expression was demonstrated in proliferating AEC in BM-treated lungs by immunofluorescence. When analyzing all six CCN family members, WISP-1 was upregulated the most 14 days after BM challenge, as analyzed by qRT-PCR. To elucidate WISP-1 function, cultured primary mouse AEC were stimulated with WISP-1 and demonstrated a 230% increase in proliferation, analyzed by 3H-thymidine incorporation. This was mediated through enhanced phosphorylation, but not expression of protein kinase B (PKB/Akt, as detected by immunoblot. Finally, increased expression of WISP-1 was detected in lung homogenates and isolated AEC from IPF patients, using qRT-PCR. Immunohistochemical analysis of WISP-1 and Ki67 verified the existence of hyperplastic and proliferative AEC expressing WISP-1 in vivo. Our study thus identifies WISP-1 as a novel regulator of AEC injury and repair, and suggests that WISP-1 is a key mediator in pulmonary fibrosis.

  20. Dietary antioxidants protect gut epithelial cells from oxidant-induced apoptosis

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    Bobrowski Paul

    2001-12-01

    Full Text Available Abstract Background The potential of ascorbic acid and two botanical decoctions, green tea and cat's claw, to limit cell death in response to oxidants were evaluated in vitro. Methods Cultured human gastric epithelial cells (AGS or murine small intestinal epithelial cells (IEC-18 were exposed to oxidants – DPPH (3 μM, H2O2 (50 μM, peroxynitrite (300 μM – followed by incubation for 24 hours, with antioxidants (10 μg/ml administered as a 1 hour pretreatment. Cell number (MTT assay and death via apoptosis or necrosis (ELISA, LDH release was determined. The direct interactions between antioxidants and DPPH (100 μM or H2O2 (50 μM were evaluated by spectroscopy. Results The decoctions did not interact with H2O2, but quenched DPPH although less effectively than vitamin C. In contrast, vitamin C was significantly less effective in protecting human gastric epithelial cells (AGS from apoptosis induced by DPPH, peroxynitrite and H2O2 (P 2O2, but green tea was more effective than cat's claw in reducing DPPH-induced apoptosis (P 2O2, and was attenuated both by cat's claw and green tea (P Conclusions These results indicate that dietary antioxidants can limit epithelial cell death in response to oxidant stress. In the case of green tea and cat's claw, the cytoprotective response exceed their inherent ability to interact with the injurious oxidant, suggestive of actions on intracellular pathways regulating cell death.

  1. Differential gene expression profiling of Actinobacillus pleuropneumoniae during induction of primary alveolar macrophage apoptosis in piglets

    NARCIS (Netherlands)

    Wang, Lei; Qin, Wanhai; Ruidong, Zhai; Liu, Shiting; Zhang, Hu; Sun, Changjiang; Feng, Xin; Gu, Jingmin; Du, Chongtao; Han, Wenyu; Langford, P. R.; Lei, Liancheng

    2015-01-01

    Actinobacillus pleura pneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages

  2. Upregulation of pirin expression by chronic cigarette smoking is associated with bronchial epithelial cell apoptosis

    Directory of Open Access Journals (Sweden)

    Zabner Joseph

    2007-02-01

    Full Text Available Abstract Background Cigarette smoke disrupts the protective barrier established by the airway epithelium through direct damage to the epithelial cells, leading to cell death. Since the morphology of the airway epithelium of smokers does not typically demonstrate necrosis, the most likely mechanism for epithelial cell death in response to cigarette smoke is apoptosis. We hypothesized that cigarette smoke directly up-regulates expression of apoptotic genes, which could play a role in airway epithelial apoptosis. Methods Microarray analysis of airway epithelium obtained by bronchoscopy on matched cohorts of 13 phenotypically normal smokers and 9 non-smokers was used to identify specific genes modulated by smoking that were associated with apoptosis. Among the up-regulated apoptotic genes was pirin (3.1-fold, p In vitro studies using human bronchial cells exposed to cigarette smoke extract (CSE and an adenovirus vector encoding the pirin cDNA (AdPirin were performed to test the direct effect of cigarette smoke on pirin expression and the effect of pirin expression on apoptosis. Results Quantitative TaqMan RT-PCR confirmed a 2-fold increase in pirin expression in the airway epithelium of smokers compared to non-smokers (p Overexpression of pirin, using the vector AdPirin, in human bronchial epithelial cells was associated with an increase in the number of apoptotic cells assessed by both TUNEL assay (5-fold, p Conclusion These observations suggest that up-regulation of pirin may represent one mechanism by which cigarette smoke induces apoptosis in the airway epithelium, an observation that has implications for the pathogenesis of cigarette smoke-induced diseases.

  3. Cigarette smoke causes caspase-independent apoptosis of bronchial epithelial cells from asthmatic donors.

    Directory of Open Access Journals (Sweden)

    Fabio Bucchieri

    Full Text Available Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.Since primary bronchial epithelial cells (PBECs from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE; cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF.Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH, but not ascorbic acid (AA, protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.

  4. The endogenous bacteria alter gut epithelial apoptosis and decrease mortality following Pseudomonas aeruginosa pneumonia.

    Science.gov (United States)

    Fox, Amy C; McConnell, Kevin W; Yoseph, Benyam P; Breed, Elise; Liang, Zhe; Clark, Andrew T; O'Donnell, David; Zee-Cheng, Brendan; Jung, Enjae; Dominguez, Jessica A; Dunne, W Michael; Burd, Eileen M; Coopersmith, Craig M

    2012-11-01

    The endogenous bacteria have been hypothesized to play a significant role in the pathophysiology of critical illness, although their role in sepsis is poorly understood. The purpose of this study was to determine how commensal bacteria alter the host response to sepsis. Conventional and germ-free (GF) C57Bl/6 mice were subjected to Pseudomonas aeruginosa pneumonia. All GF mice died within 2 days, whereas 44% of conventional mice survived for 7 days (P = 0.001). Diluting the dose of bacteria 10-fold in GF mice led to similar survival in GF and conventional mice. When animals with similar mortality were assayed for intestinal integrity, GF mice had lower levels of intestinal epithelial apoptosis but similar levels of proliferation and intestinal permeability. Germ-free mice had significantly lower levels of tumor necrosis factor and interleukin 1β in bronchoalveolar lavage fluid compared with conventional mice without changes in systemic cytokine production. Under conventional conditions, sepsis unmasks lymphocyte control of intestinal epithelial apoptosis, because sepsis induces a greater increase in gut apoptosis in Rag-1 mice than in wild-type mice. However, in a separate set of experiments, gut apoptosis was similar between septic GF Rag-1 mice and septic GF wild-type mice. These data demonstrate that the endogenous bacteria play a protective role in mediating mortality from pneumonia-induced sepsis, potentially mediated through altered intestinal apoptosis and the local proinflammatory response. In addition, sepsis-induced lymphocyte-dependent increases in gut epithelial apoptosis appear to be mediated by the endogenous bacteria.

  5. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang [Department of General Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Yang, Hua, E-mail: hwbyang@126.com [Department of General Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing 400037 (China)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. Black-Right-Pointing-Pointer The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. Black-Right-Pointing-Pointer GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. Black-Right-Pointing-Pointer GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  6. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    International Nuclear Information System (INIS)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang; Yang, Hua

    2012-01-01

    Highlights: ► Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. ► The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. ► GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. ► GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  7. Enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

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    Kathrin Mutze

    2015-08-01

    Full Text Available The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI and type II (ATII cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2 and an increase in enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker, exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC, whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

  8. Effects of biodegradable Mg–6Zn alloy extracts on apoptosis of intestinal epithelial cells

    International Nuclear Information System (INIS)

    Wang Zhanhui; Yan Jun; Li Jianan; Zheng Qi; Wang Zhigang; Zhang Xiaonong; Zhang Shaoxiang

    2012-01-01

    Highlights: ► We evaluated the effects of Mg–6Zn alloys on apoptosis of IEC-6 cells. ► The apoptosis was evaluated by investigating the expression of caspase-1 and Bcl-2. ► The IEC-6 cells displayed better cell functions in 60% or 20% extract. ► The conspicuous alkaline environment is disadvantageous to apoptosis of IEC cells. ► The excessive Mg concentration is disadvantageous to apoptosis of IEC-6 cells. - Abstract: In this study, intestinal epithelial cells (IEC)-6 were cultured in different concentration extracts of Mg–6Zn alloys for different time periods. To achieve a total of three concentrations (100%, 60% and 20% concentration), the extracts were serially diluted with Dulbecco's modified Eagle medium High Glucose to observe a dose–response relationship. We studied the indirect effects of Mg–6Zn alloys on IEC-6 cells apoptosis. The apoptosis of IEC-6 cells was measured using flow cytometry. And the apoptosis of IEC-6 cells was evaluated by investigating the expression of caspase-1and Bcl-2 using real-time polymerase chain reaction (PCR) and Western blotting tests. It was found that the levels of apoptosis in IEC-6 cells cultured in 100% Mg–6Zn alloy extracts were significantly higher than those in 60% and 20% extracts; the 100% extract can down-regulate expression of Bcl-2 after culture. The in vitro results indicated that the conspicuous alkaline environment and excessive Mg concentration, even Zn concentration caused by rapid corrosion of Mg–6Zn alloys promote IEC-6 cells apoptosis, although further experiments will be necessary to formally prove our conclusions. Therefore, the adjustment of the degradation rate is needed for using Mg–Zn alloy as a surgical suture material.

  9. Effect of topical vitamin E on ethanol-induced corneal epithelial apoptosis.

    Science.gov (United States)

    Bilgihan, Kamil; Konuk, Onur; Hondur, Ahmet; Akyürek, Nalan; Ozogul, Candan; Hasanreisoglu, Berati

    2005-01-01

    Ethanol is used to loosen the corneal epithelium before photoablation in laser subepithelial keratomileusis (LASEK). In this study, the apoptotic index of corneal epithelium after ethanol exposure and the effects of topical vitamin E were evaluated. The study was performed on 28 rabbit eyes in four groups. Group 1 comprised the controls. In group 2, 20% ethanol was applied topically for 20 seconds. In group 3, topical vitamin E was applied following 20% ethanol application. In group 4, only topical vitamin E was applied. Apoptosis was evaluated with TUNEL assay and transmission electron microscopy. Epithelial apoptosis was detected in all specimens in group 2. No apoptosis was detected in other groups except for one eye in group 1. The apoptotic index in group 2 was statistically higher than other groups (P < .001).

  10. Propofol inhibits LPS-induced apoptosis in lung epithelial cell line, BEAS-2B.

    Science.gov (United States)

    Lv, Xiang; Zhou, Xuhui; Yan, Jia; Jiang, Jue; Jiang, Hong

    2017-03-01

    Lipopolysaccharide (LPS) plays an important role in lung endothelial apoptosis which is crucial for lung fibrogenesis in ARDS progression. Reactive oxygen species (ROS) has been reported to be involved in LPS-induced lung epithelial cell apoptosis. Propofol is a commonly used intravenous anesthetic agent in clinic and it could attenuate LPS-induced epithelial cells oxidation and apoptosis. However, the mechanisms are still obscure. In this study, we examined whether and how propofol attenuates LPS-induced oxidation and apoptosis in BEAS-2B cells. Compared with control group, LPS up-regulated Pin-1, phosphatase A2 (PP2A) expression, induced p66 Shc -Ser 36 phosphorylation, and facilitated p66 Shc mitochondrial translocation, thus leading to superoxide anion (O 2 - ) generation, mitochondrial cytochrome c release, active caspase 3 over-expression and cell viability inhibition. Importantly, propofol was shown to down-regulate LPS-induced PP2A expression, limit p66 Shc mitochondrial translocation, decrease O 2 - generation, inhibit mitochondrial cytochrome c release, reduce active caspase 3 expression, and recover cells viability, while propofol had no effects on LPS-induced Pin-1 expression and p66 Shc -Ser 36 phosphorylation. Moreover, the protective effects of propofol on LPS-induced BEAS-2B cells apoptosis were similar to that of calyculin A, which is an inhibitor of PP2A. We also found that FTY720, which is an activator of PP2A, can effectively reverse the protective function of propofol. Our data illustrated that propofol could alleviate LPS-induced BEAS-2B cells oxidation and apoptosis through down-regulating PP2A expression, limiting p66 Shc -Ser 36 dephosphorylation and p66 Shc mitochondrial translocation, decreasing O 2 - generation, mitochondrial cytochrome c release, activating caspase 3 expression. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II

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    Prasse Antje

    2005-07-01

    Full Text Available Abstract The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2 and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 in TNF-α-, IFN-γ-, and IL-1β-stimulated human alveolar epithelial cells type II (AEC-II. AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-γ had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-γ, alone or in combination with IL-1β and TNF-α resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-γ>>IL-1β ≥ TNF-α. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.

  12. Bile acids induce activation of alveolar epithelial cells and lung fibroblasts through farnesoid X receptor-dependent and independent pathways.

    Science.gov (United States)

    Chen, Bi; Cai, Hou-Rong; Xue, Shan; You, Wen-Jie; Liu, Bin; Jiang, Han-Dong

    2016-08-01

    The roles of bile acid microaspiration and bile acid-activated farnesoid X receptor (FXR) in the pathogenesis of idiopathic pulmonary fibrosis (IPF) remain unclear. We hypothesized that bile acids activate alveolar epithelial cells (AECs) and lung fibroblasts, which may be regulated by FXR activation. Human AECs and normal or IPF-derived lung fibroblast cells were incubated with the three major bile acids: lithocholic acid (LCA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The AECs injury indices, epithelial-mesenchymal transition (EMT) and lung fibroblast activation were evaluated. FXR expression in IPF lungs and the roles of FXR and FXR-independent pathways in bile acid-induced profibrotic effects were also investigated. LCA, DCA and CDCA reduced cell viability and increased intracellular reactive oxygen species (ROS) production in A549 cells. They all induced EMT, as shown by enhanced α-SMA and vimentin and decreased E-cadherin levels. LCA directly induced differentiation of lung fibroblasts to myofibroblasts. All three bile acids promoted cellular migration but not proliferation of lung fibroblasts. FXR expression was upregulated in IPF lungs, and inhibition of FXR restrained the bile acid-induced EMT and lung fibroblast activation. Differentiation and proliferation were enhanced in lung fibroblasts exposed to conditioned medium from bile acid-stimulated A549 cells, which contained increased levels of profibrotic factors. TGF-β/Smad3 signaling was also involved in the bile acid-induced EMT and lung fibroblast differentiation. Bile acid microaspiration may promote the development of pulmonary fibrosis by inducing activation of AECs and lung fibroblasts via FXR-dependent and independent pathways. © 2016 Asian Pacific Society of Respirology.

  13. Environmental particulate (PM2.5 augments stiffness-induced alveolar epithelial cell mechanoactivation of transforming growth factor beta.

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    Marilyn M Dysart

    Full Text Available Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF, chronic obstructive pulmonary disease (COPD, and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor--beta (TGFβ signaling, the alveolar type II (ATII epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5 will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung

  14. Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

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    Jeanette Wagener

    Full Text Available C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.

  15. Acidic mammalian chitinase regulates epithelial cell apoptosis via a chitinolytic-independent mechanism.

    Science.gov (United States)

    Hartl, Dominik; He, Chuan Hua; Koller, Babara; Da Silva, Carla A; Kobayashi, Yasushi; Lee, Chun Geun; Flavell, Richard A; Elias, Jack A

    2009-04-15

    Acidic mammalian chitinase (AMCase) is produced during and plays an important role in the pathogenesis of Th2-mediated diseases and antiparasite responses. However, the effector responses of AMCase in these settings have not been adequately defined and the relationship(s) between its chitinolytic and other biologic properties have not been investigated. In these studies, we demonstrate that AMCase protects airway epithelial cells from Fas ligand- and growth factor withdrawal-induced apoptosis. This cytoprotection was associated with Akt phosphorylation and abrogated when the PI3K/Akt pathway was inhibited. Comparable cytoprotection was also seen in experiments comparing wild-type AMCase and mutant AMCase that lacked chitinolytic activity. Importantly, the apoptosis-inhibiting effect of enzymatically active and inactive AMCase was abrogated by treatment with allosamidin. These studies demonstrate that secreted AMCase feeds back in an autocrine and/or paracrine manner to protect pulmonary epithelial cells from growth factor withdrawal- and Fas ligand-induced apoptosis. They also demonstrate that the cytoprotection is mediated via a PI3K/Akt-dependent and allosamidin-sensitive pathway that is independent of the chitinolytic activity of this chitinase.

  16. Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells

    Science.gov (United States)

    Wiese, Katrin E; Haikala, Heidi M; von Eyss, Björn; Wolf, Elmar; Esnault, Cyril; Rosenwald, Andreas; Treisman, Richard; Klefström, Juha; Eilers, Martin

    2015-01-01

    Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells. PMID:25896507

  17. Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells.

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    Congcong Chen

    Full Text Available Androgen receptor (AR signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS, TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2 were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1 were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins

  18. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis.

    Science.gov (United States)

    Perrone, Erin E; Jung, Enjae; Breed, Elise; Dominguez, Jessica A; Liang, Zhe; Clark, Andrew T; Dunne, W Michael; Burd, Eileen M; Coopersmith, Craig M

    2012-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

  19. Overexpression of sICAM-1 in the Alveolar Epithelial Space Results in an Exaggerated Inflammatory Response and Early Death in Gram Negative Pneumonia

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    Curtis Jeffery L

    2011-01-01

    Full Text Available Abstract Background A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1 plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs, we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1 is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection. Methods Using the surfactant protein C (SPC promoter, we developed a transgenic mouse (SPC-sICAM-1 that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae. Results SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups. Conclusions We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.

  20. Characterisation of cellular adhesion reinforcement by multiple bond force spectroscopy in alveolar epithelial cells.

    Science.gov (United States)

    Nguyen, Ngoc-Minh; Angely, Christelle; Andre Dias, Sofia; Planus, Emmanuelle; Filoche, Marcel; Pelle, Gabriel; Louis, Bruno; Isabey, Daniel

    2017-07-01

    Integrin-mediated adhesion is a key process by which cells physically connect with their environment, and express sensitivity and adaptation through mechanotransduction. A critical step of cell adhesion is the formation of the first bonds which individually generate weak contacts (∼tens pN) but can sustain thousand times higher forces (∼tens nN) when associated. We propose an experimental validation by multiple bond force spectroscopy (MFS) of a stochastic model predicting adhesion reinforcement permitted by non-cooperative, multiple bonds on which force is homogeneously distributed (called parallel bond configuration). To do so, spherical probes (diameter: 6.6 μm), specifically coated by RGD-peptide to bind integrins, are used to statically indent and homogenously stretch the multiple bonds created for short contact times (2 s) between the bead and the surface of epithelial cells (A549). Using different separation speeds (v = 2, 5, 10 μm/s) and measuring cellular Young's modulus as well as the local stiffness preceding local rupture events, we obtain cell-by-cell the effective loading rates both at the global cell level and at the local level of individual constitutive bonds. Local rupture forces are in the range: f*=60-115 pN , whereas global rupture (detachment) forces reach F*=0.8-1.7 nN . Global and local rupture forces both exhibit linear dependencies with the effective loading rate, the slopes of these two linear relationships providing an estimate of the number of independent integrin bonds constituting the tested multiple bond structure (∼12). The MFS method enables to validate the reinforcement of integrin-mediated adhesion induced by the multiple bond configuration in which force is homogeneously distributed amongst parallel bonds. Local rupture events observed in the course of a spectroscopy manoeuver (MFS) lead to rupture force values considered in the literature as single-integrin bonds. Adhesion reinforcement permitted by the parallel

  1. Comparison of plasma, epithelial lining fluid, and alveolar macrophage concentrations of solithromycin (CEM-101) in healthy adult subjects.

    Science.gov (United States)

    Rodvold, Keith A; Gotfried, Mark H; Still, J Gordon; Clark, Kay; Fernandes, Prabhavathi

    2012-10-01

    The steady-state concentrations of solithromycin in plasma were compared with concomitant concentrations in epithelial lining fluid (ELF) and alveolar macrophages (AM) obtained from intrapulmonary samples during bronchoscopy and bronchoalveolar lavage (BAL) in 30 healthy adult subjects. Subjects received oral solithromycin at 400 mg once daily for five consecutive days. Bronchoscopy and BAL were carried out once in each subject at either 3, 6, 9, 12, or 24 h after the last administered dose of solithromycin. Drug concentrations in plasma, ELF, and AM were assayed by a high-performance liquid chromatography-tandem mass spectrometry method. Solithromycin was concentrated extensively in ELF (range of mean [± standard deviation] concentrations, 1.02 ± 0.83 to 7.58 ± 6.69 mg/liter) and AM (25.9 ± 20.3 to 101.7 ± 52.6 mg/liter) in comparison with simultaneous plasma concentrations (0.086 ± 0.070 to 0.730 ± 0.692 mg/liter). The values for the area under the concentration-time curve from 0 to 24 h (AUC(0-24) values) based on mean and median ELF concentrations were 80.3 and 63.2 mg · h/liter, respectively. The ratio of ELF to plasma concentrations based on the mean and median AUC(0-24) values were 10.3 and 10.0, respectively. The AUC(0-24) values based on mean and median concentrations in AM were 1,498 and 1,282 mg · h/L, respectively. The ratio of AM to plasma concentrations based on the mean and median AUC(0-24) values were 193 and 202, respectively. Once-daily oral dosing of solithromycin at 400 mg produced steady-state concentrations that were significantly (P solithromycin administration.

  2. The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems.

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    Olga D Chuquimia

    Full Text Available Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes were able to induce expression of chemokines (IP-10 and RANTES by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.

  3. Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

    Science.gov (United States)

    Nishi, Hiroyuki; Maeda, Noriko; Izumi, Shunsuke; Higa-Nakamine, Sayomi; Toku, Seikichi; Kakinohana, Manabu; Sugahara, Kazuhiro; Yamamoto, Hideyuki

    2015-02-05

    In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Ethanol alters alveolar fluid balance via Nadph oxidase (NOX signaling to epithelial sodium channels (ENaC in the lung.

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    Charles A Downs

    Full Text Available Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS. However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung.

  5. Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

    Science.gov (United States)

    Yang, Deng-Liang; Xu, Jia-Wen; Zhu, Jian-Guo; Zhang, Yi-Lin; Xu, Jian-Bang; Sun, Qing; Cao, Xiao-Nian; Zuo, Wu-Lin; Xu, Ruo-Shui; Huang, Jie-Hong; Jiang, Fu-Neng; Zhuo, Yang-Jia; Xiao, Bai-Quan; Liu, Yun-Zhong; Yuan, Dong-Bo; Sun, Zhao-Lin; He, Hui-Chan; Lun, Zhao-Rong; Zhong, Wei-De; Zhou, Wen-Liang

    2017-06-03

    Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17β-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca 2+ release from the endoplasmic reticulum, increased the mitochondrial Ca 2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca 2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP 3 ) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    Science.gov (United States)

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  7. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

    Science.gov (United States)

    Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

    2015-02-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The

  8. Apoptosis in the normal and inflamed airway epithelium: role of zinc in epithelial protection and procaspase-3 regulation.

    Science.gov (United States)

    Truong-Tran, Ai Q; Grosser, Dion; Ruffin, Richard E; Murgia, Chiara; Zalewski, Peter D

    2003-10-15

    The epithelium lining the airways is a physical barrier as well as a regulator of physiological and pathological events in the respiratory system. Damage to the epithelium by oxidants released from inflammatory cells is a critical factor in the pathogenesis of airway inflammatory diseases such as bronchial asthma. In these diseases, excessive apoptosis may be a likely mechanism responsible for damage to, and sloughing, of airway epithelial cells. Factors that increase the airway epithelium's resilience to apoptosis are likely to lessen the severity of this disease. One such factor is the dietary metal zinc. A special role for labile intracellular pools of zinc as anti-apoptotic agents in the regulation of the caspases, has emerged over the past two decades. This review focuses on caspase-inhibitory functions of zinc in airway epithelial cells, apparent abnormalities of zinc homeostasis in asthmatics and studies from the authors' laboratory which showed that zinc was strategically localized in the apical cytoplasm of airway epithelium to control caspase-3 activated apoptosis. These findings are discussed in the context of recent data from a murine model of allergic asthma, showing that loss of airway epithelial zinc was accompanied by changes in levels of both procaspase-3 and active caspase-3 and that nutritional zinc deprivation further increased airway epithelial apoptosis. We hypothesize that zinc has a protective role for the airway epithelium against oxyradicals and other noxious agents, with important implications for asthma and other inflammatory diseases where the epithelial barrier is vulnerable and compromised.

  9. A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wu Xiaoyan; Chen Peili [Department of Medicine, University of Chicago, Chicago, Illinois (United States); Sonis, Stephen T. [Division of Oral Medicine, Brigham and Women' s Hospital, Boston, Massachusetts (United States); Biomodels, Watertown, Massachusetts (United States); Lingen, Mark W. [Department of Pathology, University of Chicago, Chicago, Illinois (United States); Berger, Ann [NephRx Corporation, Kalamazoo, Michigan (United States); Toback, F. Gary, E-mail: gtoback@medicine.bsd.uchicago.edu [Department of Medicine, University of Chicago, Chicago, Illinois (United States)

    2012-07-01

    Purpose: No effective agents currently exist to treat oral mucositis (OM) in patients receiving chemoradiation for the treatment of head-and-neck cancer. We identified a novel 21-amino acid peptide derived from antrum mucosal protein-18 that is cytoprotective, mitogenic, and motogenic in tissue culture and animal models of gastrointestinal epithelial cell injury. We examined whether administration of antrum mucosal protein peptide (AMP-p) could protect against and/or speed recovery from OM. Methods and Materials: OM was induced in established hamster models by a single dose of radiation, fractionated radiation, or fractionated radiation together with cisplatin to simulate conventional treatments of head-and-neck cancer. Results: Daily subcutaneous administration of AMP-p reduced the occurrence of ulceration and accelerated mucosal recovery in all three models. A delay in the onset of erythema after irradiation was observed, suggesting that a protective effect exists even before injury to mucosal epithelial cells occurs. To test this hypothesis, the effects of AMP-p on tumor necrosis factor-{alpha}-induced apoptosis were studied in an endothelial cell line (human dermal microvascular endothelial cells) as well as an epithelial cell line (human adult low-calcium, high-temperature keratinocytes; HaCaT) used to model the oral mucosa. AMP-p treatment, either before or after cell monolayers were exposed to tumor necrosis factor-{alpha}, protected against development of apoptosis in both cell types when assessed by annexin V and propidium iodide staining followed by flow cytometry or ligase-mediated polymerase chain reaction. Conclusions: These observations suggest that the ability of AMP-p to attenuate radiation-induced OM could be attributable, at least in part, to its antiapoptotic activity.

  10. Inhibition of UV-B induced apoptosis in corneal epithelial cells by potassium channel modulators.

    Science.gov (United States)

    Ubels, John L; Schotanus, Mark P; Bardolph, Susan L; Haarsma, Loren D; Koetje, Leah R; Louters, Julienne R

    2010-02-01

    The goal of this study was to determine whether prevention of K(+) loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150-200 mJ/cm(2) UV-B demonstrated induction of apoptosis 6 h after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K(+). If this protection is due to a reduction of UV-induced K(+) loss then K(+) channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm(2) was significantly reduced when the cells were incubated in 0.3 microM BDS-I or 0.05-1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1-0.3 microM BDS-I and 0.1-1 mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 microM BDS-I and 0.01-0.05 mM quinidine. Patch-clamp recording showed activation of K(+) channels after exposure to UV-B and a decrease in outward K(+) current was observed following application of BDS-I. Quinidine did not block K(+) currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K(+) channels. The effect of the K(+) channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K(+) efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K(+)] in tears may protect the corneal epithelium from effects of ambient UV-B. Copyright 2009 Elsevier Ltd. All rights reserved.

  11. Inhibition of UV-B Induced Apoptosis in Corneal Epithelial Cells by Potassium Channel Modulators

    Science.gov (United States)

    Ubels, John L.; Schotanus, Mark P.; Bardolph, Susan L.; Haarsma, Loren D.; Koetje, Leah R.; Louters, Julienne R.

    2009-01-01

    The goal of this study was to determine whether prevention of K+ loss can protect human corneal-limbal epithelial (HCLE) cells from UV-B induced apoptosis. Immunostaining for activated caspase-3 of HCLE cells exposed to 150 – 200 mJ/cm2 UV-B demonstrated induction of apoptosis 6 hrs after exposure. The number of apoptotic cells was decreased by incubation in medium with 25 or 100 mM K+. If this protection is due to a reduction of UV induced K+ loss then K+ channel blockers should also protect HCLE cells from UV-B. Caspase-8 activity induced by exposure to UV-B at 150 mJ/cm2 was significantly reduced when the cells were incubated in 0.3 µM BDS-I or 0.05–1 mM quinidine. Caspase-3 was also activated by UV-B and a reduction in activity was observed after incubation in 0.1–0.3 µM BDS-I and 0.1–1mM quinidine. Induction of DNA fragmentation, as measured by the TUNEL assay, was decreased by treatment with 0.3 µM BDS-I and 0.01–0.05 mM quinidine. Patch-clamp recording showed activation of K+ channels after exposure to UV-B and a decrease in outward K+ current was observed following application of BDS-I. Quinidine did not block K+ currents in HCLE cells, suggesting that the protective effect of quinidine occurs by a mechanism other than via K+ channels. The effect of the K+ channel blocker BDS-1 on HCLE cells exposed to UV-B confirms that preventing K+ efflux protects corneal epithelial cells from apoptosis. This suggests the elevated [K+] in tears may protect the corneal epithelium from effects of ambient UV-B. PMID:19874821

  12. Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells

    Directory of Open Access Journals (Sweden)

    Crandall Edward D

    2005-04-01

    Full Text Available Abstract Background Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. Methods Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. Results This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. Conclusion This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease.

  13. Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis.

    Science.gov (United States)

    Zou, Tongtong; Rao, Jaladanki N; Liu, Lan; Xiao, Lan; Cui, Yu-Hong; Jiang, Zhengran; Ouyang, Miao; Donahue, James M; Wang, Jian-Ying

    2012-07-01

    Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

  14. Effect of vital dyes on retinal pigmented epithelial cell viability and apoptosis: implications for chromovitrectomy

    Science.gov (United States)

    Penha, Fernando M; Pons, Marianne; Costa, Elaine Fiod; Rodrigues, Eduardo B.; Maia, Mauricio; Marin-Castaño, Maria E; Farah, Michel Eid

    2013-01-01

    Purpose To investigate in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. Methods ARPE-19 cells were exposed to brilliant blue-BriB, evans blue-EB, bromophenol blue-BroB, indocyanine green-ICG, infracyanine green-IfCG, light green-LG, fast green-FG, indigo carmine-IC and congo red-CR. BSS was used as the control. Five different concentrations and two times were tested. Cell viability was determined by MTS assay and apoptosis by Bax expression on western blot. Results All dyes significantly reduced cell viability after 3 minutes of exposure at all concentrations (pdyes exposure, except BriB; ICG had the highest Bax expression (pdye was BriB followed by LG, IfCG, FG, CR, IC, BroB, RB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BriB was the only dye that did not induce apoptosis in ARPE-19 cells. PMID:24022718

  15. Runx3 is a key modulator during the epithelial-mesenchymal transition of alveolar type II cells in animal models of BPD.

    Science.gov (United States)

    Yang, Haiping; Fu, Jianhua; Yao, Li; Hou, Ana; Xue, Xindong

    2017-11-01

    Bronchopulmonary dysplasia (BPD) is a major challenge for premature infants; however, the underlying mechanisms remain unclear. We previously reported that epithelial-mesenchymal transition (EMT) in alveolar type II (AT2) epithelial cells influences the normal alveolar development process. In this study, we wished to examine whether Runx3 is an important factor for BPD by regulating EMT in AT2 cells. In vivo, animal models of BPD were established by placing newborn rats in hyperoxia tanks. Lung tissue and isolated AT2 cells were collected on different days following exposure to oxygen. The pathological changes in lung tissue, alveolar development and Runx3 expression were then investigated. In vitro, RLE-6TN cells were divided into 5 groups as follows: the cont-rol, Runx3, siRunx3, transforming growth factor-β1 (TGF-β1) and Runx3 + TGF-β1 groups, and the biomarkers of EMT were investigated. In the newborn rat model of BPD, Runx3 protein and mRNA levels in both lung tissue and BPD-derived AT2 cells were significantly lower than those in the control group. The correlation between Runx3 protein expression and pulmonary development indicators was analyzed; Runx3 expression positively correlated with the radial alveolar count (RAC) and the percentage of smooth muscle actin-positive secondary septa, but negatively correlated with alveolar wall thickness. EMT was observed in the RLE-6TN cells in which the Runx3 gene was knocked down and follwoing TGF-β1‑induced EMT stimulation; however, TGF-β1 failed to induce EMT in the RLE-6TN cells overexpressing Runx3. On the whole, our data indicte that low Runx3 levels may promote EMT, while high Runx3 levels inhibit TGF-β1-induced EMT. Therefore, we predict that low levels of Runx3 in BPD lung tissue may promote EMT in AT2 cells, thus affecting alveolar development.

  16. Lens epithelial cell apoptosis and intracellular Ca2+ increase in the presence of xanthurenic acid

    Directory of Open Access Journals (Sweden)

    Richter Christoph

    2002-04-01

    Full Text Available Abstract Background Xanthurenic acid is an endogenous product of tryptophan degradation by indoleamine 2,3-dioxygenase (IDO. We have previously reported that IDO is present in mammalian lenses, and xanthurenic acid is accumulated in the lenses with aging. Here, we studied the involvement of xanthurenic acid in the human lens epithelial cell physiology. Methods Human lens epithelial cells primary cultures were used. Control cells, and cells in the presence of xanthurenic acid grow in the dark. Western blot analysis and immunofluorescence studies were performed. Results In the presence of xanthurenic acid human lens epithelial cells undergo apoptosis-like cell death. In the control cells gelsolin stained the perinuclear region, whereas in the presence of 10 μM xanthurenic acid gelsolin is translocated to the cytoskeleton, but does not lead to cytoskeleton breakdown. In the same condition caspase-3 activation, and DNA fragmentation was observed. At low (5 to 10 μM of xanthurenic acid concentration, the elongation of the cytoskeleton was associated with migration of mitochondria and cytochrome c release. At higher concentrations xanthurenic acid (20 μM and 40 μM damaged mitochondria were observed in the perinuclear region, and nuclear DNA cleavage was observed. We observed an induction of calpain Lp 82 and an increase of free Ca2+ in the cells in a xanthurenic acid concentration-dependent manner. Conclusions The results show that xanthurenic acid accumulation in human lens epithelial cells disturbs the normal cell physiology and leads to a cascade of pathological events. Xanthurenic acid induces calpain Lp82 and caspases in the cells growing in the dark and can be involved in senile cataract development.

  17. A ROS-dependent and Caspase-3-mediated apoptosis in sheep bronchial epithelial cells in response to Mycoplasma Ovipneumoniae infections.

    Science.gov (United States)

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

    2017-05-01

    Mycoplasma Ovipneumoniae (M. ovipneumoniae) is a primary etiological agent of enzootic pneumonia in sheep and goats. It can enter and colonize ovine respiratory epithelial cells to establish an infection, which leads a serious cell death of epithelial cells. However, the nature of the interaction between pathogen of M. ovipneumoniae and host cells in the cell injury is currently not well understood. In this study, we investigated the epithelial cell apoptosis caused by an infection of M. ovipneumoniae in sheep primary air-liquid interface (ALI) epithelial cultures. The results showed that M. ovipneumoniae could specifically bind to ciliated cells at early stage of infection. Flow cytometric analysis demonstrated that an infection of M. ovipneumoniae induced a time-dependent cell apoptotic cell death, accompanied with an increased production of extracellular nitric oxide (NO), intracellular reactive oxygen species (ROS) production and activation of caspase-3 signaling in sheep bronchial epithelial cells. The induced cell apoptosis was further confirmed by a transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Interestingly, the M. ovipneumoniae-induced apoptosis and activation of caspase-3 were correlated with the production of ROS but not NO. Mechanistically, M. ovipneumoniae-induced cell apoptosis was mediated by a mechanism by increasing the expression of phosphorylation of p38 and pro-apoptotic proteins, and activating caspase-3, caspase-8 and poly ADP-ribose polymerase (PARP) cleavage. These results suggest a ROS-dependent and caspase-3-mediated cell apoptosis in sheep bronchial epithelial cells in response to M. ovipneumoniae infections. Copyright © 2017. Published by Elsevier B.V.

  18. Pneumocystis carinii major surface glycoprotein induces interleukin-8 and monocyte chemoattractant protein-1 release from a human alveolar epithelial cell line

    DEFF Research Database (Denmark)

    Benfield, T L; Lundgren, Bettina; Shelhamer, J H

    1999-01-01

    (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from an alveolar epithelial cell line (A549). RESULTS: Incubation of A549 cells with MSG in concentrations from 0.4 to 10 microg mL-1 for 24 h caused dose-dependent increases in IL-8 release (3.4-fold above control, P ..., suggesting that MSG stimulates A549 cells in part through carbohydrate moieties. Dexamethasone significantly inhibited MSG-induced IL-8 release in concentrations of 10-6-10-8 mol L-1 compared with control experiments (P

  19. Cadmium Induces p53-Dependent Apoptosis in Human Prostate Epithelial Cells

    Science.gov (United States)

    Aimola, Pierpaolo; Carmignani, Marco; Volpe, Anna Rita; Di Benedetto, Altomare; Claudio, Luigi; Waalkes, Michael P.; van Bokhoven, Adrie; Tokar, Erik J.; Claudio, Pier Paolo

    2012-01-01

    Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl2 and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl2 concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis. PMID:22448262

  20. Characterization of snail1 and pten transcriptional regulation by snail1: New insights into epithelial-to-mesenchymal transition and cell resistance to apoptosis

    OpenAIRE

    Escrivà Izquierdo, María

    2008-01-01

    The product of the snail1 gene (SNAIL1) is a transcriptional repressor required for triggering the epithelial-to-mesenchymal transition (EMT). SNAIL1 transcription is induced when epithelial cells are forced to acquire a mesenchymal phenotype. Furthermore, ectopic expression of snail1 in epithelial cells promotes resistance to apoptosis. In this study, we demonstrate that this resistance to ã radiation-induced apoptosis caused by Snail1 is associated with the transcriptional inhibition of PTE...

  1. Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy.

    Directory of Open Access Journals (Sweden)

    Katalin Susztak

    2005-02-01

    Full Text Available Diabetic nephropathy (DNP is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis.Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP.

  2. Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

    Science.gov (United States)

    Angely, Christelle; Nguyen, Ngoc-Minh; Andre Dias, Sofia; Planus, Emmanuelle; Pelle, Gabriel; Louis, Bruno; Filoche, Marcel; Chenal, Alexandre; Ladant, Daniel; Isabey, Daniel

    2017-08-01

    The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure 95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in

  3. Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to PulmonaryAspergillus fumigatusInfection.

    Science.gov (United States)

    Seddigh, Pegah; Bracht, Thilo; Molinier-Frenkel, Válerie; Castellano, Flavia; Kniemeyer, Olaf; Schuster, Marc; Weski, Juliane; Hasenberg, Anja; Kraus, Andreas; Poschet, Gernot; Hager, Thomas; Theegarten, Dirk; Opitz, Christiane A; Brakhage, Axel A; Sitek, Barbara; Hasenberg, Mike; Gunzer, Matthias

    2017-12-01

    The ubiquitous mold Aspergillus fumigatus threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. A. fumigatus conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during A. fumigatus infection via in vitro experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing ex vivo analyses of the cells, which encountered the mold in vivo By label-free proteome analysis of AEC II isolated from mice 24h after A. fumigatus or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA p value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with A. fumigatus This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA p value 2.91 -10 ). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, A. fumigatus infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available via Proteome

  4. Mesenchymal Stem Cell Conditioned Medium Promotes Proliferation and Migration of Alveolar Epithelial Cells under Septic Conditions In Vitro via the JNK-P38 Signaling Pathway

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    Jie Chen

    2015-11-01

    Full Text Available Background/Aims: Mesenchymal stem cell (MSC based therapies may be useful for treating acute respiratory distress syndrome (ARDS, but the underlying mechanisms are incompletely understood. We investigated the impact of human umbilical cord Wharton's jelly-derived MSC (hUC-MSC secreted factors on alveolar epithelial cells under septic conditions and determined the relevant intracellular signaling pathways. Methods: Human alveolar epithelial cells (AEC and primary human small airway epithelial cells (SAEC were subjected to lipopolysaccharide (LPS with or without the presence of hUC-MSC-conditioned medium (CM. Proliferation and migration of AEC and SAEC were determined via an MTT assay, a wound healing assay and a transwell migration assay (only for AEC. Protein phosphorylation was determined by western blot and the experiments were repeated in presence of small-molecule inhibitors. The hMSC-secretory proteins were identified by LC-MS/MS mass spectrometry. Results: MSC-CM enhanced proliferation and migration. Activation of JNK and P38, but not ERK, was required for the proliferation and migration of AEC and SAEC. Pretreatment of AEC or SAEC with SP600125, an inhibitor of JNK1 or SB200358, an inhibitor of P38, significantly reduced cell proliferation and migration. An array of proteins including TGF-beta receptor type-1, TGF-beta receptor type-2, Ras-related C3 botulinum toxin substrate 1 and Ras-related C3 botulinum toxin substrate 2 which influencing the proliferation and migration of AEC and SAEC were detected in MSC-CM. Conclusion: Our data suggest MSC promote epithelial cell repair through releasing a repertoire of paracrine factors via activation of JNK and P38 MAPK.

  5. Apoptosis in oral epithelial dysplastic lesions and oral squamous cell carcinoma: A prognostic marker.

    Science.gov (United States)

    Nambiar, Shwetha; Hegde, Veda

    2016-01-01

    Apoptotic index (AI) using light microscopy as an indirect measure to assess the significance of apoptosis as a proliferative marker in dysplastic lesions and malignant epithelial lesions of the oral cavity. (1) To quantify the apoptotic bodies/cells in oral epithelial dysplastic (OED) lesions and oral squamous cell carcinoma (OSCC). (2) To measure AI in OED and OSCC. (3) To compare AI in OED and OSCC. The proposed laboratory-based retrospective study involved the use of hematoxylin and eosin (H and E)-stained slides of previously diagnosed OED lesions and OSCC from institutional archives. This study constituted 50 cases, each of H and E-stained slides of previously diagnosed cases of OED and OSCC. AI was calculated as the number of apoptotic bodies/cells expressed as a percentage of the total number of nonapoptotic tumor/dysplastic cells counted in each case. Nonparametric tests such as Kruskal-Wallis test and Mann-Whitney test were used. There was a statistically significant increase in AI from OED to OSCC (P = 0.000). Further studies need to be undertaken to detect and understand the apoptotic mechanisms in the progression from OED to OSCC.

  6. Apoptosis in oral epithelial dysplastic lesions and oral squamous cell carcinoma: A prognostic marker

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    Shwetha Nambiar

    2016-01-01

    Full Text Available Background: Apoptotic index (AI using light microscopy as an indirect measure to assess the significance of apoptosis as a proliferative marker in dysplastic lesions and malignant epithelial lesions of the oral cavity. Aims: (1 To quantify the apoptotic bodies/cells in oral epithelial dysplastic (OED lesions and oral squamous cell carcinoma (OSCC. (2 To measure AI in OED and OSCC. (3 To compare AI in OED and OSCC. Settings and Design: The proposed laboratory-based retrospective study involved the use of hematoxylin and eosin (H and E-stained slides of previously diagnosed OED lesions and OSCC from institutional archives. Materials and Methods: This study constituted 50 cases, each of H and E-stained slides of previously diagnosed cases of OED and OSCC. AI was calculated as the number of apoptotic bodies/cells expressed as a percentage of the total number of nonapoptotic tumor/dysplastic cells counted in each case. Statistical Analysis Used: Nonparametric tests such as Kruskal–Wallis test and Mann–Whitney test were used. Results: There was a statistically significant increase in AI from OED to OSCC (P = 0.000. Conclusions: Further studies need to be undertaken to detect and understand the apoptotic mechanisms in the progression from OED to OSCC.

  7. Syndecan-1 mediates the coupling of positively charged submicrometer amorphous silica particles with actin filaments across the alveolar epithelial cell membrane.

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    Orr, Galya; Panther, David J; Cassens, Kaylyn J; Phillips, Jaclyn L; Tarasevich, Barbara J; Pounds, Joel G

    2009-04-15

    The cellular interactions and pathways of engineered submicro- and nano-scale particles dictate the cellular response and ultimately determine the level of toxicity or biocompatibility of the particles. Positive surface charge can increase particle internalization, and in some cases can also increase particle toxicity, but the underlying mechanisms are largely unknown. Here we identify the cellular interaction and pathway of positively charged submicrometer synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. Using time lapse fluorescence imaging in living cells and other quantitative imaging approaches, it is found that heparan sulfate proteoglycans play a critical role in the attachment and internalization of the particles in alveolar type II epithelial cell line (C10), a potential target cell type bearing apical microvilli. Specifically, the transmembrane heparan sulfate proteoglycan, syndecan-1, is found to mediate the initial interactions of the particles at the cell surface, their coupling with actin filaments across the cell membrane, and their subsequent internalization via macropinocytosis. The observed interaction of syndecan molecules with the particle prior to their engagement with actin filaments suggests that the particles initiate their own internalization by facilitating the clustering of the molecules, which is required for the actin coupling and subsequent internalization of syndecan. Our observations identify a new role for syndecan-1 in mediating the cellular interactions and fate of positively charged submicrometer amorphous silica particles in the alveolar type II epithelial cell, a target cell for inhaled particles.

  8. Syndecan-1 mediates the coupling of positively charged submicrometer amorphous silica particles with actin filaments across the alveolar epithelial cell membrane

    International Nuclear Information System (INIS)

    Orr, Galya; Panther, David J.; Cassens, Kaylyn J.; Phillips, Jaclyn L.; Tarasevich, Barbara J.; Pounds, Joel G.

    2009-01-01

    The cellular interactions and pathways of engineered submicro- and nano-scale particles dictate the cellular response and ultimately determine the level of toxicity or biocompatibility of the particles. Positive surface charge can increase particle internalization, and in some cases can also increase particle toxicity, but the underlying mechanisms are largely unknown. Here we identify the cellular interaction and pathway of positively charged submicrometer synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. Using time lapse fluorescence imaging in living cells and other quantitative imaging approaches, it is found that heparan sulfate proteoglycans play a critical role in the attachment and internalization of the particles in alveolar type II epithelial cell line (C10), a potential target cell type bearing apical microvilli. Specifically, the transmembrane heparan sulfate proteoglycan, syndecan-1, is found to mediate the initial interactions of the particles at the cell surface, their coupling with actin filaments across the cell membrane, and their subsequent internalization via macropinocytosis. The observed interaction of syndecan molecules with the particle prior to their engagement with actin filaments suggests that the particles initiate their own internalization by facilitating the clustering of the molecules, which is required for the actin coupling and subsequent internalization of syndecan. Our observations identify a new role for syndecan-1 in mediating the cellular interactions and fate of positively charged submicrometer amorphous silica particles in the alveolar type II epithelial cell, a target cell for inhaled particles.

  9. Prototheca zopfii isolated from bovine mastitis induced oxidative stress and apoptosis in bovine mammary epithelial cells.

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    Shahid, Muhammad; Gao, Jian; Zhou, Yanan; Liu, Gang; Ali, Tariq; Deng, Youtian; Sabir, Naveed; Su, Jingliang; Han, Bo

    2017-05-09

    Bovine protothecal mastitis results in considerable economic losses worldwide. However, Prototheca zopfii induced morphological alterations and oxidative stress in bovine mammary epithelial cells (bMECs) is not comprehensively studied yet. Therefore, the aim of this current study was to investigate the P. zopfii induced pathomorphological changes, oxidative stress and apoptosis in bMECs. Oxidative stress was assessed by evaluating catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) contents and lactate dehydrogenase (LDH) activity, while ROS generation and apoptosis was measured by confocal laser scanning microscopy. The results revealed that infection of P. zopfii genotype II (GTII) significantly changed bMECs morphology, increased apoptotic rate and MDA contents at 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control group, in time-dependent manner. LDH activity and ROS generation was also increased (p < 0.01) at 12 h and 24 h. However, SOD and CAT contents in bMECs infected with GTII were decreased (p < 0.05) at 12 h, while GPx (p < 0.01), SOD (p < 0.05) and CAT (p < 0.01) levels were reduced at 24 h. In case of GTI, only CAT and GPx activities were significantly decreased when the duration prolonged to 24 h but lesser than GTII. This suggested that GTII has more devastating pathogenic effects in bMECs, and the findings of this study concluded that GTII induced apoptosis and oxidative stress in bMECs via the imbalance of oxidant and antioxidant defenses as well as the production of intracellular ROS.

  10. The axonal guidance cue semaphorin 3C contributes to alveolar growth and repair.

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    Arul Vadivel

    Full Text Available Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C, contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O2-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.

  11. NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells

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    Duan, Wei-Xia; He, Min-Di; Mao, Lin [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China); Qian, Feng-Hua [Department of Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Li, Yu-Ming [Institute of Hepatobiliary Surgery, XinQiao Hospital, Third Military Medical University, Chongqing 400038 (China); Pi, Hui-Feng; Liu, Chuan; Chen, Chun-Hai; Lu, Yong-Hui; Cao, Zheng-Wang; Zhang, Lei; Yu, Zheng-Ping [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China); Zhou, Zhou, E-mail: lunazhou00@163.com [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China)

    2015-07-15

    With application of nano-sized nickel-containing particles (Nano-Ni) expanding, the health concerns about their adverse effects on the pulmonary system are increasing. However, the mechanisms for the pulmonary toxicity of these materials remain unclear. In the present study, we focused on the impacts of NiO nanoparticles (NiONPs) on sirtuin1 (SIRT1), a NAD-dependent deacetylase, and investigated whether SIRT1 was involved in NiONPs-induced apoptosis. Although the NiONPs tended to agglomerate in fluid medium, they still entered into the human bronchial epithelial cells (BEAS-2B) and released Ni{sup 2+} inside the cells. NiONPs at doses of 5, 10, and 20 μg/cm{sup 2} inhibited the cell viability. NiONPs' produced cytotoxicity was demonstrated through an apoptotic process, indicated by increased numbers of Annexin V positive cells and caspase-3 activation. The expression of SIRT1 was markedly down-regulated by the NiONPs, accompanied by the hyperacetylation of p53 (tumor protein 53) and overexpression of Bax (Bcl-2-associated X protein). However, overexpression of SIRT1 through resveratrol treatment or transfection clearly attenuated the NiONPs-induced apoptosis and activation of p53 and Bax. Our results suggest that the repression of SIRT1 may underlie the NiONPs-induced apoptosis via p53 hyperacetylation and subsequent Bax activation. Because SIRT1 participates in multiple biologic processes by deacetylation of dozens of substrates, this knowledge of the impact of NiONPs on SIRT1 may lead to an improved understanding of the toxic mechanisms of Nano-Ni and provide a molecular target to antagonize Nano-Ni toxicity. - Highlights: • NiONPs were taken up by BEAS-2B cells and released Ni{sup 2+}. • NiONPs produced cytotoxicity was demonstrated through an apoptotic process. • NiONPs repressed SIRT1 expression and activated p53 and Bax. • Overexpression of SIRT1 attenuated NiONPs-induced apoptosis via deacetylation p53.

  12. Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production, oxidative stress, and caspase-3 activation.

    Science.gov (United States)

    Bai, Fangfang; Ni, Bo; Liu, Maojun; Feng, Zhixin; Xiong, Qiyan; Xiao, Shaobo; Shao, Guoqing

    2013-09-15

    Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Elevated extracellular K+ inhibits apoptosis of corneal epithelial cells exposed to UV-B radiation.

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    Singleton, Katherine R; Will, David S; Schotanus, Mark P; Haarsma, Loren D; Koetje, Leah R; Bardolph, Susan L; Ubels, John L

    2009-08-01

    The goal of this study was to determine if the high [K(+)] in tears, 20-25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal-limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50-200 mJ/cm(2) followed by measurement of K(+) channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K(+) channels after UV-B exposure at 80 mJ/cm(2) or 150 mJ/cm(2) and a decrease in UV-induced K(+) efflux with increasing [K(+)](o). The UV-activated current was partially blocked by the specific K(+) channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100-200 mJ/cm(2). DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100mM K(o)(+) after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100mM K(o)(+) following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100-200 mJ/cm(2). This decrease in mitochondrial potential was prevented by 100mM K(o)(+); however, 25 or 50mM K(o)(+) provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), and this UV-activation was significantly reduced by 25-100mM K(o)(+). The data show that the physiologically relevant [K(+)](o) of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K(+)] in tears reduces loss of K(+) from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular

  14. VCP phosphorylation-dependent interaction partners prevent apoptosis in Helicobacter pylori-infected gastric epithelial cells.

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    Cheng-Chou Yu

    Full Text Available Previous studies have demonstrated that valosin-containing protein (VCP is associated with H. pylori-induced gastric carcinogenesis. By identifying the interactome of VCP overexpressed in AGS cells using a subtractive proteomics approach, we aimed to characterize the cellular responses mediated by VCP and its functional roles in H. pylori-associated gastric cancer. VCP immunoprecipitations followed by proteomic analysis identified 288 putative interacting proteins, 18 VCP-binding proteins belonged to the PI3K/Akt signaling pathway. H. pylori infection increased the interaction between Akt and VCP, Akt-dependent phosphorylation of VCP, levels of ubiquitinated proteins, and aggresome formation in AGS cells. Furthermore, phosphorylated VCP co-localized with the aggresome, bound ubiquitinated proteins, and increased the degradation of cellular regulators to protect H. pylori-infected AGS cells from apoptosis. Our study demonstrates that VCP phosphorylation following H. pylori infection promotes both gastric epithelial cell survival, mediated by the PI3K/Akt pathway, and the degradation of cellular regulators. These findings provide novel insights into the mechanisms of H. pylori infection induced gastric carcinogenesis.

  15. Puerarin protects against Staphylococcus aureus-induced injury of human alveolar epithelial A549 cells via downregulating alpha-hemolysin secretion.

    Science.gov (United States)

    Tang, Feng; Li, Wen-Hua; Zhou, Xuan; Liu, Yong-Hua; Li, Zhe; Tang, Yu-Shun; Kou, Xu; Wang, Shu-De; Bao, Min; Qu, Lian-Da; Li, Min; Li, Bing

    2014-08-01

    Alpha-hemolysin, a secreted pore-forming toxin, plays an indispensable role in the pathogenicity of Staphylococcus aureus. In this study, the antimicrobial activity of puerarin against S. aureus was investigated; as a result, puerarin showed no influence on the growth of this organism. However, hemolysis and western blotting assays showed that puerarin concentration dependently inhibited the secretion of alpha-hemolysin at low concentrations. Real-time RT-PCR assay was further employed to evaluate the transcriptional level of hla, the gene encoding alpha-hemolysin, and RNAIII, an effector molecule of the agr system. The results indicated that the RNAIII expression and subsequent hla transcription were also inhibited by puerarin in a dose-dependent manner. Furthermore, puerarin significantly prevented human alveolar epithelial A549 cells from S. aureus-induced injury. Thereby, puerarin may be considered as a potential candidate for the development of antivirulence drugs in the treatment of S. aureus-mediated infections.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Downregulation of Smac attenuates H2O2-induced apoptosis via endoplasmic reticulum stress in human lens epithelial cells.

    Science.gov (United States)

    De-Qian, Kong; Yue, Liu; Li, Li; Guangying, Zheng

    2017-07-01

    Second mitochondria-derived activator of caspases (Smac) is reported to promote apoptosis. Given the important role of apoptosis in cataract development, the aim of this study was to investigate whether Smac induces human lens epithelial cell (HLEC) apoptosis via endoplasmic reticulum stress (ERS). Smac expression was examined by immunohistochemistry in anterior lens capsules from 157 patients with age-related cataracts and 5 normal controls. The role of Smac in hydrogen peroxide (H2O2)-induced ERS and apoptosis was further evaluated using small interfering RNA knockdown in an HLEC line. Notably, Smac expression was significantly higher in patients with cataracts than in controls, but showed no association with cataract severity. Cell survival was inversely correlated with H2O2 concentration, and was most significantly affected at 200 μmol/L. Moreover, flow cytometry revealed that Smac knockdown attenuated H2O2-induced apoptosis and enhanced apoptotic- and endoplasmic reticulum-related marker expression-including that of glucose-regulated protein 78, C/EBP homologous protein, caspase 3, B-cell chronic lymphocytic leukemia/lymphoma 2-associated X, and BCL2-at the gene and protein level. Collectively, these results indicate that Smac plays an important role in ERS-induced apoptosis in HLECs, suggesting its close association with cataract development.

  18. Apoptosis of Corneal Epithelial Cells Caused by Ultraviolet B-induced Loss of K+ is Inhibited by Ba2+

    Science.gov (United States)

    Glupker, Courtney D.; Boersma, Peter M.; Schotanus, Mark P.; Haarsma, Loren D.; Ubels, John L.

    2017-01-01

    UVB exposure at ambient outdoor levels triggers rapid K+ loss and apoptosis in human corneal limbal epithelial (HCLE) cells cultured in medium containing 5.5 mM K+, but considerably less apoptosis occurs when the medium contains the high K+ concentration that is present in tears (25 mM). Since Ba2+ blocks several K+ channels, we tested whether Ba2+-sensitive K+ channels are responsible for some or all of the UVB-activated K+ loss and subsequent activation of the caspase cascade and apoptosis. Corneal epithelial cells in culture were exposed to UVB at 80 or 150 mJ/cm2. Patch-clamp recording was used to measure UVB-induced K+ currents. Caspase-activity and TUNEL assays were performed on HCLE cells exposed to UVB followed by incubation in the presence or absence of Ba2+. K+ currents were activated in HCLE cells following UVB-exposure. These currents were reversibly blocked by 5 mM Ba2+. When HCLE cells were incubated with 5 mM Ba2+ after exposure to UVB, activation of caspases-9, -8, and -3 and DNA fragmentation were significantly decreased. The data confirm that UVB-induced K+ current activation and loss of intracellular K+ leads to activation of the caspase cascade and apoptosis. Extracellular Ba2+ inhibits UVB-induced apoptosis by preventing loss of intracellular K+ when K+ channels are activated. Ba2+ therefore has effects similar to elevated extracellular K+ in protecting HCLE cells from UVB-induced apoptosis. This supports our overall hypothesis that elevated K+ in tears contributes to protection of the corneal epithelium from adverse effects of ambient outdoor UVB. PMID:27189864

  19. The proliferation, apoptosis, invasion of endothelial-like epithelial ovarian cancer cells induced by hypoxia

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    Zhu Pengfei

    2010-09-01

    Full Text Available Abstract Background Epithelial ovarian cancer is one of the most malignant cancers in women because metastasis occurs in the most of patients by the time of diagnosis. Cancer cells have strong capacity to form angiogenesis or vasculogenic mimicry, which plays the major role in its malignant phenotype. Vasculogenic mimicry might contribute to the failure of the angiogenesis-targeted therapy strategies. Under the microenvironment of the tumor, hypoxia is the most common phenomena because of the vast energy and oxygen consuming. In the present study, the endothelial-like cells induced by hypoxia from SKOV-3 and ES-2 ovarian cancer cells were harvested to investigate the changes in their biological behaviors. Methods The endothelial-like cells from SKOV-3 and ES-2 cells were harvested by laser capture microdissection. The biological behaviors of the endothelial-like cells, including proliferation, cell cycle, apoptosis, invasion and telomerase activity were determined by MTT, FCM, Transwell chamber and TRAP-ELISA methods. HIF-1α is the most important factor for the behavior changes under hypoxic condition. Some other genes relative to biological behaviors are also changes following the changes of HIF-1α. In order to elucidate the underlying mechanisms for these changes by hypoxia, the relative genes expressions including HIF-1α, CyclinD1, Flk-1, VEGF, p53 and V-src were determined by real-time PCR. Results SKOV-3 and ES-2 cells were resistant to hypoxia by adoption of proliferation, apoptosis, differentiation and invasion. Combined with other studies, the more poorly cancer cells differentiate, the more strongly cells are resistant to hypoxia, the more possible to form vasculogenic mimicry. The changes in the expression of HIF-1α, and HIF-1α-dependent VEGF, Flk-1, Cyclin D1, and HIF-1α-independent p53 have been involved in this process. Conclusions HIF-1α took an important role in the behavioral changes of SKOV-3 and ES-2 cells by hypoxia. At

  20. Paraquat induces epithelial-mesenchymal transition-like cellular response resulting in fibrogenesis and the prevention of apoptosis in human pulmonary epithelial cells.

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    Atsushi Yamada

    Full Text Available The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ poisoning. Epithelial-mesenchymal transition (EMT has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0-500 μM for 2-12 days. Short-term (2 days high-dose (>100 μM treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker, suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion. In contrast, long-term (6-12 days low-dose (30 μM treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA. The mesenchymal-like cells also secreted the extracellular matrix (ECM protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during

  1. MicroRNA-25 inhibits high glucose-induced apoptosis in renal tubular epithelial cells via PTEN/AKT pathway.

    Science.gov (United States)

    Li, Huicong; Zhu, Xiaoguang; Zhang, Junwei; Shi, Jun

    2017-12-01

    Diabetic nephropathy (DN) has become the major cause of end-stage renal disease (ESRD). It has been demonstrated that apoptosis of renal tubular epithelial cells induced by hyperglycemia contributes to the pathogenesis of DN. Recent researches have corroborated the critical roles of microRNAs (miRNAs) in the apoptosis of various types of cells including renal tubular epithelial cells. However, the eff ; ;ect of miRNAs on the hyperglycemia-induced apoptosis of renal tubular epithelial cells remains unclear. The aim of this study is to explore the eff ; ;ect of miRNAs on the hyperglycemia-induced apoptosis of renal tubular epithelial cells and its molecular mechanism. Using a miRNA microarray, miRNAs putatively associated with DN were examined in renal biopsy tissue samples from DN patients and healthy controls. Validation analysis of miR-25 level in serum samples and renal biopsy tissue samples was performed using quantitative reverse transcription PCR (qRT-PCR). Then, gain- and loss- of function experiments were performed to determine the protective roles of miR-25 in high glucose-induced damage to renal tubular epithelial cells. Furthermore, the target gene of miR-25 and the downstream signaling pathway were also investigated. Microarray analysis and qRT-PCR revealed that miR-25 was significantly downregulated in renal biopsy tissue and serum samples from DN patients. We also observed that an inverse relationship between serum miR-25 level and proteinuria in DN patients. Meanwhile, miR-25 was decreased in human kidney (HK-2) cells subjected to HG treatment in a time dependent manner. Its overexpression reduced production of reactive oxygen species (ROS), suppressed cell apoptosis in HG-induced cell damage model, which was coupled with the decreased expression of cleaved caspase-3 and activity of caspase-3. Subsequent analyses demonstrated that phosphatase and tensin homolog deleted on chromosome ten (PTEN) was a direct and functional target of miR-25, which was

  2. High proportion of granzyme B+ intraepithelial lymphocytes contributes to epithelial apoptosis in Helicobacter pylori-associated lymphocytic gastritis.

    Science.gov (United States)

    Han, Song-Hee; Joo, Mee; Kim, Kyoung-Mee

    2013-08-01

    Helicobacter pylori infection has been linked to the development of lymphocytic gastritis (LG) characterized by ≥25 intraepithelial lymphocytes (IELs) per 100 epithelial cells. We hypothesize that the changes in the subpopulation and/or cytotoxicity of IELs leading to epithelial cell apoptosis may be involved in the pathogenesis of H. pylori-associated LG. We examined IEL subpopulations and the expression of cytotoxic molecules by IELs in biopsy specimens from 36 patients with H. pylori-associated LG by immunostainings for CD3, CD4, CD8, T-cell-restricted intracellular antigen-1 (TIA-1), and granzyme B (GrB) and compared the results with those obtained from 49 patients with H. pylori-associated gastritis (HPG). To investigate whether the IEL-mediated cytotoxicity is related to the increase of epithelial apoptosis, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using ApopTag detection kit. Between LG and HPG groups, significant differences in the number of CD3+, CD4+, CD8+, TIA-1+ or GrB+ IELs, and ApopTag indices were found. Among the CD3+ IELs, the proportion of CD8+ IELs or TIA-1+ IELs did not differ between two groups. The LG group showed a selective increase in GrB-positive, phenotypically activated IELs, which was paralleled by an increase in ApopTag indices. In contrast, the HPG group showed more heterogeneous IEL subpopulations with more CD4+ IELs and less GrB+ IELs compared with the LG group, and we did not find any significant variable contributing to the epithelial apoptosis in the HPG group. This study shows that in addition to the numerical increase in the IELs, there are significant changes in the subpopulations and cytotoxicity of IELs between HPG and H. pylori-associated LG. In particular, enhanced GrB-associated cytotoxicity of the IELs in H. pylori-associated LG contributes to an increase in epithelial apoptosis. © 2013 John Wiley & Sons Ltd.

  3. Alisertib, an Aurora kinase A inhibitor, induces apoptosis and autophagy but inhibits epithelial to mesenchymal transition in human epithelial ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Ding YH

    2015-01-01

    Full Text Available Yong-Hui Ding,1,2 Zhi-Wei Zhou,2,3 Chun-Fang Ha,1 Xue-Yu Zhang,1 Shu-Ting Pan,4 Zhi-Xu He,3 Jeffrey L Edelman,2 Dong Wang,5 Yin-Xue Yang,6 Xueji Zhang,7 Wei Duan,8 Tianxin Yang,9 Jia-Xuan Qiu,4 Shu-Feng Zhou2,3 1Department of Gynecology, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 5Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 8School of Medicine, Deakin University, Waurn Ponds, Australia; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA Abstract: Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS, a selective Aurora kinase A (AURKA inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT, and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA

  4. Potential in vitro model for testing the effect of exposure to nanoparticles on the lung alveolar epithelial barrier

    Directory of Open Access Journals (Sweden)

    Raymond Derk

    2015-03-01

    Full Text Available Pulmonary barrier function plays a pivotal role in protection from inhaled particles. However, some nano-scaled particles, such as carbon nanotubes (CNT, have demonstrated the ability to penetrate this barrier in animal models, resulting in an unusual, rapid interstitial fibrosis. To delineate the underlying mechanism and specific bio-effect of inhaled nanoparticles in respiratory toxicity, models of lung epithelial barriers are required that allow accurate representation of in vivo systems; however, there is currently a lack of consistent methods to do so. Thus, this work demonstrates a well-characterized in vitro model of pulmonary barrier function using Calu-3 cells, and provides the experimental conditions required for achieving tight junction complexes in cell culture, with trans-epithelial electrical resistance measurement used as a biosensor for proper barrier formation and integrity. The effects of cell number and serum constituents have been examined and we found that changes in each of these parameters can greatly affect barrier formation. Our data demonstrate that use of 5.0 × 104 Calu-3 cells/well in the Transwell cell culture system, with 10% serum concentrations in culture media is optimal for assessing epithelial barrier function. In addition, we have utilized CNT exposure to analyze the dose-, time-, and nanoparticle property-dependent alterations of epithelial barrier permeability as a means to validate this model. Such high throughput in vitro cell models of the epithelium could be used to predict the interaction of other nanoparticles with lung epithelial barriers to mimic respiratory behavior in vivo, thus providing essential tools and bio-sensing techniques that can be uniformly employed.

  5. Role of phospholipase A₂ (PLA₂) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis.

    Science.gov (United States)

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-08-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (P keratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. Copyright © 2013. Published by Elsevier Ltd.

  6. Analysis of the Oxidative Stress Status in Nonspecific Vaginitis and Its Role in Vaginal Epithelial Cells Apoptosis

    Science.gov (United States)

    Chen, Zhaojie; Zhang, Zhen; Zhang, Haiyan; Xie, Beibei

    2015-01-01

    Nonspecific vaginitis (NSV), also named bacterial vaginosis, is one of the most common genital system diseases in women during their reproductive years. The specific pathogenic mechanism of NSV is not clear yet. Upon the balance alteration, large amount of reactive oxidant species (ROS) is generated and accumulated in the genital tract, and thus resulting in oxidative stress, which has been reported to be an important trigger of mitochondrial pathway cell apoptosis. In this study, the antioxidant secretion level and antioxidant enzyme activity in the vaginal discharge were evaluated to analyze the oxidative status in the vaginal tract of NSV patients. The effect of oxidative stress on the vaginal mucosa epithelial cell apoptosis was then studied. The role of oxidative stress on NSV development was uncovered; thus open new direction for the prevention and treatment of NSV by providing antiradical agents was revealed. PMID:26558281

  7. A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

    Science.gov (United States)

    Weisskopf, M; Schaffner, W; Jundt, G; Sulser, T; Wyler, S; Tullberg-Reinert, H

    2005-10-01

    Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

  8. The concentrations of clinafloxacin in alveolar macrophages, epithelial lining fluid, bronchial mucosa and serum after administration of single 200 mg oral doses to patients undergoing fibre-optic bronchoscopy.

    Science.gov (United States)

    Honeybourne, D; Andrews, J M; Cunningham, B; Jevons, G; Wise, R

    1999-01-01

    The concentrations of clinafloxacin were measured in serum, bronchial mucosa, alveolar macrophages and epithelial lining fluid after single 200 mg oral doses of clinafloxacin had been administered to 15 subjects who were undergoing bronchoscopy. Concentrations were measured using a microbiological assay method. Mean concentrations in serum, bronchial mucosa, alveolar macrophages and epithelial lining fluid at a mean of 1.27 h post-dose were 1.54, 2.65, 15.60 and 2.71 mg/L respectively. These site concentrations exceeded the MIC90 for common respiratory pathogens and indicate that clinafloxacin is likely to be effective in the treatment of a wide range of respiratory tract infections.

  9. Aerosol-based efficient delivery of telithromycin, a ketolide antimicrobial agent, to lung epithelial lining fluid and alveolar macrophages for treatment of respiratory infections.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Seki, Toshinobu; Morimoto, Kazuhiro

    2010-07-01

    The efficacy of aerosol-based delivery of telithromycin (TEL), as a model antimicrobial agent, for the treatment of respiratory infections was evaluated by comparison with oral administration. The aerosol formulation (0.2 mg/kg) was administered to rat lungs using a Liquid MicroSprayer. The time courses of the concentration of TEL in lung epithelial lining fluid (ELF) and alveolar macrophages (AMs) following administration of an aerosol formulation to rat lungs were markedly higher than that following the administration of an oral formulation (50 mg/kg). The time course of the concentrations of TEL in plasma following administration of the aerosol formulation was markedly lower than that in ELF and AMs. These results indicate that the aerosol formulation is more effective in delivering TEL to ELF and AMs, compared to the oral formulation, despite a low dose and it avoids distribution of TEL to the blood. In addition, the antibacterial effects of TEL in ELF and AMs following administration of the aerosol formulation were estimated by pharmacokinetics/pharmacodynamics analysis. The concentrations of TEL in ELF and the AMs time curve/minimum inhibitory concentration of TEL ratio were markedly higher than the effective values. This study indicates that an antibiotic aerosol formulation may be an effective pulmonary drug delivery system for the treatment of respiratory infections.

  10. Upregulation of Fas-Fas-L (CD95/CD95L)-mediated epithelial apoptosis--a putative role in pouchitis?

    LENUS (Irish Health Repository)

    Coffey, J C

    2012-02-03

    INTRODUCTION: Ileal pouch-anal anastomosis (IPAA) remains the gold standard for patients with refractory ulcerative colitis. Pouchitis causes considerable morbidity in 40% of patients with IPAA. This study examined the role of increased epithelial apoptosis in the etiology of pouchitis. METHODS: Following ethical approval pouch biopsies taken from patients with a history of pouchitis were compared with age-matched controls from patients who were pouchitis free. Apoptosis was detected immunohistochemically using a monoclonal antibody (M30) and terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin end labeling (TUNEL). Villous atrophy was assessed histologically and correlated with levels of apoptosis. Epithelial Fas-ligand (L) was also assessed immunohistochemically. RESULTS: A significant increase in TUNEL staining was seen at the epithelial but not at the lamina propria level for known pouchitis patients versus controls (0.091 vs 0.035; P < 0.01). Similarly, epithelial M30 immunoreactivity (0.225 vs 0.082; P < 0.05) and villous atrophy (0.035 vs 0.10; P < 0.05) were significantly increased in pouches with previous pouchitis when compared with normal pouches. Upregulation of Fas-L expression was characteristic of this epithelium. Mononuclear cells were strongly positive for Fas-L. Increased epithelial levels of apoptosis correlated with increased levels of villous atrophy. CONCLUSIONS: Our data suggest a role for elevated Fas-Fas-L (CD95-CD95L)-mediated epithelial apoptosis in the etiology of pouchitis. Increased levels of villous atrophy may result from increased apoptosis and thereby predispose to infection by otherwise apathogenic organisms.

  11. Cytotoxic and Apoptosis-Inducing Activity of Plants from the Family Asparagaceae in Relation to Human Alveolar Adenocarcinoma Cells

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    Y.N. Kamalova

    2016-06-01

    Full Text Available Cancer is known as the second major mortality cause. The number of new cases is increasing every year. Thus, it is urgent for scientists to search for alternative drugs with selective antitumor action and minimal side effects. It is known that some plant metabolites exhibit antioxidant, cytotoxic, and antitumor activity, while at the same time being less toxic than modern allopathic drugs. In this work, we have investigated the cytotoxic and apoptosis-inducing effects of extracts obtained from plants of the family Asparagaceae on A549 human lung adenocarcinoma cells. The analysis has been performed using flow cytofluorometry. If extracts showed cytotoxicity, the apoptosis-inducing action has been evaluated at the concentration of 50 μg/mL; in other cases, the analyzed concentration range was 50–300 μg/mL. On the basis of the experiments carried out, the following conclusions have been made. Extracts of the leaves and rhizomes of Sansevieria cylindrica and Sansevieria trifasciata do not possess antitumor activity. Extracts of the leaves of Polianthes tuberosa and Furcraea gigantea, which were cytotoxic at high concentrations, cause cell death at 50 μg/mL in the amount of 21.35 ± 1.86 and 15.6 ± 3.23, respectively. Extracts of Polianthes tuberosa bulbs and Yucca filamentosa leaves are able to induce apoptosis at higher concentrations. When the concentration reaches 100 μg/mL, the proportion of apoptotic cells for these plants is 45.76 ± 1.34 and 11.33 ± 0.07, respectively. The number of dead cells at the concentration of 300 μg/mL increased up to 73.33 ± 3.05 and 81.75 ± 4.07. The results have great importance for development of new drugs based on metabolites from these plant extracts.

  12. A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Jonathan M. Williams

    2013-11-01

    The gut barrier, composed of a single layer of intestinal epithelial cells (IECs held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1−/−, Tnfr2 (Tnfr2−/−, nuclear factor kappa B1 (Nfκb1−/− or Nfĸb2 (Nfĸb2−/−. Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and

  13. Lipopolysaccharide (LPS) Promotes Apoptosis in Human Breast Epithelial × Breast Cancer Hybrids, but Not in Parental Cells.

    Science.gov (United States)

    Fried, Sabrina; Tosun, Songuel; Troost, Gabriele; Keil, Silvia; Zaenker, Kurt S; Dittmar, Thomas

    2016-01-01

    Toll-like receptors (TLRs) belong to the group of pathogen recognition receptors known to play a crucial role in the innate immune system. In cancer, TLR expression is still debated controversially due to contradictory results reporting that both induction of apoptosis as well as tumor progression could depend on TLR signaling, whereby recent data rather indicate a pro-tumorigenic effect. The biological phenomenon of cell fusion has been associated with cancer progression due to findings revealing that fusion-derived hybrid cells could exhibit properties like an increased metastatogenic capacity and an increased drug resistance. Thus, M13MDA435 hybrid cell lines, which derived from spontaneous fusion events between human M13SV1-EGFP-Neo breast epithelial cells and human MDA-MB-435-Hyg breast cancer cells, were investigated. Cultivation of cells in the presence of the TLR4 ligand LPS potently induced apoptosis in all hybrid clones, but not in parental cells, which was most likely attributed to differential kinetics of the TLR4 signal transduction cascade. Activation of this pathway concomitant with NF-κB nuclear translocation and TNF-α expression was solely observed in hybrid cells. However, induction of LPS mediated apoptosis was not TNF-α dependent since TNF-α neutralization was not correlated to a decreased amount of dead cells. In addition to TNF-α, LPS also caused IFN-β expression in hybrid clones 1 and 3. Interestingly, hybrid clones differ in the mode of LPS induced apoptosis. While neutralization of IFN-β was sufficient to impair the LPS induced apoptosis in M13MDA435-1 and -3 hybrids, the amount of apoptotic M13MDA435-2 and -4 hybrid cells remained unchanged in the presence of neutralizing IFN-β antibodies. In summary, the fusion of non-LPS susceptible parental human breast epithelial cells and human breast cancer cells gave rise to LPS susceptible hybrid cells, which is in view with the cell fusion hypothesis that hybrid cells could exhibit novel

  14. Heat shock protein 90β inhibits apoptosis of intestinal epithelial cells induced by hypoxia through stabilizing phosphorylated Akt

    Directory of Open Access Journals (Sweden)

    Shuai Zhang

    2013-01-01

    Full Text Available Intestinal epithelial cell (IEC apoptosis induced by hypoxiacompromise intestinal epithelium barrier function. Both Akt andHsp90 have cytoprotective function. However, the specific roleof Akt and Hsp90β in IEC apoptosis induced by hypoxia has notbeen explored. We confirmed that hypoxia-induced apoptosiswas reduced by Hsp90β overexpression but enhanced bydecreasing Hsp90β expression. Hsp90β overexpressionenhanced BAD phosphorylation and thus reduced mitochondrialrelease of cytochrome C. Reducing Hsp90β expression hadopposite effects. The protective effect of Hsp90β againstapoptosis was negated by LY294002, an Akt inhibitor. Furtherstudy showed that Akt phosphorylation was enhanced byHsp90β, which was not due to the activation of upstream PI3Kand PDK1 but because of stabilization of pAkt via directinteraction between Hsp90β and pAkt. These results demonstratethat Hsp90β may play a significant role in protecting IECs fromhypoxia-induced apoptosis via stabilizing pAkt to phosphorylateBAD and reduce cytochrome C release. [BMB Reports 2013;46(1: 47-52

  15. Microplasma Induced Cell Morphological Changes and Apoptosis of Ex Vivo Cultured Human Anterior Lens Epithelial Cells - Relevance to Capsular Opacification.

    Directory of Open Access Journals (Sweden)

    Nina Recek

    Full Text Available Inducing selective or targeted cell apoptosis without affecting large number of neighbouring cells remains a challenge. A plausible method for treatment of posterior capsular opacification (PCO due to remaining lens epithelial cells (LECs by reactive chemistry induced by localized single electrode microplasma discharge at top of a needle-like glass electrode with spot size ~3 μm is hereby presented. The focused and highly-localized atmospheric pressure microplasma jet with electrode discharge could induce a dose-dependent apoptosis in selected and targeted individual LECs, which could be confirmed by real-time monitoring of the morphological and structural changes at cellular level. Direct cell treatment with microplasma inside the medium appeared more effective in inducing apoptosis (caspase 8 positivity and DNA fragmentation at a highly targeted cell level compared to treatment on top of the medium (indirect treatment. Our results show that single cell specific micropipette plasma can be used to selectively induce demise in LECs which remain in the capsular bag after cataract surgery and thus prevent their migration (CXCR4 positivity to the posterior lens capsule and PCO formation.

  16. Andrographolide-induced apoptosis in human renal tubular epithelial cells: Roles of endoplasmic reticulum stress and inflammatory response.

    Science.gov (United States)

    Gu, Li-Li; Zhang, Xin-Yue; Xing, Wen-Min; Xu, Jia-Dong; Lu, Hong

    2016-07-01

    Andrographolide sodium bisulfate as a kind of soluble derivative of andrographolide (AD), is obviously known to be nephrotoxicity, but AD has not been reported clearly. Our study aimed to investigate the induction of apoptosis in human renal tubular epithelial (HK-2) cells by AD and its possible mechanism. Our results demonstrated that AD (0-250μmol/L) inhibited Hk-2 cells proliferation in a dose- and time-dependent manner and induced apoptosis, accompanied by decreased of superoxide dismutase (SOD) activity and increased of malondialdehvde (MDA) content. Simultaneously, AD regulated the expression of endoplasmic reticulum (ER) molecular chaperone glucose-regulated protein 78 (GRP78/Bip) protein, elevated the expressions of C/EBP homologous protein (CHOP) and Caspase-4, indicating activation of ER stress signaling, and induced the alterative expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) proteins. It provided evidence that ER stress and inflammation would be significant mechanisms responsible for AD-induced apoptosis in addition to oxidative stress. Copyright © 2016. Published by Elsevier B.V.

  17. Uniform TiO2 nanoparticles induce apoptosis in epithelial cell lines in a size-dependent manner.

    Science.gov (United States)

    Sun, Qingqing; Ishii, Takayuki; Kanehira, Koki; Sato, Takeshi; Taniguchi, Akiyoshi

    2017-05-02

    The size of titanium dioxide (TiO 2 ) nanoparticles is a vital parameter that determines their cytotoxicity. However, most reported studies have employed irregular shapes and sizes of TiO 2 nanoparticles, as it is difficult to produce nanoparticles of suitable sizes for research. We produced good model TiO 2 nanoparticles of uniform shape and size for use in studying their cytotoxicity. In this work, spherical, uniform polyethylene glycol-modified TiO 2 (TiO 2 -PEG) nanoparticles of differing sizes (100, 200, and 300 nm) were prepared using the sol-gel method. A size-dependent decrease in cell viability was observed with increasing nanoparticle size. Furthermore, apoptosis was found to be positively associated with nanoparticle size, as evidenced by an increase in caspase-3 activity with increasing nanoparticle size. Larger nanoparticles exhibited higher cellular uptake, suggesting that larger nanoparticles more strongly induce apoptosis. In addition, the cellular uptake of different sizes of nanoparticles was energy dependent, suggesting that there are size-dependent uptake pathways. We found that 100 and 200 nm (but not 300 nm) nanoparticles were taken up via clathrin-mediated endocytosis. These results utilizing uniform nanoparticles suggest that the size-dependent cytotoxicity of nanoparticles involves active cellular uptake, caspase-3 activation, and apoptosis in the epithelial cell line (NCI-H292). These findings will hopefully aid in the future design and safe use of nanoparticles.

  18. Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells.

    Science.gov (United States)

    Wille, Aline; Gerber, Annegret; Heimburg, Anke; Reisenauer, Anita; Peters, Christoph; Saftig, Paul; Reinheckel, Thomas; Welte, Tobias; Bühling, Frank

    2004-07-01

    Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL(-/-) mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the single-chain isoform of catD. Furthermore, we found that catL-deficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catL-deficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.

  19. Chromosomal damage and apoptosis analysis in exfoliated oral epithelial cells from mouthwash and alcohol users

    Science.gov (United States)

    Rocha, Rodrigo dos Santos; Meireles, José Roberto Cardoso; de Moraes Marcílio Cerqueira, Eneida

    2014-01-01

    Chromosomal damage and apoptosis were analyzed in users of mouthwash and/or alcoholic beverages, using the micronucleus test on exfoliated oral mucosa cells. Samples from four groups of 20 individuals each were analyzed: three exposed groups (EG1, EG2 and EG3) and a control group (CG). EG1 comprised mouthwash users; EG2 comprised drinkers, and EG3 users of both mouthwashes and alcoholic beverages. Cell material was collected by gently scraping the insides of the cheeks. Then the cells were fixed in a methanol/acetic acid (3:1) solution and stained and counterstained, respectively, with Schiff reactive and fast green. Endpoints were computed on 2,000 cells in a blind test. Statistical analysis showed that chromosomal damage and apoptosis were significantly higher in individuals of groups EG1 and EG3 than in controls (p < 0.005 and p < 0.001, respectively). No significant difference in chromosomal damage and apoptosis was observed between the exposed groups. In EG2, only the occurrence of apoptosis was significantly higher than in the controls. These results suggest that mouthwashes alone or in association with alcoholic drinks induce genotoxic effects, manifested as chromosomal damage and apoptosis. They also suggest that alcoholic drinks are effective for stimulating the process of apoptosis. However, these data need to be confirmed in larger samples. PMID:25505845

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

    Science.gov (United States)

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

    2017-05-19

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

  1. Anti-IL-2 receptor antibody decreases cytokine-induced apoptosis of human renal tubular epithelial cells (TEC).

    Science.gov (United States)

    Wang, Shuang; Zhang, Zhu-Xu; Yin, Ziqin; Liu, Weihua; Garcia, Bertha; Huang, Xuyan; Acott, Philip; Jevnikar, Anthony M

    2011-07-01

    Transplant rejection is mediated by T-cell activation which is modulated by interleukin-2 (IL-2) binding to IL-2R (CD25). Monoclonal anti-IL-2 receptor antibody is used in renal transplantation to reduce rejection. Interestingly, proximal tubular epithelial cells (TEC) express CD25, similar to T cells. We have demonstrated that IL-2 induces murine TEC apoptosis through down-regulation of the caspase-8 inhibitor protein c-FLIP. Anti-CD25 antibody may be useful clinically to limit renal injury, but this has not been tested in human TEC. Human PT-2 TEC were isolated and cloned from the urine of transplant patients. Apoptosis was determined by FACS with Annexin-V FITC. Protein expression was studied using western blot, and mRNA levels by quantitative real-time (PR-PCR). We demonstrated that the morphology of a human kidney cell line (PT-2) cloned from urine was consistent with proximal TEC and expresses alkaline phosphatase, cytokeratin, vimentin, CD13, CD26, and low levels of E-cadherin. Basal IL-2 receptor (CD25) was up-regulated by IL-2/IFN-γ stimulation, and cytokine exposure induced apoptosis in a dose-dependent manner. Apoptosis with IL-2/IFN-γ was associated with increased caspase-8 activity and decreased endogenous caspase-8 inhibitor c-FLIP mRNA and protein expression. IL-2/IFN-γ-induced apoptosis could be blocked by pre-treatment of PT-2 with anti IL-2R antibody (basiliximab) but not control IgG antibody. These data demonstrate for the first time in human TEC that IL-2 and IFN-γ can induce TEC apoptosis which can be blocked by CD25 blockade antibody. These data suggest that anti-CD25 mAb might similarly attenuate inflammation-induced TEC injury in vivo. Kidney-expressed CD25 may represent a clinically important new target for attenuating early inflammatory injury in donor kidneys and preserving renal function during anti-rejection therapy.

  2. Curcumin modulates the effect of histone modification on the expression of chemokines by type II alveolar epithelial cells in a rat COPD model

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    Gan L

    2016-11-01

    Full Text Available Lixing Gan,1 Chengye Li,2 Jian Wang,1 Xuejun Guo3 1Department of Respiratory Medicine, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 2Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 3Department of Respiratory Medicine, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Background: Studies have suggested that histone modification has a positive impact on various aspects associated with the progression of COPD. Histone deacetylase 2 (HDAC2 suppresses proinflammatory gene expression through deacetylation of core histones.Objective: To investigate the effect of histone modification on the expression of chemokines in type II alveolar epithelial cells (AEC II in a rat COPD model and regulation of HDAC2 expression by curcumin in comparison with corticosteroid.Materials and methods: The rat COPD model was established by cigarette smoke exposure and confirmed by histology and pathophysioloy. AEC II were isolated and cultured in vitro from the COPD models and control animals. The cells were treated with curcumin, corticosteroid, or trichostatin A, and messenger RNA (mRNA expression of interleukin-8 (IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage inflammatory protein-2α (MIP-2α was assessed by quantitative real-time polymerase chain reaction (RT-PCR. The expression of HDAC2 was measured by Western blot. Chromatin immunoprecipitation was used to detect H3/H4 acetylation and H3K9 methylation in the promoter region of three kinds of chemokine genes (IL-8, MCP-1, and MIP-2α. Results: Compared to the control group, the mRNAs of MCP-1, IL-8, and MIP-2α were upregulated 4.48-fold, 3.14-fold, and 2.83-fold, respectively, in the AEC II from COPD model. The protein expression of HDAC2 in the AEC II from COPD model was significantly lower than from the control group (P<0

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

    Science.gov (United States)

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

    2016-11-16

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

  4. Aerosol-based efficient delivery of clarithromycin, a macrolide antimicrobial agent, to lung epithelial lining fluid and alveolar macrophages for treatment of respiratory infections.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Morimoto, Kazuhiro

    2012-04-01

    Macrolide antimicrobial agents are generally given by the oral route for the treatment of respiratory infections caused by pathogenic microorganisms infected in lung epithelial lining fluid (ELF) and alveolar macrophages (AMs). However, because macrolides distribute to many different tissues via the blood after oral administration, systemic side effects are frequently induced. In contrast with oral administration, aerosolization may be an efficient method for delivering macrolides directly to ELF and AMs. In this study, the efficacy of aerosol-based delivery of clarithromycin (CAM), as a model macrolide, for the treatment of respiratory infections was evaluated by comparison with oral administration. The aerosol formulation of CAM (0.2 mg/kg) was administered to rat lungs using a Liquid MicroSprayer(®). The oral formulation of CAM (50 mg/kg) was used for comparison. Time courses of concentrations of CAM in ELF and AMs following administration were obtained, and then the bioavailability (BA) was calculated. In addition, the area under the concentrations of CAM in ELF and AMs-time curve/minimum inhibitory concentration at which 90% of isolates ratio [area under the curve (AUC/MIC(90))] were calculated to estimate the antibacterial effects in ELF and AMs. The BA of CAM in ELF and AMs following administration of aerosol formulation were markedly greater than that following administration of oral formulation. This indicates that the aerosol formulation is more effective in delivering CAM to ELF and AMs, compared with the oral formulation, despite a low dose. The AUC/MIC(90) of CAM in ELF and AMs were markedly higher than the effective values. This indicates that the aerosol formulation could be useful for the treatment of respiratory infections caused by pathogenic microorganisms infected in ELF and AMs. This study suggests that aerosol formulation of macrolides is an effective pulmonary drug delivery system for the treatment of respiratory infections.

  5. Effect of irradiation/bone marrow transplantation on alveolar epithelial type II cells is aggravated in surfactant protein D deficient mice.

    Science.gov (United States)

    Mühlfeld, Christian; Madsen, Jens; Mackay, Rose-Marie; Schneider, Jan Philipp; Schipke, Julia; Lutz, Dennis; Birkelbach, Bastian; Knudsen, Lars; Botto, Marina; Ochs, Matthias; Clark, Howard

    2017-01-01

    Irradiation followed by bone marrow transplantation (BM-Tx) is a frequent therapeutic intervention causing pathology to the lung. Although alveolar epithelial type II (AE2) cells are essential for lung function and are damaged by irradiation, the long-term consequences of irradiation and BM-Tx are not well characterized. In addition, it is unknown whether surfactant protein D (SP-D) influences the response of AE2 cells to the injurious events. Therefore, wildtype (WT) and SP-D -/- mice were subjected to a myeloablative whole body irradiation dose of 8 Gy and subsequent BM-Tx and compared with age- and sex-matched untreated controls. AE2 cell changes were investigated quantitatively by design-based stereology. Compared with WT, untreated SP-D -/- mice showed a higher number of larger sized AE2 cells and a greater amount of surfactant-storing lamellar bodies. Irradiation and BM-Tx induced hyperplasia and hypertrophy in WT and SP-D -/- mice as well as the formation of giant lamellar bodies. The experimentally induced alterations were more severe in the SP-D -/- than in the WT mice, particularly with respect to the surfactant-storing lamellar bodies which were sometimes extremely enlarged in SP-D -/- mice. In conclusion, irradiation and BM-Tx have profound long-term effects on AE2 cells and their lamellar bodies. These data may explain some of the clinical pulmonary consequences of this procedure. The data should also be taken into account when BM-Tx is used as an experimental procedure to investigate the impact of bone marrow-derived cells for the phenotype of a specific genotype in the mouse.

  6. Andrographolide antagonizes cigarette smoke extract-induced inflammatory response and oxidative stress in human alveolar epithelial A549 cells through induction of microRNA-218.

    Science.gov (United States)

    Li, Ying-jie; Yu, Chang-hai; Li, Jing-bo; Wu, Xi-ya

    2013-12-01

    Andrographolide is a major bioactive labdane diterpenoid isolated from Andrographis paniculata and has protective effects against cigarette smoke (CS)-induced lung injury. This study was done to determine whether such protective effects were mediated through modulation of microRNA (miR)-218 expression. Therefore, we exposed human alveolar epithelial A549 cells to cigarette smoke extract (CSE) with or without andrographolide pretreatment and measured the level of glutathione, nuclear factor-kappaB (NF-κB) activation, proinflammatory cytokine production, and miR-218 expression. We found that andrographolide pretreatment significantly restored the glutathione level in CSE-exposed A549 cells, coupled with reduced inhibitor κB (IκB)-α phosphorylation and p65 nuclear translocation and interleukin (IL)-8 and IL-6 secretion. The miR-218 expression was significantly upregulated by andrographolide pretreatment. To determine the biological role of miR-218, we overexpressed and downregulated its expression using miR-218 mimic and anti-miR-218 inhibitor, respectively. We observed that miR-218 overexpression led to a marked reduction in IκB-α phosphorylation, p65 nuclear accumulation, and NF-κB-dependent transcriptional activity in CSE-treated A549 cells. In contrast, miR-218 silencing enhanced IκB-α phosphorylation and p65 nuclear accumulation in cells with andrographolide pretreatment and reversed andrographolide-mediated reduction of IL-6 and IL-8 production. In addition, depletion of miR-218 significantly reversed the upregulation of glutathione levels in A549 cells by andrographolide. Taken together, our results demonstrate that andrographolide mitigates CSE-induced inflammatory response in A549 cells, largely through inhibition of NF-κB activation via upregulation of miR-218, and thus has preventive benefits in CS-induced inflammatory lung diseases.

  7. Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

    Science.gov (United States)

    Fresta, Claudia G; Chakraborty, Aishik; Wijesinghe, Manjula B; Amorini, Angela M; Lazzarino, Giacomo; Lazzarino, Giuseppe; Tavazzi, Barbara; Lunte, Susan M; Caraci, Filippo; Dhar, Prajnaparamita; Caruso, Giuseppe

    2018-02-14

    Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

  8. Pseudomonas aeruginosa-induced apoptosis in airway epithelial cells is mediated by gap junctional communication in a JNK-dependent manner.

    Science.gov (United States)

    Losa, Davide; Köhler, Thilo; Bellec, Jessica; Dudez, Tecla; Crespin, Sophie; Bacchetta, Marc; Boulanger, Pierre; Hong, Saw See; Morel, Sandrine; Nguyen, Tuan H; van Delden, Christian; Chanson, Marc

    2014-05-15

    Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

  9. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

    Science.gov (United States)

    Silva, Julhiany de Fátima da; Vicentim, Juliana; Oliveira, Haroldo Cesar de; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; Silva, Juliana Leal Monteiro da; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-06-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

  10. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

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    Julhiany de Fátima da Silva

    2015-06-01

    Full Text Available The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

  11. Histidine deficiency attenuates cell viability in rat intestinal epithelial cells by apoptosis via mitochondrial dysfunction

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    Tatsunobu Matsui, M.S.

    2017-06-01

    Conclusions: This is the first report showing that histidine deficiency reduced cell viability and induced apoptosis in IEC-6 cells, and that a small amount of histidine supplementation prevented and improved the IEC-6 cell injury. This is a potential new clinical treatment against intestinal and/or gastric cell injury that would improve the patient's quality of life.

  12. Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation

    DEFF Research Database (Denmark)

    Seidelin, Jakob Benedict; Larsen, Sylvester; Linnemann, Dorte

    2015-01-01

    Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim...

  13. Inhibition of intestinal epithelial apoptosis improves survival in a murine model of radiation combined injury.

    Science.gov (United States)

    Jung, Enjae; Perrone, Erin E; Brahmamdan, Pavan; McDonough, Jacquelyn S; Leathersich, Ann M; Dominguez, Jessica A; Clark, Andrew T; Fox, Amy C; Dunne, W Michael; Hotchkiss, Richard S; Coopersmith, Craig M

    2013-01-01

    World conditions place large populations at risk from ionizing radiation (IR) from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy) followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA). While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01). Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01). These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

  14. Inhibition of intestinal epithelial apoptosis improves survival in a murine model of radiation combined injury.

    Directory of Open Access Journals (Sweden)

    Enjae Jung

    Full Text Available World conditions place large populations at risk from ionizing radiation (IR from detonation of dirty bombs or nuclear devices. In a subgroup of patients, ionizing radiation exposure would be followed by a secondary infection. The effects of radiation combined injury are potentially more lethal than either insult in isolation. The purpose of this study was to determine mechanisms of mortality and possible therapeutic targets in radiation combined injury. Mice were exposed to IR with 2.5 Gray (Gy followed four days later by intratracheal methicillin-resistant Staphylococcus aureus (MRSA. While either IR or MRSA alone yielded 100% survival, animals with radiation combined injury had 53% survival (p = 0.01. Compared to IR or MRSA alone, mice with radiation combined injury had increased gut apoptosis, local and systemic bacterial burden, decreased splenic CD4 T cells, CD8 T cells, B cells, NK cells, and dendritic cells, and increased BAL and systemic IL-6 and G-CSF. In contrast, radiation combined injury did not alter lymphocyte apoptosis, pulmonary injury, or intestinal proliferation compared to IR or MRSA alone. In light of the synergistic increase in gut apoptosis following radiation combined injury, transgenic mice that overexpress Bcl-2 in their intestine and wild type mice were subjected to IR followed by MRSA. Bcl-2 mice had decreased gut apoptosis and improved survival compared to WT mice (92% vs. 42%; p<0.01. These data demonstrate that radiation combined injury results in significantly higher mortality than could be predicted based upon either IR or MRSA infection alone, and that preventing gut apoptosis may be a potential therapeutic target.

  15. Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in human renal proximal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ko Eun [Department of Internal Medicine, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Eun Young [Department of Physiology, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Chang Seong; Choi, Joon Seok; Bae, Eun Hui; Ma, Seong Kwon [Department of Internal Medicine, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Kyung Keun [Department of Pharmacology, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Lee, Jong Un [Department of Physiology, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of); Kim, Soo Wan, E-mail: skimw@chonnam.ac.kr [Department of Internal Medicine, Chonnam National University Medical School, Gwangju 501-757 (Korea, Republic of)

    2013-05-10

    Highlights: •MSP/RON system is activated in rat kidney damaged by gentamicin. •MSP inhibits GM-induced cellular apoptosis and inflammation in HK-2 cells. •MSP attenuates GM-induced activation of MAPKs and NF-κB pathways in HK-2 cells. -- Abstract: The present study aimed to investigate whether macrophage-stimulating protein (MSP) treatment attenuates renal apoptosis and inflammation in gentamicin (GM)-induced tubule injury and its underlying molecular mechanisms. To examine changes in MSP and its receptor, recepteur d’origine nantais (RON) in GM-induced nephropathy, rats were injected with GM for 7 days. Human renal proximal tubular epithelial (HK-2) cells were incubated with GM for 24 h in the presence of different concentrations of MSP and cell viability was measured by MTT assay. Apoptosis was determined by flow cytometry of cells stained with fluorescein isothiocyanate-conjugated annexin V protein and propidium iodide. Expression of Bcl-2, Bax, caspase-3, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), IκB-α, and mitogen-activated protein kinases (MAPKs) was analyzed by semiquantitative immunoblotting. MSP and RON expression was significantly greater in GM-treated rats, than in untreated controls. GM-treatment reduced HK-2 cell viability, an effect that was counteracted by MSP. Flow cytometry and DAPI staining revealed GM-induced apoptosis was prevented by MSP. GM reduced expression of anti-apoptotic protein Bcl-2 and induced expression of Bax and cleaved caspase 3; these effects and GM-induced expression of COX-2 and iNOS were also attenuated by MSP. GM caused MSP-reversible induction of phospho-ERK, phospho-JNK, and phospho-p38. GM induced NF-κB activation and degradation of IκB-α; the increase in nuclear NF-κB was blocked by inhibitors of ERK, JNK, p-38, or MSP pretreatment. These findings suggest that MSP attenuates GM-induced inflammation and apoptosis by inhibition of the MAPKs

  16. The prevention of radiation-induced DNA damage and apoptosis in human intestinal epithelial cells by salvianic acid A

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    Yanjun Zhang

    2014-07-01

    Full Text Available The topic of radiation always provokes public debate, and the uses of radiation for therapeutic and other purposes have always been associated with some anxiety. Salvia miltiorrhiza Bunge has been widely used for the treatment of various diseases including cerebrovascular diseases, coronary artery diseases, and myocardial infarction. Salvianolic acid A (SAA d (+-(3,4-dihydroxyphenyl lactic acid is the principal effective, watersoluble constituent of Salvia miltiorrhiza Bunge. In our present study, radiation-induced DNA damage and apoptosis in human intestinal epithelial cells (HIEC in the presence and absence of SAA were examined. We investigated the effects of SAA on ROS formation and the activity of enzymatic antioxidants (SOD, the lipid peroxidative index and the levels of non-enzymatic antioxidant (GSH. Finally, we investigated whether the reduction of radiation-induced cell death caused by SAA might be related to mitochondria-dependent apoptosis. Present findings indicate that SAA is a promising radioprotective agent with a strong antioxidant activity. SAA exerted its protective action on the proliferative activity of HIEC cells as evidenced by decreased cytotoxicity after exposure to γ-radiation. It is possible that SAA achieved its radioprotective action, at least in part, by enhancing DNA repair and the activity of antioxidant enzymes, by scavenging ROS and by inhibiting the mitochondria-dependent apoptotic pathway.

  17. Deoxycholic Acid Could Induce Apoptosis and Trigger Gastric Carcinogenesis on Gastric Epithelial Cells by Quantitative Proteomic Analysis

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    Yanyan Shi

    2016-01-01

    Full Text Available Background. Pathologic duodenogastric reflux can induce or aggravate gastritis because of the presence of bile acids. Bile reflux has been generally considered to be associated with intestinal metaplasia and gastric cancer. However, the pathogenic mechanisms of the effects of bile acids on gastric mucosa are still unknown. Methods. To explore the mechanisms by which bile acids induce gastric mucosal lesions, we examined cell apoptosis in the gastric epithelial cell line GES-1 and investigated the changes in protein profiles of GES-1 cells in response to a bile acid deoxycholic acid using a proteomics approach. Changes in the profiles of the differently expressed proteins were analyzed using the DAVID and STRING programs. Results. We found apoptosis was significantly induced in GES-1 cells by deoxycholic acid. Using liquid chromatographic/tandem mass spectrometric (LC-MS/MS methods, 134 upregulated proteins and 214 downregulated proteins were identified in the bile acid treated GES-1 cells. Bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. Conclusion. These findings may improve the understanding of the molecular mechanisms underlying the pathogenicity of bile acids on gastric mucosa.

  18. Curcumin Inhibits Heat-Induced Apoptosis by Suppressing NADPH Oxidase 2 and Activating the Akt/mTOR Signaling Pathway in Bronchial Epithelial Cells

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    Yuan Peng

    2017-04-01

    Full Text Available Background: Heat causes bronchial epithelial cell apoptosis, which is a known factor contributing to airway damage during inhalation injury. Accumulating evidence has shown the effect of curcumin on inhibiting apoptosis. In this study, we investigated whether curcumin suppresses heat-induced apoptosis in bronchial epithelial cells and the underlying mechanism. Methods: Bronchial epithelial cell line 16HBE140 cells were incubated at either 42 °C, 47 °C, 52 °C, or 57 °C for 5 min in a cell incubator and then returned back to normal culture conditions (37 °C. An in vivo thermal inhalation injury rat model was established with a heat gun blowing hot air into the airway of rats. 16HBE140 cells and lung tissue were obtained for further study with or without curcumin treatment. Cell viability was determined by measuring the absorbance of 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT. 2',7'-dichlorofluorescein diacetate fluorescence was used as a measure of reactive oxygen species (ROS production. Levels of Bcl2, Bax, α-ATP, cleaved Poly (ADP-ribose polymerase (PARP, cleaved caspase-3, gp91phox, p47phox, p67phox, p22phox, p40phox, and Rac were determined by Western blotting. TUNEL staining was used to determine apoptosis. Results: Heat treatment triggered the apoptosis of 16HBE140 cells as shown by the increase in apoptosis molecular markers, including Bcl-2, Bax, cleaved PARP, and cleaved caspase-3. Administration of curcumin significantly inhibited apoptosis of 16HBE140 cells and suppressed the membrane translocation of NADPH oxidase 2 cytosolic components, as well as ROS production. Downregulation of Akt and mTOR phosphorylation induced by heat was also reversed by curcumin. Furthermore, we demonstrated that NADPH oxidase 2 is upstream of Akt/mTOR in heat-induced apoptosis. The protective role of curcumin on bronchial epithelia apoptosis was also confirmed in vivo by a rat inhalation injury model. Conclusion: This study

  19. Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells.

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    Hans Linde Nielsen

    Full Text Available Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05, by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001, suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6 cm/s in control, P<0.05 but showed no difference in permeability for 4 kDa FITC-dextran (FD-4. The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and

  20. Apoptosis induction of human endometriotic epithelial and stromal cells by noscapine

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    Mohammad Rasoul Khazaei

    2016-09-01

    Full Text Available Objective(s: Endometriosis is a complex gynecologic disease with unknown etiology. Noscapine has been introduced as a cancer cell suppressor. Endometriosis was considered as a cancer like disorder, The aim of present study was to investigate noscapine apoptotic effect on human endometriotic epithelial and stromal cells in vitro. Materials and Methods:In this in vitro study, endometrial biopsies from endometriosis patients (n=9 were prepared and digested by an enzymatic method (collagenase I, 2 mg/ml. Stromal and epithelial cells were separated by sequential filtration through a cell strainer and ficoll layering. The cells of each sample were divided into five groups: control (0, 10, 25, 50 and 100 micromole/liter (µM concentration of noscapine and were cultured for three different periods of times; 24, 48 and 72 hr. Cell viability was assessed by colorimetric assay. Nitric oxide (NO concentration was measured by Griess reagent. Cell death was analyzed by Acridine Orange (AO–Ethidium Bromide (EB double staining and Terminal deoxynucleotidyl transferase (TdT dUTP Nick-End Labeling (TUNEL assay. Data were analyzed by one-way ANOVA. Results: Viability of endometrial epithelial and stromal cells significantly decreased in 10, 25, 50 and 100 µM noscapine concentration in 24, 48, 72 hr (P

  1. Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jung Ar [Department of Internal Medicine, Yonsei University College of Medicine, Yonsei University Health System, Seoul 135-270 (Korea, Republic of); Chung, Jin Sil [Laboratory of Molecular Cell Biology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of); Cho, Sang-Ho [Department of Pathology, Pochon CHA University, College of Medicine, Gyeonggi-do (Korea, Republic of); Kim, Hyung Jung, E-mail: khj57@yuhs.ac.kr [Department of Internal Medicine, Yonsei University College of Medicine, Yonsei University Health System, Seoul 135-270 (Korea, Republic of); Yoo, Young Do, E-mail: ydy1130@korea.ac.kr [Laboratory of Molecular Cell Biology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713 (Korea, Republic of)

    2013-09-20

    Highlights: •Romo1 mediates oxidative stress-induced mitochondrial ROS production. •Romo1 induction by oxidative stress plays an important role in oxidative stress-induced apoptosis. •Romo1 overexpression correlates with epithelial cell death in patients with IPF. -- Abstract: Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H{sub 2}O{sub 2}) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H{sub 2}O{sub 2} treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.

  2. Giardia duodenalis assemblage-specific induction of apoptosis and tight junction disruption in human intestinal epithelial cells: effects of mixed infections.

    Science.gov (United States)

    Koh, Wan Hon; Geurden, Thomas; Paget, Tim; O'Handley, Ryan; Steuart, Robert F; Thompson, R C Andrew; Buret, Andre G

    2013-04-01

    In view of the interest in genotype-specific pathogenesis in Giardia duodenalis , the aim of the present study was to examine the effects of infection with different, or mixed, G. duodenalis assemblages on the integrity of human intestinal epithelia. To that end, human epithelial cells (HCT-8) were cultured and exposed to different G. duodenalis assemblages (A, B, and E) or a combination of these assemblages. Epithelial disruption and apoptosis were evaluated by fluorescent microscopy and apoptotic oligonucleosome quantification. The results indicate that infection with trophozoites disrupts epithelial tight junctions and induces varying degrees of enterocyte apoptosis, depending on the infecting assemblage. All disruptions were caspase-3 dependent and were more pronounced when caused by a non-host specific assemblage. Furthermore, infections by isolates in combination with isolates from another assemblage enhanced the epithelial disruption and apoptosis. Further studies in vitro and in vivo are required to confirm the mechanisms of enhanced pathogenicity of mixed or non-host specific (or both) G. duodenalis infections. Findings in the present study point to the potential pathogenic importance of intra-species polyparasitism in giardiasis.

  3. [Effect of MTRR gene on apoptosis and autophagy pathways in multiresistant epithelial ovarian cancer].

    Science.gov (United States)

    Chen, J; Wang, Q; Zhang, W; Li, L

    2016-04-25

    To explore the effect of down-regulated methionine synthase reductase(MTRR)gene on the apoptosis and autophagy pathway, and offer a possible approach for the MTRR to reverse the multi-resistant ovarian cancer. (1)The experiment was divided into 3 groups, SKOV3/DDP-MTRRi(down-regulated MTRR group), SKOV3/DDP-NC(negative control group), and SKOV3/DDP(blank control group). Different concentration of cisplatin(0, 1, 2, and 4 μg/ml)treated on 3 groups cells. The apoptosis rate was measured by flow cytometry(FCM). Autophagy was detected by immunofluorescence. Autophagy microtubule associated protein light chain 3β(LC3B)and p62 were detected by western blot. The formation of autophagosome of cells was observed by transmission electron microscope.(2)Detection of autophagy and apoptosis of SKOV3/DDP-MTRRi induced by rapamycin. The experiment was divided into 4 groups included rapamycin group(5 nmol/L rapamycin), rapamycin+cisplatin group(5 nmol/L rapamycin+ 4 μg/ml cisplatin), cisplatin group(4 μg/ml cisplatin)and blank control group. LC3B and p62 protein were detected by western blot. The survival rate cells were detected by methyl thiazolyl tetrazolium(MTT)method. The apoptosis rate was measured by FCM.(3)The 3 groups cells(SKOV3/DDP, SKOV3/DDP-NC and SKOV3/DDP-MTRRi)induced by a certain concentration of cisplatin(4 μg/ml)after 48 hours, then detecting the protein expression of caspase, Bcl-2 family in apoptosis pathway and the key proteins in phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)autophagy pathways by western blot, getting the time when the proteins' expression changed. (1)The 3 groups cells(SKOV3/DDP, SKOV3/DDP-NC, and SKOV3/DDP-MTRRi)induced by a certain concentration of cisplatin(4 μg/ml)after 48 hours, apoptosis and autophagy of 3 groups of cells were gradually increased with the increased concentration of cisplatin. The apoptosis rate of SKOV3/DDP-MTRRi cells[(26.2 ± 1.4)%]were significantly increased compared with the SKOV3/DDP-NC cells or

  4. Involvement of the Extrinsic and Intrinsic Pathways in Ultraviolet B-Induced Apoptosis of Corneal Epithelial Cells

    Science.gov (United States)

    Ubels, John L.; Glupker, Courtney D.; Schotanus, Mark P.; Haarsma, Loren D.

    2015-01-01

    The goal of this study was to elucidate the pathway by which UVB initiates efflux of K+ and subsequently apoptosis in human corneal limbal epithelial (HCLE) cells. The initial focus of the study was on the extrinsic pathway involving Fas. HCLE cells transfected with Fas siRNA were exposed to 80–150 mJ/cm2 UVB and incubated in culture medium with 5.5 mM K+. Knock down of Fas resulted in limited reduction in UVB-induced caspase-8 and -3 activity. Patch-clamp recordings showed no difference in UVB-induced normalized K+ currents between Fas transfected and control cells. Knockdown of caspase-8 had no effect on the activation of caspase-3 following UVB exposure, while a caspase-8 inhibitor completely eliminated UVB activation of caspase-3. This suggests that caspase-8 is a robust enzyme, able to activate caspase-3 via residual caspase-8 present after knockdown, and that caspase-8 is directly involved in the UVB activation of caspase-3. Inhibition of caspase-9 significantly decreased the activation of caspases-8 and -3 in response to UVB. Knockdown of Apaf-1, required for activation of caspase-9, resulted in a significant reduction in UVB-induced activation of caspases-9, -8, and -3. Knockdown of Apaf-1 also inhibited intrinsic and UVB-induced levels of apoptosis, as determined by DNA fragmentation measured by TUNEL assay. In UVB exposed cultures treated with caspase-3 inhibitor, the percentage of apoptotic cells was reduced to control levels, confirming the necessity of caspase-3 activation in DNA fragmentation. The lack of effect of Fas knockdown on K+ channel activation, as well as the limited effect on activation of caspases-8 and -3, strongly suggest that Fas and the extrinsic pathway is not of primary importance in the initiation of apoptosis in response to UVB in HCLE cells. Inhibition of caspase-8 and -3 activation following inhibition of caspase-9, as well as reduction in activation of caspases-9, -8, and -3 and DNA fragmentation in response to Apaf-1

  5. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution

    International Nuclear Information System (INIS)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz; Andre, Veronique; Le Goff, Jeremie; Sichel, Francois; Roy Saint-Georges, Francoise; Mulliez, Philippe

    2012-01-01

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% 2 /g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). 32 P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC 50 = 74.63 μg/mL and L132: LC-5-0 = 75.36 μg/mL). Exposure to desorbed particles (MA: C1= 61.11 μg/mL and L132 : C2 = 61.71 μg/mL) and B[a]P (1 μM) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that they were competent in terms of metabolic activation of PAHs. The

  6. Concentrations of garenoxacin in plasma, bronchial mucosa, alveolar macrophages and epithelial lining fluid following a single oral 600 mg dose in healthy adult subjects.

    Science.gov (United States)

    Andrews, J; Honeybourne, D; Jevons, G; Boyce, M; Wise, R; Bello, A; Gajjar, D

    2003-03-01

    A microbiological assay was used to measure concentrations of garenoxacin (BMS-284756) in plasma, bronchial mucosa (BM), alveolar macrophages (AM) and epithelial lining fluid (ELF), following a single 600 mg oral dose. Twenty-four healthy subjects were allocated into four nominal time intervals after the dose, 2.5-3.5, 4.5-5.5, 10.5-11.5 and 23.5-24.5 h. Mean concentrations in plasma, BM, AM and ELF, respectively, for the four nominal time windows were for 2.5-3.5 h 10.0 mg/L (S.D. 2.8), 7.0 mg/kg (S.D. 1.3), 106.1 mg/L (S.D. 60.3) and 9.2 mg/L (S.D. 3.6); 4.5-5.5 h 8.7 mg/L (S.D. 2.2), 6.0 mg/kg (S.D. 1.9), 158.6 mg/L (S.D. 137.4) and 14.3 mg/L (S.D. 8.2); 10.5-11.5 h 6.1 mg/L (S.D. 1.9), 4.0 mg/kg (S.D. 1.4), 76.0 mg/L (S.D. 47.7) and 7.9 mg/L (S.D. 4.6); and 23.5-24.5 h 2.1 mg/L (S.D. 0.5), 1.7 mg/kg (S.D. 0.7), 30.7 mg/L (S.D. 12.9) and 3.3 mg/L (S.D. 2.3). Concentrations at all sites exceeded MIC(90)s for the common respiratory pathogens Haemophilus influenzae (0.03 mg/L), Moraxella catarrhalis (0.015 mg/L) and Streptococcus pneumoniae (0.06 mg/L). These data suggest that garenoxacin should be effective in the treatment of community-acquired pneumonia and chronic obstructive pulmonary disease.

  7. [Prolonged exposure to crystalline silica Min-U-Sil-5 influences apoptosis or extracellular matrix genes expression in human bronchial epithelial cells].

    Science.gov (United States)

    Gambelunghe, A; Antognelli, C; Murgia, N; dell'Omo, M; Talesa, V N; Muzi, G

    2011-01-01

    Crystalline silica (Min-U-Sil-5) induces oxidative stress in human bronchial epithelial cells (BEAS-2B), through the intracellular accumulation of ROS that cause oxidative damage leading to the degradation of extracellular matrix (ECM) proteins and to the loss of cell adhesion molecules inducing apoptosis and genotoxic damage. This paper briefly summarizes some of the recent findings from our laboratories with emphasis on the molecular events by which the cronic and cumulative exposure to crystalline silica can induce cellular damage that promotes changes in extracellular matrix and in apoptosis gene expression.

  8. Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

    Science.gov (United States)

    Kunsmann, Lisa; Greune, Lilo; Bauwens, Andreas; Zhang, Wenlan; Kuczius, Thorsten; Kim, Kwang Sik; Mellmann, Alexander; Schmidt, M. Alexander; Karch, Helge

    2013-01-01

    Enterohemorrhagic Escherichia coli (EHEC) strains cause diarrhea and hemolytic uremic syndrome resulting from toxin-mediated microvascular endothelial injury. EHEC hemolysin (EHEC-Hly), a member of the RTX (repeats-in-toxin) family, is an EHEC virulence factor of increasingly recognized importance. The toxin exists as free EHEC-Hly and as EHEC-Hly associated with outer membrane vesicles (OMVs) released by EHEC during growth. Whereas the free toxin is lytic towards human endothelium, the biological effects of the OMV-associated EHEC-Hly on microvascular endothelial and intestinal epithelial cells, which are the major targets during EHEC infection, are unknown. Using microscopic, biochemical, flow cytometry and functional analyses of human brain microvascular endothelial cells (HBMEC) and Caco-2 cells we demonstrate that OMV-associated EHEC-Hly does not lyse the target cells but triggers their apoptosis. The OMV-associated toxin is internalized by HBMEC and Caco-2 cells via dynamin-dependent endocytosis of OMVs and trafficked with OMVs into endo-lysosomal compartments. Upon endosome acidification and subsequent pH drop, EHEC-Hly is separated from OMVs, escapes from the lysosomes, most probably via its pore-forming activity, and targets mitochondria. This results in decrease of the mitochondrial transmembrane potential and translocation of cytochrome c to the cytosol, indicating EHEC-Hly-mediated permeabilization of the mitochondrial membranes. Subsequent activation of caspase-9 and caspase-3 leads to apoptotic cell death as evidenced by DNA fragmentation and chromatin condensation in the intoxicated cells. The ability of OMV-associated EHEC-Hly to trigger the mitochondrial apoptotic pathway in human microvascular endothelial and intestinal epithelial cells indicates a novel mechanism of EHEC-Hly involvement in the pathogenesis of EHEC diseases. The OMV-mediated intracellular delivery represents a newly recognized mechanism for a bacterial toxin to enter host cells in

  9. Comparative study of the effects of PM1-induced oxidative stress on autophagy and surfactant protein B and C expressions in lung alveolar type II epithelial MLE-12 cells.

    Science.gov (United States)

    Bai, Ru; Guan, Longfei; Zhang, Wei; Xu, Jinxia; Rui, Wei; Zhang, Fang; Ding, Wenjun

    2016-12-01

    There is a strong link between smaller air pollution particles and a range of serious health conditions. Thus, there is a need for understanding the impacts of airborne fine particulate matter (PM) with an aerodynamic diameter of PM1) on lung alveolar epithelial cells. In the present study, mouse lung epithelial type II cell MLE-12 cells were used to examine the intracellular oxidative responses and the surfactant protein expressions after exposure to various concentrations of PM1 collected from an urban site and a steel-factory site (referred as uPM1 and sPM1 hereafter, respectively). Physicochemical characterization of PM1 was performed by using scanning electron microscopy and transmission electron microscopy. Cytotoxicity and autophagy induced by PM1 were assessed by using comprehensive approaches after MLE-12 cells were exposed to different concentrations of PM1 for various times. Expression of surfactant proteins B and C in MLE-12 cells was determined by Western blotting. All of the tested PM1 induced cytotoxicity evidenced by significant decrease of cell viability and increase of lactate dehydrogenase (LDH) release in a time- and concentration-dependent manner in the exposed cells compared with the unexposed cells. A similar pattern of increase of intercellular reactive oxygen species (ROS) generation and decrease of superoxide dismutase (SOD) and catalase (CAT) activities was also observed. PM1-induced autophagy was evidenced by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and increased levels of beclin1. Data from Western blotting showed significant decrease of surfactant protein B and C expressions. Relatively high concentrations of transition metals, including Fe, Cu and Mn, may be responsible for the higher toxicity of sPM1 compared with uPM1. Moreover, pretreatment with N-acetylcysteine (NAC) or Chelex (a metal chelating agent, which removes a large suite of metals from PM1) prevented the increase of

  10. Methoxyphenyl chalcone sensitizes aggressive epithelial cancer to cisplatin through apoptosis induction and cancer stem cell eradication.

    Science.gov (United States)

    Su, Yu-Kai; Huang, Wen-Chien; Lee, Wei-Hwa; Bamodu, Oluwaseun Adebayo; Zucha, Muhammad Ary; Astuti, Indwiani; Suwito, Heri; Yeh, Chi-Tai; Lin, Chien-Min

    2017-05-01

    Current standard chemotherapy for late stage ovarian cancer is found unsuccessful due to relapse after completing the regimens. After completing platinum-based chemotherapy, 70% of patients develop relapse and resistance. Recent evidence proves ovarian cancer stem cells as the source of resistance. Therefore, treatment strategy to target both cancer stem cells and normal stem cells is essential. In this study, we developed a novel chalcone derivative as novel drug candidate for ovarian cancer treatment. We found that methoxyphenyl chalcone was effective to eliminate ovarian cancer cells when given either as monotherapy or in combination with cisplatin. We found that cell viability of ovarian cancer cells was decreased through apoptosis induction. Dephosphorylation of Bcl2-associated agonist of cell death protein was increased after methoxyphenyl chalcone treatment that led to activation of caspases. Interestingly, this drug also worked as a G2/M checkpoint modulator with alternative ways of DNA damage signal-evoking potential that might work to increase response after cisplatin treatment. In addition, methoxyphenyl chalcone was able to suppress autophagic flux and stemness regulator in ovarian spheroids that decreased their survival. Therefore, combination of methoxyphenyl chalcone and cisplatin showed synergistic effects. Taken together, we believe that our novel compound is a promising novel therapeutic agent for effective clinical treatment of ovarian cancer.

  11. Detection of epithelial apoptosis in pelvic ileal pouches for ulcerative colitis and familial adenomatous polyposis

    Directory of Open Access Journals (Sweden)

    Velloso Lício A

    2010-01-01

    Full Text Available Abstract Background Ileal pouch-anal anastomosis (IPAA is the surgical procedure of choice for patients with refractory ulcerative colitis (UC and for familial adenomatous polyposis (FAP with many rectal polyps. Pouchitis is one of the more frequent complications after IPAA in UC patients; however, it is rare in FAP. Objective Evaluate pro-apoptotic activity in endoscopically and histological normal mucosa of the ileal pouch in patients with UC and FAP. Methods Eighteen patients (nine with UC and nine with FAP with J pouch after total rectocolectomy were studied. Biopsies were obtained from the mucosa of the pouch and from normal ileum. The specimens were snap-frozen and the expressions of Bax and Bcl-2 were determined by immunoblot of protein extracts and by immunohistochemistry analysis. FADD, Caspase-8, APAF-1 and Caspase-9 were evaluated by immunoprecipitation and immunoblot. Results Patients with UC had significantly higher protein levels of Bax and APAF-1, Caspase-9 than patients with FAP, but were similar to controls. The expressions of Bcl-2 and FADD, Caspase-8 were similar in the groups. Immunohistochemistry for Bax showed less intensity of immunoreactions in FAP than in UC and Controls. Bcl-2 immunostaining was similar among the groups. Conclusion Patients with FAP present lower levels of pro-apoptotic proteins in all methods applied, even in the absence of clinical and endoscopic pouchitis and dysplasia in the histological analysis. These findings may explain a tendency of up-regulation of apoptosis in UC patients, resulting in higher rates of progression to pouchitis in these patients, which could correlate with mucosal atrophy that occurs in inflamed tissue. However, FAP patients had low pro-apoptotic activity in the mucosa, and it could explain the tendency to low cell turn over and presence of adenomas in this syndrome.

  12. In vitro effects of water-pipe smoke condensate on the endocytic activity of Type II alveolar epithelial cells (A549 with bacillus Calmette–Guérin

    Directory of Open Access Journals (Sweden)

    Ian M Adcock

    2016-01-01

    Conclusion: WPC exposure increased epithelial cells' permeability and death and enhanced their capacity for macropinocytosis. Our in vitro data suggest possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacteria. Further studies will be conducted to understand the mechanism of action of WPC.

  13. Distinction Between Cell Proliferation and Apoptosis Signals Regulated by Brain-Derived Neurotrophic Factor in Human Periodontal Ligament Cells and Gingival Epithelial Cells.

    Science.gov (United States)

    Kashiwai, Kei; Kajiya, Mikihito; Matsuda, Shinji; Ouhara, Kazuhisa; Takeda, Katsuhiro; Takata, Takashi; Kitagawa, Masae; Fujita, Tsuyoshi; Shiba, Hideki; Kurihara, Hidemi

    2016-07-01

    Previously, we reported that brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration by inducing periodontal ligament cell proliferation in vivo. In addition, the down growth of gingival epithelial cells, which comprises a major obstacle to the regeneration, was not observed. However, the underlying molecular mechanism is still unclear. Therefore, this study aimed to investigate the effect of BDNF on cell proliferation and apoptosis in human periodontal ligament (HPL) cells and human gingival epithelial cells (OBA9 cells) and to explore the molecular mechanism in vitro. HPL cells dominantly expressed a BDNF receptor, TrkB, and BDNF increased cell proliferation and ERK phosphorylation. However, its proliferative effect was diminished by a MEK1/2 inhibitor (U0126) and TrkB siRNA transfection. Otherwise, OBA9 cells showed a higher expression level of p75, which is a pan-neurotrophin receptor, than that of HPL cells. BDNF facilitated not cell proliferation but cell apoptosis and JNK phosphorylation in OBA9 cells. A JNK inhibitor (SP600125) and p75 siRNA transfection attenuated the BDNF-induced cell apoptosis. Moreover, OBA9 cells pretreated with SP600125 or p75 siRNA showed cell proliferation by BDNF stimulation, though it was reduced by U0126 and TrkB siRNA. Interestingly, overexpression of p75 in HPL cells upregulated cell apoptosis and JNK phosphorylation by BDNF treatment. These results indicated that TrkB-ERK signaling regulates BDNF-induced cell proliferation, whereas p75-JNK signaling plays roles in cell apoptotic and cytostatic effect of BDNF. Overall, BDNF activates periodontal ligament cells proliferation and inhibits the gingival epithelial cells growth via the distinct pathway. J. Cell. Biochem. 117: 1543-1555, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  14. Berberine activates Nrf2 nuclear translocation and inhibits apoptosis induced by high glucose in renal tubular epithelial cells through a phosphatidylinositol 3-kinase/Akt-dependent mechanism.

    Science.gov (United States)

    Zhang, Xiuli; Liang, Dan; Lian, Xu; Jiang, Yan; He, Hui; Liang, Wei; Zhao, Yue; Chi, Zhi-Hong

    2016-06-01

    Apoptosis of tubular epithelial cells is a major feature of diabetic kidney disease, and hyperglycemia triggers the generation of free radicals and oxidant stress in tubular cells. Berberine (BBR) is identified as a potential anti-diabetic herbal medicine due to its beneficial effects on insulin sensitivity, glucose metabolism and glycolysis. In this study, the underlying mechanisms involved in the protective effects of BBR on high glucose-induced apoptosis were explored using cultured renal tubular epithelial cells (NRK-52E cells) and human kidney proximal tubular cell line (HK-2 cells). We identified the pivotal role of phosphatidylinositol 3-kinase (PI3K)/Akt in BBR cellular defense mechanisms and revealed the novel effect of BBR on nuclear factor (erythroid-derived 2)-related factor-2 (Nrf2) and heme oxygenase (HO)-1 in NRK-52E and HK-2 cells. BBR attenuated reactive oxygen species production, antioxidant defense (GSH and SOD) and oxidant-sensitive proteins (Nrf2 and HO-1), which also were blocked by LY294002 (an inhibitor of PI3K) in HG-treated NRK-52E and HK-2 cells. Furthermore, BBR improved mitochondrial function by increasing mitochondrial membrane potential. BBR-induced anti-apoptotic function was demonstrated by decreasing apoptotic proteins (cytochrome c, Bax, caspase3 and caspase9). All these findings suggest that BBR exerts the anti-apoptosis effects through activation of PI3K/Akt signal pathways and leads to activation of Nrf2 and induction of Nrf2 target genes, and consequently protecting the renal tubular epithelial cells from HG-induced apoptosis.

  15. Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

    Energy Technology Data Exchange (ETDEWEB)

    Parra, E.R.; Pincelli, M.S. [Departamento de Patologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Teodoro, W.R.; Velosa, A.P.P. [Disciplina de Reumatologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil); Martins, V.; Rangel, M.P.; Barbas-Filho, J.V.; Capelozzi, V.L. [Departamento de Patologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP (Brazil)

    2014-06-04

    Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.

  16. Advanced glycation end products induce human corneal epithelial cells apoptosis through generation of reactive oxygen species and activation of JNK and p38 MAPK pathways.

    Directory of Open Access Journals (Sweden)

    Long Shi

    Full Text Available Advanced Glycation End Products (AGEs has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA induced Human telomerase-immortalized corneal epithelial cells (HUCLs apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE. AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.

  17. Pulmonary alveolar proteinosis

    Science.gov (United States)

    PAP; Alveolar proteinosis; Pulmonary alveolar phospholipoproteinosis; Alveolar lipoproteinosis phospholipidosis ... PAP is unknown. In others, it occurs with lung infection or an immune problem. It also can ...

  18. TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract.

    Science.gov (United States)

    Li, Guoxing; Song, Huiyang; Chen, Lei; Yang, Weihua; Nan, Kaihui; Lu, Peirong

    2017-07-01

    Age-related cataract is among the most common chronic disorders of ageing and the apoptosis of lens epithelial cells contributes to non-congenital cataract development. We amid to explore the role of TUG1 and miR-421 in the age-related cataract. The expression level of TUG1, miR-421 and caspase-3 were detected by RT-qPCR. The apoptotic-related protein, caspase-3, Bax and blc-2 were analyzed by western blot. We performed ultraviolet (UV) irradiation to induce SAR01/04 cell apoptosis which was analyzed by flow cytometry. RIP pull-down and luciferase reporter assay were used to verified the combination and regulating among TUG1, miR-421 and caspase-3. Here, we observed that the expression level of TUG1 and caspase-3 in the anterior lens capsules of age-related cataract were significantly higher and miR-421 was significantly lower than that in the normal anterior lens capsules. The apoptosis-related protein, caspase-3, Bax and blc-2 were abnormal expression in the anterior lens capsules of age-related cataract tissue. Our data showed that the expression level of TUG1 and caspase-3 and cell apoptosis rate in SAR01/04 cells treated with UV irradiation was remarkably higher than that in the control. TUG1 negatively regulated miR-421 expression and promoted UV irradiation-induced SAR01/04 cell apoptosis. However, miR-421 inhibitor and pcDNA-caspase-3 could reverse the action of the SRA01/04 cell apoptosis by si-TUG1, which suggested TUG1 promoted UV irradiation-induced apoptosis through downregulating miR-421 expression. Furthermore, this study confirmed TUG1 could been in combination with miR-421, and TUG1 and caspase-3 were both a directly target of miR-421. TUG1 modulated lens epithelial cell apoptosis through miR-421/caspase-3 axis. These findings will offer a novel insight into the pathogenesis of cataract. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Role of phospholipase A2 (PLA2) inhibitors in attenuating apoptosis of the corneal epithelial cells and mitigation of Acanthamoeba keratitis

    Science.gov (United States)

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2013-01-01

    The aim of this study is to determine if the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites induces apoptosis of corneal epithelial cells through a cytosolic phospholipase A2α (cPLA2α)-mediated pathway. The efficacy of cPLA2α inhibitors to provide protection against Acanthamoeba keratitis was examined in vivo. Chinese hamster corneal epithelial (HCORN) cells were incubated with or without MIP-133. MIP-133 induces significant increase in cPLA2α and macrophage inflammatory protein-2 (MIP-2/CXCL2) levels from corneal cells. Moreover, cPLA2α inhibitors, MAFP (Methyl-arachidonyl fluorophosphonate) and AACOCF3 (Arachidonyl trifluoromethyl ketone), significantly reduce cPLA2α and CXCL2 from these cells (Pkeratitis as compared with control animals. Collectively, the results indicate that cPLA2α is involved in MIP-133 induced apoptosis of corneal epithelial cells, polymorphonuclear neutrophil infiltration, and production of CXCL2. Moreover, cPLA2α inhibitors can be used as a therapeutic target in Acanthamoeba keratitis. PMID:23792108

  20. Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

    Directory of Open Access Journals (Sweden)

    Liu Feng-Lin

    2012-10-01

    Full Text Available Abstract Background Lipoteichoic acid (LTA, a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS, could induce surfactant protein-A (SP-A production in human alveolar epithelial (A549 cells. Objectives In this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms. Methods A549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF-κB, extracellular signal-regulated kinase 1/2 (ERK1/2, and mitogen-activated/extracellular signal-regulated kinase kinase (MEK1 were determined. Results Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA. Conclusions Therefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

  1. Lipoxin A4 promotes lung epithelial repair whilst inhibiting fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Shengxing Zheng

    2016-10-01

    Full Text Available Therapy that promotes epithelial repair whilst protecting against fibroproliferation is critical for restoring lung function in acute and chronic respiratory diseases. Primary human alveolar type II cells were used to model the effects of lipoxin A4 in vitro upon wound repair, proliferation, apoptosis and transdifferention. Effects of lipoxin A4 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also assessed. Lipoxin A4 promoted type II cell wound repair and proliferation, blocked the negative effects of soluble Fas ligand/tumour necrosis factor α upon cell proliferation, viability and apoptosis, and augmented the epithelial cell proliferative response to bronchoaveolar lavage fluid (BALF from acute respiratory distress syndrome (ARDS. In contrast, Lipoxin A4 reduced fibroblast proliferation, collagen production and myofibroblast differentiation induced by transforming growth factor β and BALF from ARDS. The effects of Lipoxin A4 were phosphatidylinositol 3′-kinase dependent and mediated via the lipoxin A4 receptor. Lipoxin A4 appears to promote alveolar epithelial repair by stimulating epitheial cell wound repair, proliferation, reducing apoptosis and promoting trans-differentiation of alveolar type II cells into type I cells. Lipoxin A4 reduces fibroblast proliferation, collagen production and myofibroblast differentiation. These data suggest that targeting lipoxin actions may be a therapeutic strategy for treating the resolution phase of ARDS.

  2. [Effects of heme oxygenase-1/carbon monoxide pathway on the mitochondrial fusion in rat alveolar epithelial type II cells stimulated by lipopolysaccharide].

    Science.gov (United States)

    Jia, Haojuan; Shi, Jia; Dong, Shu'an; Zhang, Yuan; Yu, Jianbo

    2018-03-01

    To investigate the effects of heme oxygenase-1/carbon monoxide (HO-1/CO) pathway on mitochondrial fusion in rat alveolar epithelial type II cells (AEC II) stimulated by lipopolysaccharide (LPS). Once the cultured in vitro rat AEC II cells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups (n = 5 each). RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECII cells. The cells in carbon monoxide-releasing molecule-2 (CORM-2, in vitro CO release agent) + LPS group (CL group) and Hemin (HO-1 inducer) + LPS group (HL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-IX (ZnPP-IX, HO-1 inhibitor) + LPS group (ZL group) was pretreated with 10 μmol/L ZnPP-IX for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 + ZnPP-IX + LPS group (CZL group) and Hemin + ZnPP-IX + LPS group (HZL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were determined by enzyme linked immunosorbent assay (ELISA), the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (OPA1) were determined by Western Blot. Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 (ng/L): 48.6±3.7, 48.4±3.1 vs. 58.7±2.5; TNF-α (ng/L): 40.7±5.3, 39.4±4.3 vs. 51.8±5

  3. Suppression of Apoptosis by Basement Membrane Requires three-dimensional Tissue Organization and Withdrawal from the Cell Cycle

    Energy Technology Data Exchange (ETDEWEB)

    Boudreau, N.; Werb, Z.; Bissell, M.J.

    1995-12-28

    The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce {beta}-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the G1 cyclin kinase inhibitor p21/WAF-I and positive proliferative signals including c-myc and cyclin Dl were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor a and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-{beta}1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.

  4. Impaired nuclear factor erythroid 2-related factor 2 expression increases apoptosis of airway epithelial cells in patients with chronic obstructive pulmonary disease due to cigarette smoking.

    Science.gov (United States)

    Yamada, Kazuhiro; Asai, Kazuhisa; Nagayasu, Fumihiro; Sato, Kanako; Ijiri, Naoki; Yoshii, Naoko; Imahashi, Yumiko; Watanabe, Tetsuya; Tochino, Yoshihiro; Kanazawa, Hiroshi; Hirata, Kazuto

    2016-02-09

    Cigarette smoking-induced oxidative stress is known to be a key mechanism in COPD pathogenesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a central transcription factor that regulates the antioxidant defense system. The aim of this study was to compare Nrf2 expression in COPD subjects and control subjects, and to determine the role of Nrf2 in protecting against oxidative stress-induced apoptosis. We enrolled 8 COPD subjects and 7 control subjects in this study. We performed bronchial brushing by bronchoscopy and obtained bronchial epithelial cells from the airways. Nrf2 expression in bronchial epithelial cells was evaluated by real-time PCR and Western blotting. We examined the effect of 10 or 15 % cigarette smoke extract (CSE) induced A549 cells apoptosis using a time-lapse cell imaging assay with caspase-3/7 activation detecting reagent and performed Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling assay for confirming A549 cells apoptosis. We also examined the effects of Nrf2 knockdown and, 0.1, 0.5, and 1.0 mM N-acetyl cysteine on CSE-induced apoptosis. Statistical analyses were performed using t-test, paired t-test or an analysis of variance followed by the Tukey-Kramer method. Nrf2 mRNA expression in COPD subjects was significantly lower than that in control subjects and Nrf2 mRNA were negatively correlated with pack year. Nrf2 protein in COPD subjects was significantly lower than that in control subjects. CSE-induced A549 cells apoptosis was increased in a time-, concentration-dependent manner, and was significantly increased by Nrf2 knockdown. N-acetyl cysteine significantly ameliorated CSE-induced apoptosis. Nrf2 expression was lower in COPD patients than in control subjects. Nrf2 might have a protective role against apoptosis caused by CSE-induced oxidative stress. These results suggest an involvement of Nrf2 in COPD and administration of antioxidants to patients with COPD might be a basic therapeutic option.

  5. Human MCF10A mammary epithelial cells undergo apoptosis following actin depolymerization that is independent of attachment and rescued by Bcl-2.

    Science.gov (United States)

    Martin, S S; Leder, P

    2001-10-01

    Many tumor cells are impaired in adhesion-regulated apoptosis, which contributes to their metastatic potential. However, suppression of this apoptotic pathway in untransformed cells is not mediated only by adhesion to the extracellular matrix but also through the resulting ability to spread and adopt a distinct morphology. Since cell spreading is dependent on the integrity of the actin microfilament cytoskeleton, we sought to determine if actin depolymerization was sufficient to induce apoptosis, even in the presence of continuous attachment. For this study, we used a human mammary epithelial cell line (MCF10A), which is immortalized but remains adhesion dependent for survival. Treatment of MCF10A cells with latrunculin-A (LA), an inhibitor of actin polymerization, rapidly led to disruption of the actin cytoskeleton and caused cell rounding but preserved attachment. Initiation of apoptosis in LA-treated MCF10A cells was detected by mitochondrial localization of the Bax apoptotic protein, which was prevented by overexpression of Bcl-2. DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage in LA-treated MCF10A cells indicated progression to the execution phase of apoptosis. The MDA-MB-453 cell line, which was derived from a metastatic human mammary tumor, was resistant to PARP cleavage and loss of viability in response to actin depolymerization. Stable overexpression of Bcl-2 in the untransformed MCF10A cells was able to recapitulate the resistance to apoptosis found in the tumor cell line. We demonstrate that inhibition of actin polymerization is sufficient to stimulate apoptosis in attached MCF10A cells, and we present a novel role for Bcl-2 in cell death induced by direct disruption of the actin cytoskeleton.

  6. Inhibition of invasion and induction of apoptosis by curcumin in H-ras-transformed MCF10A human breast epithelial cells.

    Science.gov (United States)

    Kim, M S; Kang, H J; Moon, A

    2001-08-01

    Curcumin, a dietary pigment in turmeric, posseses anti-carcinogenic and anti-metastatic properties. The present study was conducted to study in vitro chemopreventive effects of curcumin in transformed breast cells. Here, we show that curcumin inhibits H-ras-induced invasive phenotype in MCF10A human breast epithelial cells (H-ras MCF10A) and downregulates matrix metalloproteinase (MMP)-2 dose-dependently. Curcumin exerted cytotoxic effect on H-ras MCF10A cells in a concentration-dependent manner. Curcumin-induced cell death was mainly due to apoptosis in which a prominent downregulation of Bcl-2 and upregulation of Bax were involved. We also suggest a possible involvement of caspase-3 in curcumin-induced apoptosis. Curcumin treatment resulted in the production of reactive oxygen species (ROS) in H-ras MCF10A cells. Apoptotic event by curcumin was significantly inhibited by pretreatment of an antioxidant N-acetyl-L-cysteine (NAC), suggesting redox signaling as a mechanism responsible for curcumin-induced apoptosis in H-ras MCF10A cells. Taken together, our results demonstrate that curcumin inhibits invasion and induces apoptosis, proving the chemopreventive potential of curcumin.

  7. Autocrine production of TGF-β confers resistance to apoptosis after an epithelial-mesenchymal transition process in hepatocytes: Role of EGF receptor ligands

    International Nuclear Information System (INIS)

    Castillo, Gaelle del; Murillo, Miguel M.; Alvarez-Barrientos, Alberto; Bertran, Esther; Fernandez, Margarita; Sanchez, Aranzazu; Fabregat, Isabel

    2006-01-01

    Transforming growth factor-beta (TGF-β) induces apoptosis in fetal rat hepatocytes. However, a subpopulation of these cells survives, concomitant with changes in phenotype, reminiscent of an epithelial-mesenchymal transition (EMT). We have previously suggested that EMT might confer cell resistance to apoptosis (Valdes et al., Mol. Cancer Res., 1: 68-78, 2002). However, the molecular mechanisms responsible for this resistance are not explored yet. In this work, we have isolated and subcultured the population of hepatocytes that suffered the EMT process and are resistant to apoptosis (TGF-β-treated fetal hepatocytes: TβT-FH). We prove that they secrete mitogenic and survival factors, as analyzed by the proliferative and survival capacity of conditioned medium. Inhibition of the epidermal growth factor receptor (EGFR) sensitizes TβT-FH to die after serum withdrawal. TβT-FH expresses high levels of transforming growth factor-alpha (TGF-α) and heparin-binding EGF-like growth factor (HB-EGF) and shows constitutive activation of the EGFR pathway. A blocking anti-TGF-α antibody restores the capacity of cells to die. TGF-β, which is expressed by TβT-FH, mediates up-regulation of TGF-α and HB-EGF expression in those cells. In summary, results suggest that an autocrine loop of TGF-β confers resistance to apoptosis after an EMT process in hepatocytes, through the increase in the expression of EGFR ligands

  8. Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

    Science.gov (United States)

    Ni, B; Bai, F F; Wei, Y; Liu, M J; Feng, Z X; Xiong, Q Y; Hua, L Z; Shao, G Q

    2015-09-25

    Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

  9. Relaxin attenuates aristolochic acid induced human tubular epithelial cell apoptosis in vitro by activation of the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Xie, Xiang-Cheng; Zhao, Ning; Xu, Qun-Hong; Yang, Xiu; Xia, Wen-Kai; Chen, Qi; Wang, Ming; Fei, Xiao

    2017-06-01

    Aristolochic acid nephropathy remains a leading cause of chronic kidney disease (CKD), however few treatment strategies exist. Emerging evidence has shown that H2 relaxin (RLX) possesses powerful antifibrosis and anti-apoptotic properties, therefore we aimed to investigate whether H2 relaxin can be employed to reduce AA-induced cell apoptosis. Human proximal tubular epithelial (HK-2) cells exposed to AA-I were treated with or without administration of H2 RLX. Cell viability was examined using the WST-8 assay. Apoptotic morphologic alterations were observed using the Hoechst 33342 staining method. Apoptosis was detected using flow cytometry. The expression of caspase 3, caspase 8, caspase 9, ERK1/2, Bax, Bcl-2, and Akt proteins was determined by Western blot. Co-treatment with RLX reversed the increased apoptosis observed in the AA-I only treated group. RLX restored expression of phosphorylated Akt which found to be decreased in the AA-I only treated cells. RLX co-treatment led to a decrease in the Bax/Bcl-2 ratio as well as the cleaved form of caspase-3 compared to the AA-I only treated cells. This anti-apoptotic effect of RLX was attenuated by co-administration of the Akt inhibitor LY294002. The present study demonstrated H2 RLX can decrease AA-I induced apoptosis through activation of the PI3K/Akt signaling pathway.

  10. Alveolar development and disease.

    Science.gov (United States)

    Whitsett, Jeffrey A; Weaver, Timothy E

    2015-07-01

    Gas exchange after birth is entirely dependent on the remarkable architecture of the alveolus, its formation and function being mediated by the interactions of numerous cell types whose precise positions and activities are controlled by a diversity of signaling and transcriptional networks. In the later stages of gestation, alveolar epithelial cells lining the peripheral lung saccules produce increasing amounts of surfactant lipids and proteins that are secreted into the airspaces at birth. The lack of lung maturation and the associated lack of pulmonary surfactant in preterm infants causes respiratory distress syndrome, a common cause of morbidity and mortality associated with premature birth. At the time of birth, surfactant homeostasis begins to be established by balanced processes involved in surfactant production, storage, secretion, recycling, and catabolism. Insights from physiology and engineering made in the 20th century enabled survival of newborn infants requiring mechanical ventilation for the first time. Thereafter, advances in biochemistry, biophysics, and molecular biology led to an understanding of the pulmonary surfactant system that made possible exogenous surfactant replacement for the treatment of preterm infants. Identification of surfactant proteins, cloning of the genes encoding them, and elucidation of their roles in the regulation of surfactant synthesis, structure, and function have provided increasing understanding of alveolar homeostasis in health and disease. This Perspective seeks to consider developmental aspects of the pulmonary surfactant system and its importance in the pathogenesis of acute and chronic lung diseases related to alveolar homeostasis.

  11. [Pulmonary apoptosis and necrosis in hyperoxia-induced acute mouse lung injury].

    Science.gov (United States)

    Zhang, Xiang-feng; Foda, Hussein D

    2004-07-01

    To investigate the pathways to cell death in hyperoxia-induced lung injury and the functional significance of apoptosis in vivo in response to hyperoxia. Seventy-two mice were exposed in sealed cages > 98% oxygen (for 24 - 72 h) or room air, and the severity of lung injury and epithelium sloughing was evaluated. The extent and location of apoptosis in injured lung tissues were studied by terminal transferase dUTP end labeling assay (TUNEL), reverse transcript-polymerase chain reaction (RT-PCR) and immunohistochemistry. Hyperoxia caused acute lung injury; the hyperoxic stress resulted in marked epithelium sloughing. TUNEL assay exhibited increased apoptosis index both in alveolar epithelial cells and bronchial epithelial cells in sections from mice after 48 h hyperoxia compared with their control group (0.51 +/- 0.10, 0.46 +/- 0.08 verse 0.04 +/- 0.02, 0.02 +/- 0.01). This was accompanied by increased expression of caspase-3 mRNA in lung tissues after 48 h hyperoxia compared with their control group (0.53 +/- 0.09 verse 0.34 +/- 0.07), the expression was higher at 72 h of hyperoxia (0.60 +/- 0.08). Immunohistochemistry study showed caspase-3 protein was located in cytoplasm and nuclei of airway epithelial cells, alveolar epithelial cells and macrophage in hyperoxia mice. The expression of caspase-3 protein in airway epithelium significantly increased at 24 h of hyperoxia compared with their control group (41.62 +/- 3.46 verse 15.86 +/- 1.84), the expression level was highest at 72 h of hyperoxia (55.24 +/- 6.80). Both apoptosis and necrosis contribute to cell death during hyperoxia. Apoptosis plays an important role in alveolar damage and cell death from hyperoxia.

  12. Alveolar epithelial cells (A549) exposed at the air-liquid interface to diesel exhaust: First study in TNO's powertrain test center

    NARCIS (Netherlands)

    Kooter, I.M.; Alblas, M.J.; Jedynska, A.D.; Steenhof, M.; Houtzager, M.M.G.; Ras, M.G. van

    2013-01-01

    Air–liquid interface (ALI) exposures enable in vitro testing ofmixtures of gases and particles such as diesel exhaust (DE). The main objective of this study was to investigate the feasibility of exposing human lung epithelial cells at the ALI to complete DE generated by a heavy-duty truck in the

  13. Compound 13, an α1-selective small molecule activator of AMPK, inhibits Helicobacter pylori-induced oxidative stresses and gastric epithelial cell apoptosis

    International Nuclear Information System (INIS)

    Zhao, Hangyong; Zhu, Huanghuang; Lin, Zhou; Lin, Gang; Lv, Guoqiang

    2015-01-01

    Half of the world's population experiences Helicobacter pylori (H. pylori) infection, which is a main cause of gastritis, duodenal and gastric ulcer, and gastric cancers. In the current study, we investigated the potential role of compound 13 (C13), a novel α1-selective small molecule activator of AMP-activated protein kinase (AMPK), against H. pylori-induced cytotoxicity in cultured gastric epithelial cells (GECs). We found that C13 induced significant AMPK activation, evidenced by phosphorylation of AMPKα1 and ACC (acetyl-CoA carboxylase), in both primary and transformed GECs. Treatment of C13 inhibited H. pylori-induced GEC apoptosis. AMPK activation was required for C13-mediated GEC protection. Inhibition of AMPK kinase activity by the AMPK inhibitor Compound C, or silencing AMPKα1 expression by targeted-shRNAs, alleviated C13-induced GEC protective activities against H. pylori. Significantly, C13 inhibited H. pylori-induced reactive oxygen species (ROS) production in GECs. C13 induced AMPK-dependent expression of anti-oxidant gene heme oxygenase (HO-1) in GECs. Zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP), two HO-1 inhibitors, not only suppressed C13-mediated ROS scavenging activity, but also alleviated its activity in GECs against H. pylori. Together, these results indicate that C13 inhibits H. pylori-induced ROS production and GEC apoptosis through activating AMPK–HO–1 signaling. - Highlights: • We synthesized compound 13 (C13), a α1-selective small molecule AMPK activator. • C13-induced AMPK activation requires α1 subunit in gastric epithelial cells (GECs). • C13 enhances Helicobacter pylori-induced pro-survival AMPK activation to inhibit GEC apoptosis. • C13 inhibits H. pylori-induced reactive oxygen species (ROS) production in GECs. • AMPK-heme oxygenase (HO-1) activation is required for C13-mediated anti-oxidant activity

  14. Compound 13, an α1-selective small molecule activator of AMPK, inhibits Helicobacter pylori-induced oxidative stresses and gastric epithelial cell apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Hangyong; Zhu, Huanghuang; Lin, Zhou; Lin, Gang; Lv, Guoqiang, E-mail: lvguoqiangwuxivip@163.com

    2015-08-07

    Half of the world's population experiences Helicobacter pylori (H. pylori) infection, which is a main cause of gastritis, duodenal and gastric ulcer, and gastric cancers. In the current study, we investigated the potential role of compound 13 (C13), a novel α1-selective small molecule activator of AMP-activated protein kinase (AMPK), against H. pylori-induced cytotoxicity in cultured gastric epithelial cells (GECs). We found that C13 induced significant AMPK activation, evidenced by phosphorylation of AMPKα1 and ACC (acetyl-CoA carboxylase), in both primary and transformed GECs. Treatment of C13 inhibited H. pylori-induced GEC apoptosis. AMPK activation was required for C13-mediated GEC protection. Inhibition of AMPK kinase activity by the AMPK inhibitor Compound C, or silencing AMPKα1 expression by targeted-shRNAs, alleviated C13-induced GEC protective activities against H. pylori. Significantly, C13 inhibited H. pylori-induced reactive oxygen species (ROS) production in GECs. C13 induced AMPK-dependent expression of anti-oxidant gene heme oxygenase (HO-1) in GECs. Zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP), two HO-1 inhibitors, not only suppressed C13-mediated ROS scavenging activity, but also alleviated its activity in GECs against H. pylori. Together, these results indicate that C13 inhibits H. pylori-induced ROS production and GEC apoptosis through activating AMPK–HO–1 signaling. - Highlights: • We synthesized compound 13 (C13), a α1-selective small molecule AMPK activator. • C13-induced AMPK activation requires α1 subunit in gastric epithelial cells (GECs). • C13 enhances Helicobacter pylori-induced pro-survival AMPK activation to inhibit GEC apoptosis. • C13 inhibits H. pylori-induced reactive oxygen species (ROS) production in GECs. • AMPK-heme oxygenase (HO-1) activation is required for C13-mediated anti-oxidant activity.

  15. Selenoprotein R Protects Human Lens Epithelial Cells against D-Galactose-Induced Apoptosis by Regulating Oxidative Stress and Endoplasmic Reticulum Stress.

    Science.gov (United States)

    Dai, Jie; Liu, Hongmei; Zhou, Jun; Huang, Kaixun

    2016-02-10

    Selenium is an essential micronutrient for humans. Much of selenium's beneficial influence on health is attributed to its presence within 25 selenoproteins. Selenoprotein R (SelR), known as methionine sulfoxide reductase B1 (MsrB1), is a selenium-dependent enzyme that, like other Msrs, is required for lens cell viability. In order to investigate the roles of SelR in protecting human lens epithelial (hLE) cells against damage, the influences of SelR gene knockdown on d-galactose-induced apoptosis in hLE cells were studied. The results showed that both d-galactose and SelR gene knockdown by siRNA independently induced oxidative stress. When SelR-gene-silenced hLE cells were exposed to d-galactose, glucose-regulated protein 78 (GRP78) protein level was further increased, mitochondrial membrane potential was significantly decreased and accompanied by a release of mitochondrial cytochrome c. At the same time, the apoptosis cells percentage and the caspase-3 activity were visibly elevated in hLE cells. These results suggested that SelR might protect hLE cell mitochondria and mitigating apoptosis in hLE cells against oxidative stress and endoplasmic reticulum (ER) stress induced by d-galactose, implying that selenium as a micronutrient may play important roles in hLE cells.

  16. Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway

    International Nuclear Information System (INIS)

    Li Qin; Suen, T.-C.; Sun Hong; Arita, Adriana; Costa, Max

    2009-01-01

    Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells

  17. 7,8-Dihydroxyflavone ameliorates high-glucose induced diabetic apoptosis in human retinal pigment epithelial cells by activating TrkB.

    Science.gov (United States)

    Yu, Xiaoyi; Liu, Qiuhong; Wang, Xiaochuan; Liu, Hong; Wang, Yan

    2018-01-01

    In diabetic retinopathy, prolonged high-level blood glucose induced significant impairments among various retinal tissues, including retinal pigment epithelial (RPE) cells. In an in vitro model of human RPE cells, we evaluated whether 7,8-Dihydroxyflavone (DHF) may effectively prevent high glucose-induced diabetic apoptosis among human RPE cells. ARPE-19 cells, a Human RPE cell line, were treated with d-glucose (50 mM) to induce apoptosis in vitro. Prior to glucose, ARPE-19 cells were pre-incubated with various concentrations of DHF. The effect of DHF on d-glucose-induced apoptosis was examined by TUNEL assay, in a concentration-dependent manner. The biological effects of DHF on Caspase-9 (Casp-9) and TrkB signaling pathways in d-glucose-injured ARPE-19 cells were evaluated by qRT-PCR and western blot (WB) assays. A TrkB antagonist, K252a, was also applied in DHF and d-glucose treated ARPE-19 cells. Possible effect of K252a blocking TrkB signaling pathway, thus reversing DHF-modulated apoptosis prevention was also examined by TUNEL and WB assays. DHF ameliorated d-glucose-induced diabetic apoptosis in ARPE-19 cells. Apoptotic factor Casp-9, at both mRNA and protein levels, were drastically inhibited by DHF in d-glucose-injured ARPE-19 cells. Also, DHF activated TrkB signaling pathway through phosphorylation. K252a dramatically reversed the preventive effect of DHF on d-glucose-induced apoptosis in ARPE-19 cells. Further investigation showed that K252a functioned through de-activating or de-phosphorylating TrkB signaling pathway. This work demonstrates that DHF, through activation of TrkB signaling pathway, has a preventive function in d-glucose-induced apoptosis in PRE cells in diabetic retinopathy. Copyright © 2017. Published by Elsevier Inc.

  18. Surfactant Proteins-A and -D Attenuate LPS-Induced Apoptosis in Primary Intestinal Epithelial Cells (IECs).

    Science.gov (United States)

    Zhang, Linlin; Meng, Qinghe; Yepuri, Natesh; Wang, Guirong; Xi, Xiuming; Cooney, Robert N

    2018-01-01

    SP-A/D KO mice with sepsis demonstrate more severe lung, kidney, and gut injury/apoptosis than WT controls. We hypothesize SP-A and SP-D directly regulate lipopolysaccharide (LPS)-induced P38 mitogen-activated protein kinase (MAPK) activation and gut apoptosis during sepsis. Primary IECs were established from SP-A/D KO or C57BL/6 WT mice, stimulated with LPS and harvested at 24 h. IECs from WT mice were treated with SP-A, SP-D, or vehicle for 20 h, then LPS for 24 h. Apoptosis, cleaved caspase-3 levels and the ratio of BAX/Bcl-2 were assayed. The role of P38 MAPK was examined using the P38 MAPK-agonist U46619 and inhibitor SB203580 in LPS-treated cells. p-P38 MAPK/t-P38 MAPK, TLR4, and CD14 were measured by Western Blot. LPS-induced apoptosis, caspase-3 levels, BAX/Bcl-2, and p-P38/t-P38 MAPK were increased in SP-A/D KO IECs. SP-A and SP-D attenuate LPS-induced increase in apoptosis, cleaved caspase-3, BAX/Bcl-2, and p-P38/t-P38 MAPK in WT IECs. U46619 increased apoptosis, caspase-3, and BAX/Bcl-2 in IECs which was attenuated by SP-A/D. SB203580 attenuates the LPS-induced increase in apoptosis, caspase-3, and BAX/Bcl-2 in WT IECs. Addition of SP-A or SP-D to SB203580 completely ameliorates LPS-induced apoptosis. The LPS-induced increase in TLR4 and CD14 expression is greater in IECs from SP-A/D KO mice and treatment of WT IECs with SP-A or SP-D prevents the LPS-induced increase in TLR4 and CD14. SP-A and SP-D attenuate LPS-induced increases in apoptosis, caspase-3, and BAX/Bcl-2 in IECs. Attenuation of LPS-induced activation of TLR4 and P38 MAPK signaling pathways represents potential mechanisms for the protective effects of SP-A/D on apoptosis.

  19. Retinal pigmented epithelial cells cytotoxicity and apoptosis through activation of the mitochondrial intrinsic pathway: role of indocyanine green, brilliant blue and implications for chromovitrectomy.

    Directory of Open Access Journals (Sweden)

    Fernando M Penha

    Full Text Available To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE cell line.ARPE-19 cells were exposed to brilliant blue (BriB, methyl blue (MetB, acid violet (AcV and indocyanine green (ICG. Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL and two exposure times (3 and 30 min were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process.ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01. BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein.The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

  20. Retinal Pigmented Epithelial Cells Cytotoxicity and Apoptosis through Activation of the Mitochondrial Intrinsic Pathway: Role Of Indocyanine Green, Brilliant Blue and Implications for Chromovitrectomy

    Science.gov (United States)

    Penha, Fernando M.; Pons, Marianne; Costa, Elaine Fiod; Barros, Nilana Meza Tenório; Rodrigues, Eduardo B.; Cardoso, Emmerson Badaró; Dib, Eduardo; Maia, Mauricio; Marin-Castaño, Maria E.; Farah, Michel Eid

    2013-01-01

    Purpose To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. Methods ARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process. Results ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein. Conclusions The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process. PMID:23675521

  1. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-05-01

    Full Text Available Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH, apoptosis analysis by annexin V and propidium iodide (PI double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM and transmission electron microscope (TEM, mitochondrial transmembrane potential (ΔΨm assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05 release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01 and casepase-3 (p < 0.05 levels, significantly (p < 0.01 increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of

  2. [Effect of Polydatin on Epithelial-Mesenchymal Transition of Human Alveolar Epithelium A549 Cells Induced by TGF-β1].

    Science.gov (United States)

    Yang, Jun-chao; Xu, Lu; Song, Kang; Wang, Yuan; Gao, Run-di; Chen, Rui-lin; Cao, Yu

    2016-04-01

    To explore the effect of polydatin on the growth of TGF-β₁induced humanalveolar epithelium A549 cells and the mechanism of polydatin for inhibiting the process of epithelial-mesenchymal transition (EMT). A549 cells in vitro cultured were randomly divided into five groups, i.e., the blank group, the control group, the low dose polydatin group, the middle dose polydatin group, the high dose polydatin group. Common culture fluid was added in A549 cells of the blank group. Five ng/mLTGF-β₁contained culture fluid was added in A549 cells of the control group. 50, 100, and 150 μmol/mL of polydatin plus 5 ng/mL TGF-β₁contained culture fluid was added in A549 cells of low, middle, and high dosepolydatin groups, respectively. Morphological changes were observed and recorded at different time points. The optimal concentration of polydatin was determined by MTT method. Protein and mRNA expressions of E-cad epithelial cell marker) and Vimentin (mesenchymal cell marker) were detected by Western blot and Real-time PCR. Under inverted phase contrast microscope, A549 cells turned from previous pebble shape to fusiform shape after intervened by polydatin and TGF-β1. The intercellular space was enlargedand the intercellular connection became loose. These phenomena were more obviously seen in the control group. A549 cells were more satiated in low, middle, and high dose polydatin groups than in the control group. The EMT inhibition was most obviously seen in the middle dose polydatin group at 48 h. Protein and mRNA expressions of E-cad showed an overall descending tendency after intervened by polydatin and TGF-β1 (P A549 cells time- and dose-dependently. It also played roles in inhibiting pulmonary fibrosis.

  3. The soybean peptide lunasin promotes apoptosis of mammary epithelial cells via induction of tumor suppressor PTEN: similarities and distinct actions from soy isoflavone genistein.

    Science.gov (United States)

    Pabona, John Mark P; Dave, Bhuvanesh; Su, Ying; Montales, Maria Theresa E; de Lumen, Ben O; de Mejia, Elvira G; Rahal, Omar M; Simmen, Rosalia C M

    2013-01-01

    Breast cancer is the leading cause of cancer deaths in women. Diet and lifestyle are major contributing factors to increased breast cancer risk. While mechanisms underlying dietary protection of mammary tumor formation are increasingly elucidated, there remains a dearth of knowledge on the nature and precise actions of specific bioactive components present in foods with purported health effects. The 43-amino acid peptide lunasin (LUN) is found in soybeans, is bioavailable similar to the isoflavone genistein (GEN), and thus may mediate the beneficial effects of soy food consumption. Here, we evaluated whether LUN displays common and distinct actions from those of GEN in non-malignant (mouse HC11) and malignant (human MCF-7) mammary epithelial cells. In MCF-7 cells, LUN up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced nuclear PTEN localization, similar to that shown for GEN in mammary epithelial cells. LUN-induced cellular apoptosis, akin to GEN, was mediated by PTEN, but unlike that for GEN, was p53-independent. LUN promoted E-cadherin and β-catenin non-nuclear localization similar to GEN, but unlike GEN, did not influence the proliferative effects of oncogene Wnt1 on HC11 cells. Further, LUN did not recapitulate GEN inhibitory effects on expansion of the cancer stem-like/progenitor population in MCF-7 cells. Results suggest the concerted actions of GEN and LUN on cellular apoptosis for potential mammary tumor preventive effects and highlight whole food consumption rather than intake of specific dietary supplements with limited biological effects for greater health benefits.

  4. Increased alveolar soluble Annexin V promotes lung inflammation and fibrosis

    OpenAIRE

    Buckley, S.; Shi, W.; Xu, W.; Frey, M.R.; Moats, R.; Pardo, A.; Selman, M.; Warburton, D.

    2015-01-01

    The causes underlying the self-perpetuating nature of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal disease, remain unknown. We hypothesized that alveolar soluble Annexin V contributes to lung fibrosis, based on the observation that human IPF BALF containing high Annexin V levels promoted fibroblast involvement in alveolar epithelial wound healing that was reduced when Annexin V was depleted from the BALF.

  5. Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis

    NARCIS (Netherlands)

    van der Toorn, Marco; Slebos, Dirk-Jan; de Bruin, Harold G.; Leuvenink, Henri G.; Bakker, Stephan J. L.; Gans, Rijk O. B.; Koeter, Gerard H.; van Oosterhout, Antoon J. M.; Kauffman, Henk F.

    Increased lung cell apoptosis and necrosis occur in patients with chronic obstructive pulmonary disease ( COPD). Mitochondria are crucially involved in the regulation of these cell death processes. Cigarette smoke is the main risk factor for development of COPD. We hypothesized that cigarette smoke

  6. Oral and Fecal Campylobacter concisus Strains induce Barrier dysfunction by Apoptosis in HT-29/B6 Intestinal Epithelial Cells

    DEFF Research Database (Denmark)

    Nielsen, Hans Linde; Nielsen, Henrik Ib; Ejlertsen, Tove

    in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29...

  7. Excess iodine promotes apoptosis of thyroid follicular epithelial cells by inducing autophagy suppression and is associated with Hashimoto thyroiditis disease.

    Science.gov (United States)

    Xu, Chengcheng; Wu, Fei; Mao, Chaoming; Wang, Xuefeng; Zheng, Tingting; Bu, Ling; Mou, Xiao; Zhou, Yuepeng; Yuan, Guoyue; Wang, Shengjun; Xiao, Yichuan

    2016-12-01

    The incidence of the autoimmune thyroid disease Hashimoto thyroiditis (HT) has increased in recent years, and increasing evidence supports the contribution of excess iodine intake to thyroid disease. In this study, we examined the status of autophagy and apoptosis in thyroid tissues obtained from patients with HT, and we determined the effects of excessive iodine on the autophagy and apoptosis of thyroid follicular cells (TFCs) in an attempt to elucidate the effects of excess iodine on HT development. Our results showed decreases in the autophagy-related protein LC3B-II, and increases in caspase-3 were observed in thyroid tissues from HT patients. Interestingly, the suppression of autophagy activity in TFCs was induced by excess iodine in vitro, and this process is mediated through transforming growth factor-β1 downregulation and activation of the Akt/mTOR signaling pathway. In addition, excess iodine induced autophagy suppression and enhanced reactive oxygen species (ROS) production and apoptosis of TFCs, which could be rescued by the activation of autophagy. Taken together, our results demonstrated that excess iodine contributed to autophagy suppression and apoptosis of TFCs, which could be important factors predisposing to increased risk of HT development. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Cystogenesis in ARPKD results from increased apoptosis in collecting duct epithelial cells of Pkhd1 mutant kidneys

    International Nuclear Information System (INIS)

    Hu, Bo; He, Xiusheng; Li, Ao; Qiu, Qingchao; Li, Cunxi; Liang, Dan; Zhao, Ping; Ma, Jie; Coffey, Robert J.; Zhan, Qimin; Wu, Guanqing

    2011-01-01

    Mutations in the PKHD1 gene result in autosomal recessive polycystic kidney disease (ARPKD) in humans. To determine the molecular mechanism of the cystogenesis in ARPKD, we recently generated a mouse model for ARPKD that carries a targeted mutation in the mouse orthologue of human PKHD1. The homozygous mutant mice display hepatorenal cysts whose phenotypes are similar to those of human ARPKD patients. By littermates of this mouse, we developed two immortalized renal collecting duct cell lines with Pkhd1 and two without. Under nonpermissive culture conditions, the Pkhd1 -/- renal cells displayed aberrant cell-cell contacts and tubulomorphogenesis. The Pkhd1 -/- cells also showed significantly reduced cell proliferation and elevated apoptosis. To validate this finding in vivo, we examined proliferation and apoptosis in the kidneys of Pkhd1 -/- mice and their wildtype littermates. Using proliferation (PCNA and Histone-3) and apoptosis (TUNEL and caspase-3) markers, similar results were obtained in the Pkhd1 -/- kidney tissues as in the cells. To identify the molecular basis of these findings, we analyzed the effect of Pkhd1 loss on multiple putative signaling regulators. We demonstrated that the loss of Pkhd1 disrupts multiple major phosphorylations of focal adhesion kinase (FAK), and these disruptions either inhibit the Ras/C-Raf pathways to suppress MEK/ERK activity and ultimately reduce cell proliferation, or suppress PDK1/AKT to upregulate Bax/caspase-9/caspase-3 and promote apoptosis. Our findings indicate that apoptosis may be a major player in the cyst formation in ARPKD, which may lead to new therapeutic strategies for human ARPKD.

  9. MicroRNA-184 acts as a potential diagnostic and prognostic marker in epithelial ovarian cancer and regulates cell proliferation, apoptosis and inflammation.

    Science.gov (United States)

    Qin, Chong-Zhen; Lou, Xiao-Ya; Lv, Qiao-Li; Cheng, Lin; Wu, Na-Yiyuan; Hu, Lei; Zhou, Hong-Hao

    2015-10-01

    MicroRNA-184 (miR-184) is found to be significantly deregulated in human cancers associated with tumorigenesis and progression. In this study, we aimed to investigate the role and mechanism of miR-184 expression in epithelial ovarian cancer (EOC). Relative expression of miR-184 was measured by quantificational real-time polymerase chain reaction assay (qRT-PCR) in 80 EOC patients. Kaplan-Meier curve and the log-rank test were conducted to detect the prognostic value of miR-184. Function assays including cell proliferation, apoptosis and inflammation were further explored in vitro. We found that miR-184 was down-regulated in EOC tissues and cell lines compared with paired non-cancerous tissues and IOSE, respectively. Moreover, miR-184 was expressed at significantly lower levels in late-stage (III/IV) EOC tissues. Cox regression multivariate analysis indicated that miR-184 and FIGO stage were independent prognostic indicators for EOC patients. Patients with high miR-184 level achieved significantly a higher 5-year survival rate compared with low level group (P 184 over-expression could suppress EOC cell proliferation as well as inflammation and induce apoptosis in vitro. Altogether, our results suggest that miR-184 together with pathologic diagnosis is critical for prognosis determination in EOC patients and help select treatment strategy.

  10. Polyol pathway-dependent osmotic and oxidative stresses in aldose reductase-mediated apoptosis in human lens epithelial cells: role of AOP2.

    Science.gov (United States)

    Kubo, E; Urakami, T; Fatma, N; Akagi, Y; Singh, Dhirendra P

    2004-02-20

    Aldose reductase (AR) has been implicated as a major contributor to the pathogenesis of diabetic cataracts. AR activation generates osmotic and oxidative stresses via the polyol pathway and induces cell death signals. Antioxidant protein 2 (AOP2) protects cells from oxidative stress. We investigated the effect of AR overexpression on polyol accumulation and on hyperglycemic oxidative stress and osmotic stress, as well as the effects of these stresses on human lens epithelial cell (hLEC) survival. hLECs overexpressing the AR became apoptotic during hyperglycemia and showed elevated levels of intracellular polyols. Glutathione and AOP2 levels were significantly decreased in these cells. Interestingly, supply of AOP2 and/or the AR inhibitor fidarestat protected the cells against hyperglycemia-induced death. Overexpression of AR increased osmotic and oxidative stresses, resulting in increased apoptosis in hLECs. Because AOP2 protects hyperglycemia-induced hLEC apoptosis, this molecule may have the potential to prevent hyperglycemia-mediated complications in diabetes.

  11. Measurement of Regional and Global Pulmonary Clearance of 99mTc-DTPA (Demethylamitriptylene-Acetate): An Index of Alveolar Epithelial Permeability

    International Nuclear Information System (INIS)

    Vaskova, Olivija

    1996-01-01

    The main purpose of this study has been introduction of a new method for alveoli-capillary permeability evaluation. Many reports pointed out to the altered transit of soluble particles through this barrier. From pathophysiological aspect the main interest is the elucidation of permeability's alteration in different pulmonary pathology. We decided to use for lung epithelial permeability measurements 99m Tc-DTPA inhaled aerosols and sequential assessment of its lung clearance. The aerosols were obtained using oxygen flow nebulizers with aerosols' generators Ultra Vent (Malinkrodt) and Venticis II (CIS bio international) that enabled as to get submicron particles. Oxygen flow between 9 and 11 liters per minute was used. Optimum images were obtained with 1480 MBq of inhaled aerosols at least 2 to 3 minutes. DTPA that was used for aerosols labeling had been produced in our Department and the results were compared with DTPA provided by CIS bio international. High correlation between both agents was proven. During the whole study ex tamper prepared radiopharmaceuticals were used and quality control was done using paper chromatography method. Acquisition was done in sitting position with gamma camera interfaced to a ADAC and Scintiview. The measurements lasted for 20 minutes. Data were stored on 64x64 matrices. Regions of interest over both lungs were drown and each one was divided in three segments: apical, medial, and basal. Using computer program curves of 99m Tc-DTPA lung clearance were derived. From the obtained time activity curves half-time of the global and the regional lung clearance was assessed. In the control group comprised of 32 healthy volunteers (non-smokers) we had got values, used after works as reference range. Our normal values for global clearance are: 68±5,5 min. for left whole lung, 68,1±6,5 min for right whole lung, and 49±7,7 min for apical, 66,9±8 min for middle, and 75,9±6,4 min basal regional lung clearance, and they are in keeping with the

  12. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    Science.gov (United States)

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  13. DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis.

    Science.gov (United States)

    Oya, Elisabeth; Ovrevik, Johan; Arlt, Volker M; Nagy, Eszter; Phillips, David H; Holme, Jørn A

    2011-11-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound.

  14. The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

    International Nuclear Information System (INIS)

    Kim, Han-Soo; Kim, Ju Won; Gang, Jingu; Wen, Jing; Koh, Sang Seok; Koh, Jong Sung; Chung, Hyun-Ho; Song, Si Young

    2006-01-01

    LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21 CIP1/WAF1 , RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21 CIP1/WAF1 and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21 CIP1/WAF1 and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells

  15. Lactobacillus rhamnosus induced epithelial cell apoptosis, ameliorates inflammation and prevents colon cancer development in an animal model.

    Science.gov (United States)

    Gamallat, Yaser; Meyiah, Abdo; Kuugbee, Eugene D; Hago, Ahmed Musa; Chiwala, Gift; Awadasseid, Annoor; Bamba, Djibril; Zhang, Xin; Shang, Xueqi; Luo, Fuwen; Xin, Yi

    2016-10-01

    Probiotics have been suggested as prophylactic measure in colon carcinogenesis. This study aimed at determining the potential prophylactic activity of Lactobacillus rhamnosus GG CGMCC 1.2134 (LGG) strain on colorectal carcinogenesis via measuring its effect on Nuclear factor kappa B (NFκB) inflammatory pathway and apoptosis. 64 Sprague Dawley rats were grouped into four as follows; Group 1 (Healthy control), Group 2 (LGG), Group 3 (cancer control Dimethyl hydrazine (DMH)) and Group 4 (LGG+DMH). LGG was administered orally to LGG and LGG+DMH groups. Colon carcinogenesis was chemically induced in LGG+DMH and DMH groups by weekly injection of 40mg/kg DMH. Animals were sacrificed after 25 weeks of experiment and tumor characteristics assessed. The change in expression of NFκB-p65, COX-2, TNFα, Bcl-2, Bax, iNOS, VEGFα, β-catenin, Casp3 and p53 were evaluated by western blotting and qRT-PCR. LGG treatment significantly reduced tumor incidence, multiplicity and volume in LGG+DMH treatment group compared to DMH cancer control group. Also, LGG treatment reduced the expression of β-catenin and the inflammatory proteins NFκB-p65, COX-2 and TNFα; the anti-apoptotic protein Bcl-2, but increased the expression of the pro-apoptotic proteins Bax, casp3 and p53 compared with DMH group. LGG have a potential protection effect against colon carcinogenesis; inducing apoptosis and ameliorating inflammation, and may hold a promise as bio-therapeutic dietary agent. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  16. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

    Directory of Open Access Journals (Sweden)

    Qui JX

    2015-01-01

    critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT, and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for prostate cancer.Keywords: EMT, proteomics, SILAC

  17. APO-9′-Fucoxanthinone Extracted from Undariopsis peteseniana Protects Oxidative Stress-Mediated Apoptosis in Cigarette Smoke-Exposed Human Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jun-Ho Jang

    2016-07-01

    Full Text Available Long-term cigarette smoking increases the risk for chronic obstructive pulmonary disease (COPD, characterized by irreversible expiratory airflow limitation. The pathogenesis of COPD involves oxidative stress and chronic inflammation. Various natural marine compounds possess both anti-oxidant and anti-inflammatory properties, but few have been tested for their efficacy in COPD models. In this study, we conducted an in vitro screening test to identify natural compounds isolated from various brown algae species that might provide protection against cigarette smoke extract (CSE-induced cytotoxicity. Among nine selected natural compounds, apo-9′-fucoxanthinone (Apo9F exhibited the highest protection against CSE-induced cytotoxicity in immortalized human bronchial epithelial cells (HBEC2. Furthermore, the protective effects of Apo9F were observed to be associated with a significant reduction in apoptotic cell death, DNA damage, and the levels of mitochondrial reactive oxygen species (ROS released from CSE-exposed HBEC2 cells. These results suggest that Apo9F protects against CSE-induced DNA damage and apoptosis by regulating mitochondrial ROS production.

  18. Concentrations in plasma, epithelial lining fluid, alveolar macrophages and bronchial mucosa after a single intravenous dose of 1.6 mg/kg of iclaprim (AR-100) in healthy men.

    Science.gov (United States)

    Andrews, J; Honeybourne, D; Ashby, J; Jevons, G; Fraise, A; Fry, P; Warrington, S; Hawser, S; Wise, R

    2007-09-01

    A validated microbiological assay was used to measure concentrations of iclaprim (AR-100) in plasma, bronchial mucosa (BM), alveolar macrophages (AM) and epithelial lining fluid (ELF) after a single 1.6 mg/kg intravenous 60 min iv infusion of iclaprim. Male volunteers were randomly allocated to three nominal sampling time intervals 1-2 h (Group A), 3-4 h (Group B) and 5.5-7.0 h (Group C) after the start of the drug infusion. Mean iclaprim concentrations in plasma, BM, AM and ELF, respectively, were for Group A 0.59 mg/L (SD 0.18), 0.51 mg/kg (SD 0.17), 24.51 mg/L (SD 21.22) and 12.61 mg/L (SD 7.33); Group B 0.24 mg/L (SD 0.05), 0.35 mg/kg (SD 0.17), 7.16 mg/L (SD 1.91) and 6.38 mg/L (SD 5.17); and Group C 0.14 mg/L (SD 0.05), no detectable level in BM, 5.28 mg/L (SD 2.30) and 2.66 mg/L (SD 2.08). Iclaprim concentrations in ELF and AM exceeded the MIC(90) for penicillin-susceptible Streptococcus pneumoniae (MIC90 0.06 mg/L), penicillin-intermediate S. pneumoniae (MIC90 2 mg/L), penicillin-resistant S. pneumoniae (MIC90 4 mg/L) for 7, 7 and 4 h, respectively, and Chlamydia pneumoniae (MIC90 0.5 mg/L) for 7 h. Mean iclaprim concentrations in ELF exceeded the MIC90 for Haemophilus influenzae (MIC90 4 mg/L) and Moraxella catarrhalis (MIC90 8 mg/L) for up to 4 and 2 h, respectively; in AM the MIC90 was exceeded for up to 7 h. Furthermore, the MIC90 for methicillin-resistant Staphylococcus aureus of 0.12 mg/L was exceeded at all sites for up to 7 h. These data suggest that iclaprim reaches lung concentrations that should be effective in the treatment of community-acquired pneumonia.

  19. TNF-α-Induced cPLA2 Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

    Science.gov (United States)

    Lin, Chih-Chung; Lin, Wei-Ning; Cho, Rou-Ling; Wang, Chen-yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A2 (cPLA2) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA2 expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA2 mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE2 secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE2 ELISA kit. These TNF-α-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47phox, TRAF2, NIK, IKKα, IKKβ, or p65. TNF-α markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47phox. In addition, TNF-α also stimulated p47phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47phox complex formation in HPAEpiCs, which were attenuated by a TNF-α neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated IKKα/β and NF-κB p65 phosphorylation, NF-κB (p65) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-α-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKα/β pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA2 by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway. PMID:27932980

  20. Bardoxolone methyl induces apoptosis and autophagy and inhibits epithelial-to-mesenchymal transition and stemness in esophageal squamous cancer cells

    Directory of Open Access Journals (Sweden)

    Wang YY

    2015-02-01

    Full Text Available Yan-Yang Wang,1,2 Yin-Xue Yang,3 Ren Zhao,1 Shu-Ting Pan,2,4 Hong Zhe,1 Zhi-Xu He,5 Wei Duan,6 Xueji Zhang,7 Tianxin Yang,8 Jia-Xuan Qiu,4 Shu-Feng Zhou2,51Department of Radiation Oncology, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, People’s Republic of China; 6School of Medicine, Deakin University, Waurn Ponds, VIC, Australia; 7Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 8Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USAAbstract: Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl, B-cell lymphoma 2 (Bcl-2

  1. Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma.

    Science.gov (United States)

    Inoue, H; Hashimura, M; Akiya, M; Chiba, R; Saegusa, M

    2017-02-14

    Anaplastic lymphoma kinase (ALK), which is a receptor tyrosine kinase, is essentially and transiently expressed in the developing nervous system. Recently, the deregulated expression of full-length ALK has been observed in some primary solid tumors, but little is known about its involvement in the tumorigenesis of uterine carcinosarcomas (UCSs). Here we examined the functional role of the ALK gene in UCSs. Regulation and function of the ALK gene were assessed using two endometrial carcinoma cell lines. Expression of ALK and its related molecules were also investigated using clinical samples of UCSs. In cell lines, ALK promoter activity was significantly increased by transfection of Sox11 and N-myc, which are known to contribute to neuronal properties. Cells stably overexpressing full-length ALK showed an enhancement of EMT properties mediated by TGF-β1 and HGF, along with an increase in phosphorylated (p) Akt and nuclear p65. Overexpression of p65 also led to transactivation of Twist1 gene, known as an EMT inducer. Finally, treatment of the stable ALK-overexpressing cells with doxorubicin resulted in inhibition of apoptosis with progressive increase in the expression ratio of both pAkt and bcl2 relative to total Akt and bax, respectively. In clinical samples, strong cytoplasmic ALK immunoreactivity and mRNA signals without rearrangement or amplification of the ALK locus were frequently observed in UCSs, particularly in the sarcomatous components. Further, ALK IHC score was found to be positively correlated with Sox11, N-myc, Twist1, and bcl2 scores. ALK-related signal cascades containing Akt, NF-κB, Twist1, and bcl2 may participate in initial signaling for divergent sarcomatous differentiation driven from carcinomatous components in UCSs through induction of the EMT process and inhibition of apoptotic features.

  2. Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months

    Energy Technology Data Exchange (ETDEWEB)

    Pinkerton, K.E.; Dodge, D.E.; Cederdahl-Demmler, J.; Wong, V.J.; Peake, J.; Haselton, C.J.; Mellick, P.W.; Singh, G.; Plopper, C.G. (Univ. of California, Davis (United States))

    1993-03-01

    The effects of exposure to 1.0 ppm of ozone for twenty months were studied in male Fischer 344 rats. Light microscopic, morphometric, and immunohistological approaches were used to determine the distribution and degree of differentiation of ciliated and nonciliated bronchiolar epithelial (Clara) cells lining alveolar ducts of the central acinus, a primary target of ozone-induced lung injury. Alveolar duct pathways extending beyond the level of the most proximal alveolar outpocketing of terminal bronchioles were isolated in longitudinal profile. The distance that ciliated and nonciliated bronchiolar epithelial (Clara) cells projected down each alveolar duct pathway was determined by placing concentric arcs radiating outward from a single reference point at the level of the first alveolar outpocketing. A high degree of heterogeneity in the magnitude of bronchiolar epithelial cell extension into alveolar ducts was noted for each isolation and animal. Age-matched control animals also demonstrated variation in the degree of bronchiolar epithelial cell extension down alveolar ducts. In animals exposed to ozone, a striking similarity was noted by scanning electron microscopy in the surface characteristics of cells lining both terminal bronchioles and alveolar ducts. The presence of Clara cell secretory protein in cells of bronchioles and alveolar ducts was also detected immunohistochemically and visualized using confocal laser scanning microscopy in the reflectance mode. Well-differentiated ciliated and nonciliated bronchiolar epithelial cells were found lining alveolar septal tips and alveoli up to a depth of 1,000 mu into the pulmonary acinus after 20 months of exposure to ozone. No evidence of inflammation was present in alveolar ducts, suggesting that epithelial cell transformations in alveolar ducts is a natural consequence of lifetime exposures to oxidant gases.

  3. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li JP

    2015-02-01

    , but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl and B-cell lymphoma 2 (Bcl-2, but increased the expression of Bcl-2-associated X protein (Bax and p53-upregulated modulator of apoptosis (PUMA, and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK and extracellular signal-regulated kinases 1 and 2 (Erk1/2 and inhibited the activation of protein kinase B (Akt/mammalian target of rapamycin (mTOR signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E

  4. [The influence of EphA2 overexpression on proliferation and apoptosis of human lens epithelial cells exposed to high-concentration dexamethasone in vitro].

    Science.gov (United States)

    Ma, C X; Zheng, G Y

    2018-02-11

    Objective: To construct lentiviral-mediated EphA2 overexpression vectors, transfect them into human lens epithelial cells (HLE-B3) in vitro , and investigate the effect of EphA2 gene overexpression on the proliferation and apoptosis of HLE-B3 exposed to high-concentration dexamethasone. Methods: Experimental Study. The pCDH-CMV- MCS-EF1-RFP plasmid was set up by the digestion of NOTⅠand XbaⅠ double restriction enzyme and ligation of CE ligase, and then the plasmid was transformed into DH10B cells. Seven clons were picked for enzymatic digestion and the clons with correct results were chosen for sequencing. The 293 T/17 cells were co-transfected with the pCDH-CMV-MCS-EF1-RFP-EphA2 and the packaging mixture by Lipofectamine 2000. At different multiplicities of infection (MOI=20, 50, 100, and 200) after 72-hour infection, we observed the expression of RFP and morphological changes of HLE-B3 by an inverted fluorescence microscope, and calculated the transfection efficiency through the flow cytometry. EphA2 protein expression was detected by Western blot. The following experiments were divided into four groups: normal control group (group A), EphA2 overexpression vector transfection group (group B), HLE-B3 cells exposed to dexamethasone group (group C) and EphA2 overexpression vector transfection HLE-B3 cells exposed to dexamethasone group (group D). Statistical analysis method was single factor or two factors variance analysis. Cell survival rate was detected by the Cell Counting Kit-8 assay. Cell apoptosis index was detected by Tunel. Results: Restriction enzyme digestion and sequencing indicated that EphA2 cDNA fragment was successfully inserted in the vector. The infection efficiency was up to 38.6%±3.9%, 49.2%±4.2%, 79.5%±5.5% and 80.2%±6.0% when the MOI was 20, 50, 100 and 200, respectively. There was statistically significant difference ( F= 2 600.8, P= 0.001) among the four groups and between any two groups except between the MOI=100 group and MOI=200

  5. Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

    Science.gov (United States)

    Laresgoiti, Usua; Rao, Chandrika; Brady, Jane L.; Richardson, Rachel V.; Batchen, Emma J.; Chapman, Karen E.

    2016-01-01

    Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity. PMID:27578791

  6. Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Bakke-McKellep, Anne Marie; Penn, Michael H; Salas, Patricia Mora; Refstie, Ståle; Sperstad, Sigmund; Landsverk, Thor; Ringø, Einar; Krogdahl, Ashild

    2007-04-01

    Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

  7. Mechanical ventilation-induced apoptosis in newborn rat lung is mediated via FasL/Fas pathway.

    Science.gov (United States)

    Kroon, Andreas A; Delriccio, Veronica; Tseu, Irene; Kavanagh, Brian P; Post, Martin

    2013-12-01

    Mechanical ventilation induces pulmonary apoptosis and inhibits alveolar development in preterm infants, but the molecular basis for the apoptotic injury is unknown. The objective was to determine the signaling mechanism(s) of ventilation (stretch)-induced apoptosis in newborn rat lung. Seven-day-old rats were ventilated with room air for 24 h using moderate tidal volumes (8.5 ml/kg). Isolated fetal rat lung epithelial and fibroblast cells were subjected to continuous cyclic stretch (5, 10, or 17% elongation) for up to 12 h. Prolonged ventilation significantly increased the number of apoptotic alveolar type II cells (i.e., terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling and anti-cleaved caspase-3 immunochemistry) and was associated with increased expression of the apoptotic mediator Fas ligand (FasL). Fetal lung epithelial cells, but not fibroblasts, subjected to maximal (i.e., 17%, but not lesser elongation) cyclic stretch exhibited increased apoptosis (i.e., nuclear fragmentation and DNA laddering), which appeared to be mediated via the extrinsic pathway (increased expression of FasL and cleaved caspase-3, -7, and -8). The intrinsic pathway appeared not to be involved [minimal mitochondrial membrane depolarization (JC-1 flow analysis) and no activation of caspase-9]. Universal caspases inhibition and neutralization of FasL abrogated the stretch-induced apoptosis. Prolonged mechanical ventilation induces apoptosis of alveolar type II cells in newborn rats and the mechanism appears to involve activation of the extrinsic death pathway via the FasL/Fas system.

  8. ResolvinD1 stimulates epithelial wound repair and inhibits TGF-β-induced EMT whilst reducing fibroproliferation and collagen production

    Science.gov (United States)

    Zheng, Shengxing; Wang, Qian; D'Souza, Vijay; Bartis, Dom; Dancer, Rachel; Parekh, Dhruv; Gao, Fang; Lian, Qingquan; Jin, Shengwei; Thickett, David R

    2018-01-01

    Acute and chronic inflammatory lung diseases are often associated with epithelial cell injury/loss and fibroproliferative responses. ResolvinD1 (RvD1) is biosynthesized during the resolution phase of inflammatory response and exerts potent anti-inflammatory and promotes resolution of inflammatory lung diseases. The aim of this study was to investigate whether RvD1 exerts protective effects on alveolar epithelial cell function/differentiation and protects against fibroproliferative stimuli. Primary human alveolar type II cells were used to model the effects of RvD1 in vitro upon wound repair, proliferation, apoptosis, transdifferentiation, and epithelial–mesenchymal transition (EMT). Effects of RvD1 upon primary human lung fibroblast proliferation, collagen production, and myofibroblast differentiation were also examined. RvD1 promoted alveolar type II (ATII) cell wound repair and proliferation. RvD1 protected ATII cells against sFas-ligand/TNF-α-induced apoptosis and inhibition on cell proliferation and viability. RvD1 promoted ATII cells transdifferentiation. Moreover, we demonstrate that RvD1 inhibited EMT in response to TGF-β. Furthermore RvD1 inhibited human lung fibroblast proliferation, collagen production, and myofibroblast differentiation induced by both TGF-β and bronchoalveolar lavage fluid from acute respiratory distress syndrome (ARDS) patients. The effects of RvD1 were PI3-kinase dependent and mediated via the resolvin receptor. RvD1 seems to promote alveolar epithelial repair by stimulating ATII cells wound repair, proliferation, reducing apoptosis, and inhibiting TGF-β-induced EMT. While RvD1 reduced fibroproliferation, collagen production, and myofibroblast differentiation. Together, these results suggest a potential new therapeutic strategy for preventing and treating chronic diseases (such as idiopathic pulmonary fibrosis) as well as the fibroproliferative phase of ARDS by targeting RvD1 actions that emphasizes natural resolution signaling

  9. Relative Roles of the Epithelial and Stromal Tissue Compartment(s) in Mediating the Actions of Relaxin and Estrogen on Cell Proliferation and Apoptosis in the Mouse Lower Reproductive Tract

    Science.gov (United States)

    YAO, LIJUAN; AGOULNIK, ALEXANDER I.; COOKE, PAUL S.; MELING, DARYL D.; SHERWOOD, O. DAVID

    2009-01-01

    Relaxin and estrogen are secreted by the ovary during the second half of pregnancy in rats and mice. Relaxin promotes marked growth of the lower reproductive tract in both species. Relaxin promotes accumulation of epithelial and stromal cells in the cervix and vagina by both stimulating cell proliferation and inhibiting apoptosis. Estrogen acting through estrogen receptor α (ERα) plays an essential permissive role in relaxin’s actions. A fundamental step toward understanding the actions of relaxin and estrogen is to identify the tissue compartments that initiate their effects. Limited studies using either antibodies to human relaxin receptor (LGR7, RXFP1) or an IRES-LacZ reporter cassette in the LGR7 gene revealed relaxin receptors in subepithelial stroma cells and smooth muscle cells but not in epithelial cells in rodent vaginal and/or cervical tissues. ERα has been reported in both stromal and epithelial compartments in rodent reproductive tract. This chapter describes ongoing studies that use relaxin bioactivity as a means of identifying the tissue compartment(s) that initiates the actions of relaxin and estrogen on the lower reproductive tract. Specifically, a tissue separation/recombination methodology in combination with LGR7-knockout mice was initially used to obtain functional evidence that stromal LGR7 is both necessary and sufficient to promote proliferation and inhibit apoptosis in both stromal and epithelial cells in mouse cervix and vagina. The tissue separation/recombination method is currently being used in conjunction with ERα-knockout mice to determine if the obligatory permissive effect of estrogen on relaxin-induced cell proliferation occurs through stromal and/or epithelial ERα. PMID:19416172

  10. Apoptosis in Pneumovirus Infection

    Directory of Open Access Journals (Sweden)

    Reinout A. Bem

    2013-01-01

    Full Text Available Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs.

  11. Repopulation of denuded tracheal grafts with alveolar type II cells

    International Nuclear Information System (INIS)

    Johnson, N.F.

    1988-01-01

    Repopulation of denuded heterotopic tracheal grafts with populations of specific epithelial cell types is one approach to study the differentiation potential of various cell types. This technique has been adopted to delineate the differentiation pathways of alveolar type II cells isolated from rat lungs. Under the conditions of this experiment, the reestablished epithelial lining was alveolar-like, however, ultrastructural analysis of the cells showed them to be like Clara cells. These preliminary results suggest that the secretary cells of the lung parenchyma and terminal airways may share a common ancestry. (author)

  12. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    International Nuclear Information System (INIS)

    Kalayarasan, Srinivasan; Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam

    2013-01-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells

  13. Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

    Energy Technology Data Exchange (ETDEWEB)

    Kalayarasan, Srinivasan, E-mail: kalaivasanbio@gmail.com; Sriram, Narayanan; Soumyakrishnan, Syamala; Sudhandiran, Ganapasam, E-mail: sudhandiran@yahoo.com

    2013-09-01

    Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis. Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells.

  14. Proteinosis alveolar pulmonar Pulmonary alveolar proteinosis

    Directory of Open Access Journals (Sweden)

    Concepción Sánchez Infante

    2011-12-01

    Full Text Available La proteinosis alveolar pulmonar es una enfermedad respiratoria crónica, caracterizada por alteración en el metabolismo del surfactante, lo que determina su acumulación anormal en el espacio alveolar. Es una enfermedad extremadamente rara. Se han reportado solamente 500 casos en la literatura. Se describió por primera vez en 1958. Se presenta un caso de proteinosis alveolar pulmonar en un lactante de 2 meses, con desnutrición proteico energética, que ingresa por dificultad respiratoria e hipoxemia, y, con imágenes radiológicas de tipo retículo-nodulillar, en vidrio deslustrado, en el cual se plantea inicialmente el diagnóstico de bronconeumonía. Ante la evolución desfavorable y no respuesta al tratamiento, se realizó un estudio para descartar enfermedades pulmonares crónicas. El paciente fallece y se confirma el diagnóstico por anatomía patológica. Se realiza una revisión del tema.The pulmonary alveolar proteinosis is a chronic respiratory disease characterized by surfactant metabolism alteration determining its abnormal accumulation in the alveolar space. It is a disease very rare and in literature only 500 cases have been reported; it was described for the first time in 1958. This is a case presentation of pulmonary alveolar proteinosis in an infant aged 2 months with energetic protein malnutrition admitted due to respiratory difficulty and hypoxemia and with radiologic images of the reticulonodulillary, in frosting glass, where initially is made the diagnosis of bronchopneumonia. In the face of unfavorable evolution and no response to treatment, a study was conducted to rule out chronic pulmonary diseases. Patient died confirming the diagnosis according to the pathologic anatomy. A review on subject is carried out.

  15. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hua; Shen, Shuijie [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States); Chen, Xiaoyan; Zhong, Dafang [Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203 (China); Zheng, Jiang, E-mail: jiang.zheng@seattlechildrens.org [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States)

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  16. Alveolar but not intravenous S-ketamine inhibits alveolar sodium transport and lung fluid clearance in rats

    NARCIS (Netherlands)

    Berger, Marc M.; Pitzer, Bernhard; Zügel, Stefanie; Wieland, Catharina W.; Vlaar, Alexander P.; Schultz, Marcus J.; Dahan, Albert; Bärtsch, Peter; Hollmann, Markus W.; Mairbäurl, Heimo

    2010-01-01

    BACKGROUND: S-ketamine is frequently used for analgosedation, especially during sepsis and cardiovascular instability. Because S-ketamine blocks voltage-gated sodium (Na+) channels in neurons and skeletal muscle, it is conceivable that S-ketamine also blocks alveolar epithelial Na+ channels that are

  17. The Vibrio alginolyticus T3SS effectors, Val1686 and Val1680, induce cell rounding, apoptosis and lysis of fish epithelial cells.

    Science.gov (United States)

    Zhao, Zhe; Liu, Jinxin; Deng, Yiqin; Huang, Wen; Ren, Chunhua; Call, Douglas R; Hu, Chaoqun

    2018-01-01

    Vibrio alginolyticus is a Gram-negative bacterium that is an opportunistic pathogen of both marine animals and people. Its pathogenesis likely involves type III secretion system (T3SS) mediated induction of rapid apoptosis, cell rounding and osmotic lysis of infected eukaryotic cells. Herein, we report that effector proteins, Val1686 and Val1680 from V. alginolyticus, were responsible for T3SS-mediated death of fish cells. Val1686 is a Fic-domain containing protein that not only contributed to cell rounding by inhibiting Rho guanosine triphosphatases (GTPases), but was requisite for the induction of apoptosis because the deletion mutant (Δval1686) was severely weakened in its ability to induce cell rounding and apoptosis in fish cells. In addition, Val1686 alone was sufficient to induce cell rounding and apoptosis as evidenced by the transfection of Val1686 into fish cells. Importantly, the Fic-domain essential for cell rounding activity was equally important to activation of apoptosis of fish cells, indicating that apoptosis is a downstream event of Val1686-dependent GTPase inhibition. V. alginolyticus infection likely activates JNK and ERK pathways with sequential activation of caspases (caspase-8/-10, -9 and -3) and subsequent apoptosis. Val1680 contributed to T3SS-dependent lysis of fish cells in V. alginolyticus, but did not induce autophagy as has been reported for its homologue (VopQ) in V. parahaemolyticus. Together, Val1686 and Val1680 work together to induce apoptosis, cell rounding and cell lysis of V. alginolyticus-infected fish cells. These findings provide new insights into the mechanism of cell death caused by T3SS of V. alginolyticus.

  18. Evaluation of Apoptosis in the Lung Tissue of Sulfur Mustard-exposed Individuals.

    Science.gov (United States)

    Mosayebzadeh, Marjan; Ghazanfari, Tooba; Delshad, Alireza; Akbari, Hassan

    2016-08-01

    Lung exposure to sulfur mustard (SM) results in pulmonary complications, which is the main cause of long-term disability and morbidity. Up to now, the precise mechanisms of SM-induced lung complications has not been identified. The aim of this study was to evaluate apoptosis in the lung tissue of SM-exposed individuals. The study was performed on archived lung paraffin-embedded tissue specimens of21 patients suffering from pulmonary complications due to previous SM exposure and 9 unexposed patients who had undergone lung resections for another lung disease. Evaluation of apoptosis in paraffin-embedded lung tissue sections was performed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assays. TUNEL-positive apoptotic features and caspase-3 expression of specimens were significantly higher in the SM-exposed group compared with the control group. This result demonstrates higher apoptosis rate in the SM-exposed group. Furthermore, the majority of positive cells consisted of alveolar epithelial cells in both methods. In conclusion,it seems that exposure to SM may result in increased apoptosis in respiratory epithelium. More studies are needed to evaluate the role of apoptosis in SM-induced lung complications in order to design new and effective therapeutic protocols.

  19. Oxidant-mediated epithelial cell injury in idiopathic pulmonary fibrosis.

    OpenAIRE

    Cantin, A M; North, S L; Fells, G A; Hubbard, R C; Crystal, R G

    1987-01-01

    Lung inflammatory cells of patients with idiopathic pulmonary fibrosis (IPF) were evaluated for their ability to injure 51Cr-labeled AKD alveolar epithelial cells in the presence and absence of IPF alveolar epithelial lining fluid (ELF). The IPF cells were spontaneously releasing exaggerated amounts of superoxide (O.2) and hydrogen peroxide (H2O2) compared with normal (P less than 0.02). Cytotoxicity of the AKD cells was markedly increased when the IPF inflammatory cells were incubated with a...

  20. Pulmonary alveolar microlithiasis

    Directory of Open Access Journals (Sweden)

    Surender Kashyap

    2013-01-01

    Full Text Available Pulmonary alveolar microlithiasis (PAM is a rare, chronic lung disease with bilateral intra-alveolar calcium and phosphate deposition throughout the lung parenchyma with predominance to lower and midzone. Although, etiology and pathogenesis of PAM is not fully understood, the mutation in SLC34A2 gene that encodes a sodium-phosphate co-transporter in alveolar type II cells resulting in the accumulation and forming of microliths rich in calcium phosphate (due to impaired clearance are considered to be the cause of the disease. Chest radiograph and high-resolution CT of thorax are nearly pathognomonic for diagnosing PAM. HRCT demonstrates diffuse micronodules showing slight perilobular predominance resulting in calcification of interlobular septa. Patients with PAM are asymptomatic till development of hypoxemia and cor-pulmonale. No therapy has been proven to be beneficial except lung transplantation.

  1. Protective effect of aminophylline against cigarette smoke extract-induced apoptosis in human lung fibroblasts (MRC-5 cells).

    Science.gov (United States)

    Kim, Yu J; Kim, Ju-Young; Yoon, Jin Y; Kyung, Sun Y; Lee, Sang P; Jeong, Sung H; Moon, Chanil; Park, Jeong-Woong

    2011-07-01

    Cigarette smoking is the principal cause of chronic obstructive pulmonary disease (COPD), especially emphysema, which is characterized by alveolar wall destruction and airspace enlargement. Apoptosis of lung structural cells is involved in the pathogenesis of COPD. Xanthine derivatives (aminophylline or theophylline) have been used for the treatment of COPD as a bronchodilator. But the effects of xanthine derivatives on apoptosis of the lung structural cells remain poorly understood, even though it is known that theophylline protects against ultraviolet irradiation-induced cell death in corneal epithelial cells. This study was designed to determine whether aminophylline would protect against cigarette smoke extract (CSE)-induced apoptosis in lung fibroblasts. We demonstrated that aminophylline protected against apoptosis of MRC-5 cells at a relatively lower therapeutic range (10 μg/ml), resulting in a significant increase in cell viability occurring at 20% concentration after 8-hr exposure. Annexin staining decreased from 68 ± 4% of the control to 12 ± 2% of aminophylline (10 μg/ml) pre-treatment after 20% CSE exposure for 12 hr (p MRC-5 cells after exposure to 20% CSE for 12 hr compared with control and high levels of aminophylline (>50 μg/ml) pre-treatment. These findings suggest that aminophylline protected apoptosis of MRC-5 cells through the inactivation of caspases 3 and 8 and could be an effective agent to reduce cigarette smoking-induced lung structural cell apoptosis. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

  2. Asymmetric [14C]albumin transport across bullfrog alveolar epithelium

    International Nuclear Information System (INIS)

    Kim, K.J.; LeBon, T.R.; Shinbane, J.S.; Crandall, E.D.

    1985-01-01

    Bullfrog lungs were prepared as planar sheets and bathed with Ringer solution in Ussing chambers. In the presence of a constant electrical gradient (20, 0, or -20 mV) across the tissue, 14 C-labeled bovine serum albumin or inulin was instilled into the upstream reservoir and the rate of appearance of the tracer in the downstream reservoir was monitored. Two lungs from the same animal were used to determine any directional difference in tracer fluxes. An apparent permeability coefficient was estimated from a relationship between normalized downstream radioactivities and time. Results showed that the apparent permeability of albumin in the alveolar to pleural direction across the alveolar epithelial barrier is 2.3 X 10(-7) cm/s, significantly greater (P less than 0.0005) than that in the pleural to alveolar direction (5.3 X 10(-8) cm/s) when the tissue was short circuited. Permeability of inulin, on the other hand, did not show any directional dependence and averaged 3.1 X 10(-8) cm/s in both directions. There was no effect on radiotracer fluxes permeabilities of different electrical gradients across the tissue. Gel electrophoretograms and corresponding radiochromatograms suggest that the large and asymmetric isotope fluxes are not primarily due to digestion or degradation of labeled molecules. Inulin appears to traverse the alveolar epithelial barrier by simple diffusion through hydrated paracellular pathways. On the other hand, [ 14 C]albumin crosses the alveolar epithelium more rapidly than would be expected by simple diffusion. These asymmetric and large tracer fluxes suggest that a specialized mechanism is present in alveolar epithelium that may be capable of helping to remove albumin from the alveolar space

  3. Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium.

    OpenAIRE

    Felder Marcel; Sallin Pauline; Barbe Laurent; Haenni Beat; Gazdhar Amiq; Geiser Thomas; Guenat Olivier

    2012-01-01

    We present a microfluidic epithelial wound healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system a technique that is reported here ...

  4. Eukaryotic translation initiation factor 2 subunit α (eIF2α) inhibitor salubrinal attenuates paraquat-induced human lung epithelial-like A549 cell apoptosis by regulating the PERK-eIF2α signaling pathway.

    Science.gov (United States)

    Wang, Rui; Sun, Da-Zhuang; Song, Chun-Qing; Xu, Yong-Min; Liu, Wei; Liu, Zhi; Dong, Xue-Song

    2018-02-01

    Paraquat (PQ), as one of the most widely used herbicides in the world, can cause severe lung damage in humans and animals. This study investigated the underlying molecular mechanism of PQ-induced lung cell damage and the protective role of salubrinal. Human lung epithelial-like A549 cells were treated with PQ for 24h and were pre-incubated with salubrinal for 2h, followed by 500μM of PQ treatment. Silencing eIF2α gene of the A549 cells with siRNA interference method was conducted. Cell morphology, cell viability, apoptosis and caspase-3 activity were assessed by different assays accordingly thereafter. The expression of PERK, p-PERK, ATF6, c-ATF6, IRE1α, p-IRE1α, CHOP, GRP78, p-eIF2α and β-actin was assayed by western blot. The data showed that PQ significantly reduced A549 cell viability, changed cell morphology, induced cell apoptosis and significantly upregulated the levels of GRP78, CHOP, p-PERK, c-ATF6 and p-IRE1α. However, 30μM salubrinal could attenuate the effects of PQ on damages to A549 cells through upregulating p-eIF2α. In contrast, knocking down eIF2α gene inhabited the effects of salubrinal. These results suggest that PQ-induced A549 cell apoptosis involved endoplasmic reticulum (ER) stress, specially the PERK-eIF2α pathway. Salubrinal attenuated A549 cells from PQ-induced damages through regulation of the PERK-eIF2α signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Technical note: a noninvasive method for measuring mammary apoptosis and epithelial cell activation in dairy animals using microparticles extracted from milk.

    Science.gov (United States)

    Pollott, G E; Wilson, K; Jerram, L; Fowkes, R C; Lawson, C

    2014-01-01

    Milk production from dairy animals has been described in terms of 3 processes: the increase in secretory cell numbers in late pregnancy and early lactation, secretion rate of milk per cell, and the decline in cell numbers as lactation progresses. This latter process is thought to be determined by the level of programmed cell death (apoptosis) found in the animal. Until now, apoptosis has been measured by taking udder biopsies, using magnetic resonance imaging scans, or using animals postmortem. This paper describes an alternative, noninvasive method for estimating apoptosis by measuring microparticles in milk samples. Microparticles are the product of several processes in dairy animals, including apoptosis. Milk samples from 12 Holstein cows, at or past peak lactation, were collected at 5 monthly samplings. The samples (n=57) were used to measure the number of microparticles and calculate microparticle density for 4 metrics: annexin V positive and merocyanine 540 dye positive, for both and total particles, in both whole milk (WM) and spun milk. Various measures of milk production were also recorded for the 12 cows, including daily milk yield, fat and protein percentage in the milk, somatic cell count, and the days in milk when the samples were taken. A high correlation was found between the 4 WM microparticle densities and days in milk (0.46 to 0.64), and a moderate correlation between WM microparticle densities and daily milk yield (-0.33 to -0.44). No significant relationships were found involving spun milk samples, somatic cell count, or fat and protein percentage. General linear model analyses revealed differences between cows for both level of microparticle density and its rate of change in late lactation. Persistency of lactation was also found to be correlated with the WM microparticle traits (-0.65 to -0.32). As apoptosis is likely to be the major contributor to microparticle numbers in late lactation, this work found a noninvasive method for estimating

  6. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  7. Diffuse bronchiolo-alveolar carcinoma in a dog.

    Science.gov (United States)

    Bertazzolo, W; Zuliani, D; Pogliani, E; Caniatti, M; Bussadori, C

    2002-06-01

    An eight-year-old female German wirehaired pointer was presented with signs of respiratory distress. Clinical examination, laboratory results, thoracic radiography and echocardiography indicated the presence of a diffuse interstitial lung disease with secondary appropriate erythrocytosis, pulmonary hypertension and cor pulmonale. Transthoracic fine needle aspiration biopsy of the lung suggested malignant epithelial neoplasia. A primary lung cancer with an unusually diffuse distribution of miliary/micronodular lesions was found at postmortem examination. Histological diagnosis was bronchiolo-alveolar carcinoma. Bronchiolo-alveolar carcinoma can occasionally occur in a diffuse fashion involving most or all of the lung parenchyma. In man, diffuse bronchiolo-alveolar carcinoma is considered a great imitator of other, more common diffuse interstitial forms of lung disease. This case report indicates that it is also a differential diagnosis to consider in dogs.

  8. Helicobacter pylori CagA Suppresses Apoptosis through Activation of AKT in a Nontransformed Epithelial Cell Model of Glandular Acini Formation

    Directory of Open Access Journals (Sweden)

    Gabriela Vallejo-Flores

    2015-01-01

    Full Text Available H. pylori infection is the most important environmental risk to develop gastric cancer, mainly through its virulence factor CagA. In vitro models of CagA function have demonstrated a phosphoprotein activity targeting multiple cellular signaling pathways, while cagA transgenic mice develop carcinomas of the gastrointestinal tract, supporting oncogenic functions. However, it is still not completely clear how CagA alters cellular processes associated with carcinogenic events. In this study, we evaluated the capacity of H. pylori CagA positive and negative strains to alter nontransformed MCF-10A glandular acini formation. We found that CagA positive strains inhibited lumen formation arguing for an evasion of apoptosis activity of central acini cells. In agreement, CagA positive strains induced a cell survival activity that correlated with phosphorylation of AKT and of proapoptotic proteins BIM and BAD. Anoikis is a specific type of apoptosis characterized by AKT and BIM activation and it is the mechanism responsible for lumen formation of MCF-10A acini in vitro and mammary glands in vivo. Anoikis resistance is also a common mechanism of invading tumor cells. Our data support that CagA positive strains signaling function targets the AKT and BIM signaling pathway and this could contribute to its oncogenic activity through anoikis evasion.

  9. Proteinosis alveolar pulmonar

    Directory of Open Access Journals (Sweden)

    Concepción Sánchez Infante

    2011-12-01

    Full Text Available La proteinosis alveolar pulmonar es una enfermedad respiratoria crónica, caracterizada por alteración en el metabolismo del surfactante, lo que determina su acumulación anormal en el espacio alveolar. Es una enfermedad extremadamente rara. Se han reportado solamente 500 casos en la literatura. Se describió por primera vez en 1958. Se presenta un caso de proteinosis alveolar pulmonar en un lactante de 2 meses, con desnutrición proteico energética, que ingresa por dificultad respiratoria e hipoxemia, y, con imágenes radiológicas de tipo retículo-nodulillar, en vidrio deslustrado, en el cual se plantea inicialmente el diagnóstico de bronconeumonía. Ante la evolución desfavorable y no respuesta al tratamiento, se realizó un estudio para descartar enfermedades pulmonares crónicas. El paciente fallece y se confirma el diagnóstico por anatomía patológica. Se realiza una revisión del tema.

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

    Science.gov (United States)

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

    2016-10-01

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

  11. PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.

    Science.gov (United States)

    Boelens, Mirjam C; Nethe, Micha; Klarenbeek, Sjoerd; de Ruiter, Julian R; Schut, Eva; Bonzanni, Nicola; Zeeman, Amber L; Wientjens, Ellen; van der Burg, Eline; Wessels, Lodewyk; van Amerongen, Renée; Jonkers, Jos

    2016-08-23

    Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Anthocyanin‑rich blackcurrant extract inhibits proliferation of the MCF10A healthy human breast epithelial cell line through induction of G0/G1 arrest and apoptosis.

    Science.gov (United States)

    Nanashima, Naoki; Horie, Kayo; Chiba, Mitsuru; Nakano, Manabu; Maeda, Hayato; Nakamura, Toshiya

    2017-11-01

    Blackcurrants (Ribes nigrum L., Grossulariaceae) possess a high content of anthocyanin polyphenols, which have been demonstrated to exhibit beneficial effects on health due to their antioxidant and anticarcinogenic prope-rties. The present study investigated novel functions of anthocyanin‑rich blackcurrant extracts (BCEs) in a healthy mammary epithelial cell line, MCF10A. The percentages of viable cells were 85, 75, 53 and 31% following exposure to 50, 100, 200 and 400 µg/ml BCE, respectively. The half‑maximal response concentration of BCE was 237.7 µg/ml. Microarray and Ingenuity® Pathway Analysis demonstrated that BCE downregulated cell cycle signaling, including upstream genes with mitotic roles such as polo‑like kinase signaling. BCE increased the number of cells in the G0/G1 phase and decreased the number of cells in the S and G2/M phases. Alkaline comet assays demonstrated that 50 and 100 µg/ml BCE induced DNA damage in a dose‑dependent manner. Cultures treated with 0, 50, and 100 µg/ml BCE contained 4.6, 13.4 and 16.0% apoptotic cells, respectively. As compared with the untreated cultures (1.9%), the number of necrotic cells increased in the 100 µg/ml BCE‑treated cultures (from 1.9 to 4.3%) but not in the 50 µg/ml BCE‑treated cultures. Reverse transcription‑quantitative polymerase chain reaction analysis demonstrated that BCE reduced mRNA expression of the genomic caretaker lysine‑specific demethylas  5B (KDM5B). The results suggested that blackcurrant anthocyanins may act as cell arrest and death inducers via KDM5B downregulation in healthy breast cells.

  13. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  14. Chronic Alcohol Ingestion Worsens Survival and Alters Gut Epithelial Apoptosis and Cd8+ T Cell Function after Pseudomonas Aeruginosa Pneumonia-Induced Sepsis.

    Science.gov (United States)

    Klingensmith, Nathan J; Fay, Katherine T; Lyons, John D; Chen, Ching-Wen; Otani, Shunsuke; Liang, Zhe; Chihade, Deena B; Burd, Eileen M; Ford, Mandy L; Coopersmith, Craig M

    2018-04-16

    Mortality is higher in septic patients with a history of alcohol use disorder than in septic patients without a history of chronic alcohol usage. We have previously described a model of chronic alcohol ingestion followed by sepsis from cecal ligation and puncture in which alcohol-fed septic mice have higher mortality than water-fed septic mice, associated with altered gut integrity and increased production of TNF and IFNγ by splenic CD4 T cells without alterations in CD8 T cell function. The purpose of this study was to determine whether this represents a common host response to the combination of alcohol and sepsis by creating a new model in which mice with chronic alcohol ingestion were subjected to a different model of sepsis. C57Bl/6 mice were randomized to receive either alcohol or water for 12 weeks and then subjected to Pseudomonas aeruginosa pneumonia. Mice were sacrificed either 24 hours after the onset of sepsis or followed for survival. Alcohol-fed septic mice had significantly higher 7-day mortality than water-fed septic mice (96% vs 58%). This was associated with a 5-fold increase in intestinal apoptosis in alcohol-fed septic animals, accompanied by an increase in the pro-apoptotic protein Bax. Serum IL-6 levels were higher and IL-2 levels were lower in alcohol-fed septic mice. In contrast, CD8 T cell frequency was lower in alcohol-fed mice than water-fed septic mice, associated with increased production of IFNγ and TNF in stimulated splenocytes. No significant differences were noted in CD4 T cells, lung injury or bacteremia. Mice with chronic alcohol ingestion thus have increased mortality regardless of their septic insult, associated with changes in both the gut and the immune system.

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

    Science.gov (United States)

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

    2014-09-11

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

  16. Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium.

    Science.gov (United States)

    Felder, Marcel; Sallin, Pauline; Barbe, Laurent; Haenni, Beat; Gazdhar, Amiq; Geiser, Thomas; Guenat, Olivier

    2012-02-07

    We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).

  17. Hemorragia alveolar associada a nefrite lúpica Alveolar hemorrhage associated with lupus nephritis

    Directory of Open Access Journals (Sweden)

    Ricardo Henrique de Oliveira Braga Teixeira

    2003-12-01

    Full Text Available Hemorragia alveolar, como causa de insuficiência respiratória, é pouco freqüente, com diversas etiologias possíveis. Entre elas, o lúpus eritematoso sistêmico, que se apresenta geralmente como síndrome pulmão-rim, possui alta morbimortalidade. Acredita-se que a patogênese da microangiopatia, tanto renal como pulmonar, esteja associada ao depósito de imunocomplexos, que ativariam as vias de apoptose celular. Relatam-se dois casos de pacientes com nefrite lúpica que evoluíram com hemorragia alveolar associada à insuficiência respiratória necessitando de ventilação mecânica com evoluções totalmente distintas frente às terapias farmacológicas. O achado de anticorpos antimembrana basal em um dos casos evidencia a multiplicidade de mecanismos fisiopatológicos possivelmente envolvidos, que poderiam justificar as respostas heterogêneas frente aos tratamentos disponíveis.Alveolar hemorrhage leading to respiratory failure is uncommon. Various etiologies have been reported, including systemic lupus erythematosus, which generally presents as pulmonary-renal syndrome. It is believed that the pathogenesis of microangiopathy is related to deposits of immune complexes that lead to activation of cellular apoptosis. The authors report two cases of alveolar hemorrhage and respiratory failure, both requiring mechanical ventilation. The two cases had opposite outcomes after pharmacological therapy. The presence of anti-glomerular basement membrane antibodies in one of the cases demonstrates the multiplicity of physiopathological mechanisms that may be involved. This multiplicity of mechanisms provides a possible explanation for the heterogeneous responses to the available treatments.

  18. Impact of SMOFLipid on Pulmonary Alveolar Development in Newborn Guinea Pigs.

    Science.gov (United States)

    Lavoie, Jean-Claude; Mohamed, Ibrahim; Nuyt, Anne-Monique; Elremaly, Wesam; Rouleau, Thérèse

    2018-03-13

    Parenteral nutrition (PN) is associated with bronchopulmonary dysplasia in premature infants. In animals, PN leads to alveolar loss following stimulation of apoptosis by oxidative stress (oxidized redox potential). Peroxides and aldehydes generated in PN can induce hypo-alveolarization. The implication of peroxides, which is reduced by light protection, is demonstrated. The implication of aldehydes from omega-6 fatty acids oxidation is expected. The hypothesis is that composition and light exposure of PN influences bronchopulmonary dysplasia development. Since SMOFLipid (SMOF) contains a lower amount of omega-6 fatty acids than Intralipid (IL), the aim was to compare, the impacts of PN compounded with SMOF or IL, photo-protected or not, on alveolar development. Three-day-old Guinea pigs received PN, photo-protected or not, made with SMOF or IL through a jugular vein catheter. After 4 days, lungs were sampled for determinations of redox potential of glutathione, apoptosis (caspase-3, caspase-8, and caspase-9) and alveolarization index (histology: number of intercepts/mm). Compared with IL, SMOF induces a greater oxidation of redox potential (-200 ± 1 versus [vs] -205 ± 1 mV), apoptosis (caspase-3: 0.27 ± 0.04 vs 0.16 ± 0.02; caspase-9: 0.47 ± 0.03 vs 0.30 ± 0.03), and a lower alveolarization index (27.2 ± 0.8 vs 30.0 ± 0.9). Photo-protection prevented activation of caspase-9 and was statistically without effect on redox potential, caspase-3, and alveolarization index. In our model, SMOF is pro-oxidant and induces hypo-alveolarization following exaggerated apoptosis. These results highlight the need for further studies before introducing SMOFLipid in standard neonatal care. (Nutr Clin Pract. 2018;xx:xx-xx). © 2018 American Society for Parenteral and Enteral Nutrition.

  19. Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: Comparison with conventional monocultures

    Directory of Open Access Journals (Sweden)

    Stauber Roland

    2011-01-01

    Full Text Available Abstract Background To date silica nanoparticles (SNPs play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities. They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures. Methods Thus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release, and TER (Transepithelial Electrical Resistance measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release and apoptosis markers were investigated. Results Regarding toxic effects (viability, membrane integrity, TER the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-05-18

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

  1. The development and plasticity of alveolar type 1 cells

    Science.gov (United States)

    Yang, Jun; Hernandez, Belinda J.; Martinez Alanis, Denise; Narvaez del Pilar, Odemaris; Vila-Ellis, Lisandra; Akiyama, Haruhiko; Evans, Scott E.; Ostrin, Edwin J.; Chen, Jichao

    2016-01-01

    Alveolar type 1 (AT1) cells cover >95% of the gas exchange surface and are extremely thin to facilitate passive gas diffusion. The development of these highly specialized cells and its coordination with the formation of the honeycomb-like alveolar structure are poorly understood. Using new marker-based stereology and single-cell imaging methods, we show that AT1 cells in the mouse lung form expansive thin cellular extensions via a non-proliferative two-step process while retaining cellular plasticity. In the flattening step, AT1 cells undergo molecular specification and remodel cell junctions while remaining connected to their epithelial neighbors. In the folding step, AT1 cells increase in size by more than 10-fold and undergo cellular morphogenesis that matches capillary and secondary septa formation, resulting in a single AT1 cell spanning multiple alveoli. Furthermore, AT1 cells are an unexpected source of VEGFA and their normal development is required for alveolar angiogenesis. Notably, a majority of AT1 cells proliferate upon ectopic SOX2 expression and undergo stage-dependent cell fate reprogramming. These results provide evidence that AT1 cells have both structural and signaling roles in alveolar maturation and can exit their terminally differentiated non-proliferative state. Our findings suggest that AT1 cells might be a new target in the pathogenesis and treatment of lung diseases associated with premature birth. PMID:26586225

  2. p53 alterations in atypical alveolar hyperplasia of the human lung

    NARCIS (Netherlands)

    Slebos, R. J.; Baas, I. O.; Clement, M. J.; Offerhaus, G. J.; Askin, F. B.; Hruban, R. H.; Westra, W. H.

    1998-01-01

    Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. We have previously shown that AAHs are neoplastic epithelial proliferations that often harbor activating mutations

  3. Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.

    Science.gov (United States)

    Antoniak, S; Tatsumi, K; Hisada, Y; Milner, J J; Neidich, S D; Shaver, C M; Pawlinski, R; Beck, M A; Bastarache, J A; Mackman, N

    2016-06-01

    Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection. © 2016 International Society on Thrombosis and Haemostasis.

  4. Calcitonin gene-related peptide promotes the wound healing of human bronchial epithelial cells via PKC and MAPK pathways.

    Science.gov (United States)

    Zhou, Yong; Zhang, Min; Sun, Guo-Ying; Liu, Yong-Ping; Ran, Wen-Zhuo; Peng, Li; Guan, Cha-Xiang

    2013-06-10

    Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide derived from the calcitonin gene. CGRP is widely distributed in the central and peripheral neuronal systems. In the lung, CGRP could modulate dendritic cell function, stimulate proliferation of alveolar epithelial cells and mediate lung injury in mice. In this study, we investigated the effect of CGRP on the wound healing of human bronchial epithelial cells (HBECs) in vitro. The results showed that CGRP accelerated the recovery of wound area of monolayer HBECs in a dose-dependent manner. CGRP inhibited the lipopolysaccharide-induced apoptosis in HBECs. The percentage of S phase and G2/M phase was increased in HBECs after CGRP treatment. CGRP upregulated the expression of Ki67 in a dose-dependent manner. Some pathway inhibitors were used to investigate the signal pathway in which CGRP was involved. We found out that PKC pathway inhibitor (H-7) and MAPK pathway inhibitor (PD98059) could partially attenuate the effect of CGRP, which indicated that CGRP might promote the wound healing of HBECs via PKC and/or MAPK dependent pathway by accelerating migration and proliferation, and inhibiting apoptosis. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution; Adduits encombrants a l'ADN dans des cocultures de cellules pulmonaires humaines exposees a une pollution atmospherique particulaire

    Energy Technology Data Exchange (ETDEWEB)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz [Universite Lille Nord de France - Lille (France); Unite de Chimie Environnementale et Interactions sur le Vivant, MREI, Universite du Littoral Cote d' Opale, Dunkerque (France); Andre, Veronique; Le Goff, Jeremie; Sichel, Francois [GRECAN, Universite de Caen Basse-Normandie et centre Francois Baclesse, Caen (France); Roy Saint-Georges, Francoise; Mulliez, Philippe [Service de Pneumologie, Hopital Saint-Philibert, GHICL, Lille (France)

    2012-01-15

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% < 2.5{mu}.m), specific surface area (1 m{sup 2}/g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). {sup 32}P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC{sub 50} = 74.63 {mu}g/mL and L132: LC-5-0 = 75.36 {mu}g/mL). Exposure to desorbed particles (MA: C1= 61.11 {mu}g/mL and L132 : C2 = 61.71 {mu}g/mL) and B[a]P (1 {mu}M) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that

  6. Apoptosis in rat gastric antrum: Evidence that regulation by food intake depends on nitric oxide synthase

    DEFF Research Database (Denmark)

    Cao, Bao-Hong; Mortensen, Kirsten; Tornehave, Ditte

    2000-01-01

    The turnover of the epithelium of the gastrointestinal tract is regulated by a balance between cell multiplication and cell loss. We examined the effects of starvation on apoptosis in endocrine and other epithelial cells of rat antropyloric mucosa. Apoptosis was determined by the TUNEL reaction...... epithelial apoptosis during starvation. The close paracrine relation between somatostatin cells and gastrin cells suggests that the former regulates apoptosis of the latter through release of NO....

  7. Multiple cellular responses to serotonin contribute to epithelial homeostasis.

    Directory of Open Access Journals (Sweden)

    Vaibhav P Pai

    2011-02-01

    Full Text Available Epithelial homeostasis incorporates the paradoxical concept of internal change (epithelial turnover enabling the maintenance of anatomical status quo. Epithelial cell differentiation and cell loss (cell shedding and apoptosis form important components of epithelial turnover. Although the mechanisms of cell loss are being uncovered the crucial triggers that modulate epithelial turnover through regulation of cell loss remain undetermined. Serotonin is emerging as a common autocrine-paracine regulator in epithelia of multiple organs, including the breast. Here we address whether serotonin affects epithelial turnover. Specifically, serotonin's roles in regulating cell shedding, apoptosis and barrier function of the epithelium. Using in vivo studies in mouse and a robust model of differentiated human mammary duct epithelium (MCF10A, we show that serotonin induces mammary epithelial cell shedding and disrupts tight junctions in a reversible manner. However, upon sustained exposure, serotonin induces apoptosis in the replenishing cell population, causing irreversible changes to the epithelial membrane. The staggered nature of these events induced by serotonin slowly shifts the balance in the epithelium from reversible to irreversible. These finding have very important implications towards our ability to control epithelial regeneration and thus address pathologies of aberrant epithelial turnover, which range from degenerative disorders (e.g.; pancreatitis and thyrioditis to proliferative disorders (e.g.; mastitis, ductal ectasia, cholangiopathies and epithelial cancers.

  8. Alveolar bone resorption after tooth extraction

    OpenAIRE

    Dimova, Cena; Popovski, Stipica

    2012-01-01

    Alveolar ridge resorption has long been considered an unavoidable consequence of tooth extraction. Atrophy of the alveolar bone may cause significant esthetic and surgical problems in implantation, as well as at prosthetic and restorative dentistry. Alveolar ridge prophylaxis immediately upon tooth extraction may reduce such sequelae for both, the treating dentist and the patient. Attempts to reduce alveolar bone resorption have included the placement of natural roots, root analogues, and...

  9. Intravascular bronchio-alveolar tumor

    International Nuclear Information System (INIS)

    Mata, J.M.; Caceres, J.; Prat, J.; Lopez, J.I.; Velilla, O.

    1991-01-01

    In 1975 Dail and Liebow described the clinical and pathological characteristics of a pulmonary tumor which they dominated intravascular bronchio-alveolar tumor (IVBAT). Our aim is to acquaint radiologists with the existence of this tumor by describing the radiologic findings in 2 patients with IVBAT, 1 with hepatic involvement ant the other with pulmonary osteoarthropathy. (author). 7 refs.; 2 figs

  10. Human alveolar echinococcosis in Kyrgyzstan.

    Science.gov (United States)

    Usubalieva, Jumagul; Minbaeva, Gulnara; Ziadinov, Iskender; Deplazes, Peter; Torgerson, Paul R

    2013-07-01

    Human echinococcosis is a reportable disease in Kyrgyzstan. Between 1995 and 2011, human alveolar echinococcosis increased from 60 cases per year. The origins of this epidemic, which started in 2004, may be linked to the socioeconomic changes that followed the dissolution of the former Soviet Union.

  11. True Fibroma of Alveolar Mucosa

    Directory of Open Access Journals (Sweden)

    Shankargouda Patil

    2014-01-01

    Full Text Available Benign fibrous overgrowths are often found in the oral cavity, almost always being reactive/irritational in nature. However, benign mesenchymal neoplasms of the fibroblasts are extremely uncommon. Here we report a case of “True Fibroma of Alveolar Mucosa” for its rarity.

  12. Rosmarinic acid potentiates carnosic acid induced apoptosis in lung fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sana Bahri

    Full Text Available Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA and rosmarinic acid (RA was reported to cure bleomycin-(BLM-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vitamine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts.

  13. Alveolar macrophage dysregulation in Hermansky-Pudlak syndrome type 1.

    Science.gov (United States)

    Rouhani, Farshid N; Brantly, Mark L; Markello, Thomas C; Helip-Wooley, Amanda; O'Brien, Kevin; Hess, Richard; Huizing, Marjan; Gahl, William A; Gochuico, Bernadette R

    2009-12-01

    Individuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown. To investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied. Forty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed. Concentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Expression profiling of apoptosis-related genes in enterocytes isolated from patients with ulcerative colitis

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole H

    2013-01-01

    in normal and inflamed colonic epithelial cells. An apoptosis-specific gene array expression profiling system of 96 genes was used to determine the expression profile of apoptosis-related genes. Epithelial cells isolated from three patients with active ulcerative colitis were pooled and compared to pooled...

  16. Pulmonary alveolar microlithiasis in children

    International Nuclear Information System (INIS)

    Schmidt, H.; Loercher, U.; Kitz, R.; Zielen, S.; Ahrens, P.; Koenig, R.

    1996-01-01

    Two asymptomatic Turkish sibs are presented, a 4-year-old boy and his 7-year-old sister, with pulmonary alveolar microlithiasis (PAM) confirmed by transbronchial lung biopsy and bronchoalveolar lavage. Chest radiographs and high resolution CT demonstrated wide-spread intra-alveolar calcifications in both lungs. The lesions were sharply defined and less than 1 mm in diameter. CT documented a high concentration of microliths along the bronchovascular bundles, the intralobular fissue and the (sub)pleural lung parenchyma. The combination of bronchoalveolar lavage and roentgenographic appearance in high resolution CT are characteristic and pathognomonic, and can confirm the diagnosis. The more severe changes in the elder sib and the radiographic controls suggest that the pulmonary disease may be progressive in our patients. The described family of consanguineous, unaffected parents with two affected and one healthy child confirmed the autosomal recessive inheritance of PAM (McKusick 265100). In addition, the affected girl had autosomal recessive Waardenburg-anophthalmia syndrome (McKusick 206920), raising the question of whether this is a chance occurrence or possibly a contiguous gene syndrome. (orig.)

  17. Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles

    International Nuclear Information System (INIS)

    Chuan Junlan; Li Yanzhen; Yang Likai; Sun Xun; Zhang Qiang; Gong Tao; Zhang Zhirong

    2013-01-01

    The present study aimed at developing a drug delivery system targeting the densest site of tuberculosis infection, the alveolar macrophages (AMs). Rifampicin (RFP)-loaded solid lipid nanoparticles (RFP-SLNs) with an average size of 829.6 ± 16.1 nm were prepared by a modified lipid film hydration method. The cytotoxicity of RFP-SLNs to AMs and alveolar epithelial type II cells (AECs) was examined using MTT assays. The viability of AMs and AECs was above 80 % after treatment with RFP-SLNs, which showed low toxicity to both AMs and AECs. Confocal Laser Scanning Microscopy was employed to observe the interaction between RFP-SLNs and both AMs and AECs. After incubating the cells with RFP-SLNs for 2 h, the fluorescent intensity in AMs was more and remained longer (from 0.5 to 12 h) when compared with that in AECs (from 0.5 to 8 h). In vitro uptake characteristics of RFP-SLNs in AMs and AECs were also investigated by detection of intracellular RFP by High performance liquid chromatography. Results showed that RFP-SLNs delivered markedly higher RFP into AMs (691.7 ng/mg in cultured AMs, 662.6 ng/mg in primary AMs) than that into AECs (319.2 ng/mg in cultured AECs, 287.2 ng/mg in primary AECs). Subsequently, in vivo delivery efficiency and the selectivity of RFP-SLNs were further verified in Sprague–Dawley rats. Under pulmonary administration of RFP-SLNs, the amount of RFP in AMs was significantly higher than that in AECs at each time point. Our results demonstrated that solid lipid nanoparticles are a promising strategy for the delivery of rifampicin to alveolar macrophages selectively.

  18. Diffuse Alveolar Hemorrhage Associated with Warfarin Therapy

    OpenAIRE

    Kaya, Bülent; Yildiz, Ibrahim; Baha, Reshat Mehmet; Zeytun, Neslihan Ebru Eryaşar; Yetisgen, Azize

    2015-01-01

    Diffuse alveolar hemorrhage (DAH) is a life-threatening clinical pathologic syndrome caused by a variety of diseases. We report a case of DAH related to therapy of warfarin use. In this case report, we present the diffuse alveolar hemorrhage case as a rare and life-threatening complication of warfarin.

  19. Diffuse Alveolar Hemorrhage Associated with Warfarin Therapy.

    Science.gov (United States)

    Kaya, Bülent; Yildiz, Ibrahim; Baha, Reshat Mehmet; Zeytun, Neslihan Ebru Eryaşar; Yetisgen, Azize

    2015-01-01

    Diffuse alveolar hemorrhage (DAH) is a life-threatening clinical pathologic syndrome caused by a variety of diseases. We report a case of DAH related to therapy of warfarin use. In this case report, we present the diffuse alveolar hemorrhage case as a rare and life-threatening complication of warfarin.

  20. Diffuse Alveolar Hemorrhage Associated with Warfarin Therapy

    Directory of Open Access Journals (Sweden)

    Bülent Kaya

    2015-01-01

    Full Text Available Diffuse alveolar hemorrhage (DAH is a life-threatening clinical pathologic syndrome caused by a variety of diseases. We report a case of DAH related to therapy of warfarin use. In this case report, we present the diffuse alveolar hemorrhage case as a rare and life-threatening complication of warfarin.

  1. CT quantification of pulmonary alveolar microlithiasis

    International Nuclear Information System (INIS)

    Wurche, K.D.; Kubale, R.; Vallee, D.; Ostertag, H.

    1987-01-01

    Pulmonary alveolar microlithiasis is a rare, familial disease with massive symmetrical intra-alveolar calcium deposition. Conventional CT findings and CT measurements with a dual energy technique were carried out in a 26-year-old patient suffering from this disease. The importance of the findings in the differential diagnosis and for estimating the progression and prognosis of the disease is discussed. (orig.) [de

  2. Antioxidant macromolecules in the epithelial lining fluid of the normal human lower respiratory tract.

    OpenAIRE

    Cantin, A M; Fells, G A; Hubbard, R C; Crystal, R G

    1990-01-01

    We hypothesized that the alveolar structures may contain extracellular macromolecules with antioxidant properties to defend against oxidants. To evaluate this 51Cr-labeled human lung fibroblasts (HFL-1) and cat lung epithelial cells (AKD) were exposed to a H2O2-generating system and alveolar epithelial lining fluid (ELF) from healthy nonsmokers was tested for its ability to protect the lung cells from H2O2-mediated injury. The ELF provided marked antioxidant protection, with most from a H2O-s...

  3. Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae.

    Directory of Open Access Journals (Sweden)

    Li Bin

    Full Text Available Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

  4. Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Bin, Li; Luping, Du; Bing, Sun; Zhengyu, Yu; Maojun, Liu; Zhixin, Feng; Yanna, Wei; Haiyan, Wang; Guoqing, Shao; Kongwang, He

    2014-01-01

    Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod)-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

  5. Effect of Alveolar Segmental Sandwich Osteotomy on Alveolar Height: A Preliminary Study.

    Science.gov (United States)

    Mehta, Karan S; Prasad, Kavitha; Shetty, Vibha; Ranganath, Krishnappa; Lalitha, R M; Dexith, Jayashree; Munoyath, Sejal K; Kumar, Vineeth

    2017-12-01

    Bone loss following extraction is maximum in horizontal dimension. Height is also reduced which is pronounced on the buccal aspect. Various surgical procedures are available to correct the bone volume viz. GBR, onlay bone grafting, alveolar distraction and sandwich osteotomy. Sandwich osteotomy has been found to increase the vertical alveolar bone height successfully. The objective of the study was to assess the effect of alveolar segmental sandwich osteotomy on alveolar height and crestal width. A prospective study was undertaken from December 2012 to August 2014. Seven patients with 12 implant sites with a mean age of 36 years were recruited. All seven patients with 12 implant sites underwent alveolar segmental sandwich osteotomy and interpositional bone grafting. Alveolar bone height was assessed radiographically preoperatively, immediate post-op, and at 3 months post-op. Alveolar bone width was assessed radiographically preoperatively and at 3 months post-op. Statistical significance was inferred at p  Sandwich osteotomy can be used as an alternative technique to increase alveolar bone height prior to implant placement. Moderate alveolar deficiency can be predictably corrected by this technique.

  6. Alveolar ridge augmentation by osteoinduction in rats

    DEFF Research Database (Denmark)

    Pinholt, E M; Bang, G; Haanaes, H R

    1990-01-01

    The purpose of this study was to evaluate bone substitutes for alveolar ridge augmentation by osteoinduction. Allogenic, demineralized, and lyophilized dentin and bone was tested for osteoinductive properties in order to establish an experimental model for further studies. Implantations were...

  7. Iron dysregulation combined with aging prevents sepsis-induced apoptosis.

    Science.gov (United States)

    Javadi, Pardis; Buchman, Timothy G; Stromberg, Paul E; Turnbull, Isaiah R; Vyas, Dinesh; Hotchkiss, Richard S; Karl, Irene E; Coopersmith, Craig M

    2005-09-01

    Sepsis, iron loading, and aging cause independent increases in gut epithelial and splenic apoptosis. It is unknown how their combination will affect apoptosis and systemic cytokine levels. Hfe-/- mice (a murine homologue of hemochromatosis) abnormally accumulate iron in their tissues. Aged (24-26 months) or mature (16-18 months) Hfe-/- mice and wild type (WT) littermates were subjected to cecal ligation and puncture (CLP) or sham laparotomy. Intestine, spleen, and blood were harvested 24 h later and assessed for apoptosis and cytokine levels. Gut epithelial and splenic apoptosis were low in both aged septic and sham Hfe-/- mice, regardless of the amount of iron in their diet. Mature septic WT mice had increased apoptosis compared to age-matched sham WT mice. Mature septic Hfe-/- mice had similar levels of intestinal cell death to age-matched septic WT mice but higher levels of splenic apoptosis. Apoptosis was significantly lower in septic aged Hfe-/- mice than septic mature Hfe-/- animals. Interleukin-6 was elevated in septic aged Hfe-/- mice compared to sham mice. Although sepsis, chronic iron dysregulation, and aging each increase gut and splenic apoptosis, their combination yields cell death levels similar to sham animals despite the fact that aged Hfe-/- mice are able to mount an inflammatory response following CLP and mature Hfe-/- mice have elevated sepsis-induced apoptosis. Combining sepsis with two risk factors that ordinarily increase cell death and increase mortality in CLP yields an apoptotic response that could not have been predicted based upon each element in isolation.

  8. The emphysematous lung is abnormally sensitive to TRAIL-mediated apoptosis

    Directory of Open Access Journals (Sweden)

    Milot Julie

    2011-08-01

    Full Text Available Abstract Background Alveolar apoptosis is increased in the emphysematous lung. However, mechanisms involved are not fully understood. Recently, we demonstrated that levels of TRAIL receptor 1 and 2, levels of p53, and Bax/Bcl-xL ratio were elevated in the lung of subjects with emphysema, despite smoking cessation. Thus, we postulate that due to chronic pulmonary oxidative stress, the emphysematous lung would be abnormally sensitive to TRAIL-mediated apoptosis. Methodology A549 cells were exposed to rTRAIL, cigarette smoke extract, and/or H2O2 prior to caspase-3 activity measurement and annexin V staining assessment. In addition, freshly resected lung samples were obtained from non-emphysematous and emphysematous subjects and exposed ex vivo to rTRAIL for up to 18 hours. Lung samples were harvested and levels of active caspase-3 and caspase-8 were measured from tissue lysates. Results Both cigarette smoke extract and H2O2 were able to sensitize A549 cells to TRAIL-mediated apoptosis. Moreover, following exposure to rTRAIL, caspase-3 and -8 were activated in lung explants from emphysematous subjects while being decreased in lung explants from non-emphysematous subjects. Significance of the study Alveolar sensitivity to TRAIL-mediated apoptosis is strongly increased in the emphysematous lung due to the presence of oxidative stress. This might be a new mechanism leading to increased alveolar apoptosis and persistent alveolar destruction following smoking cessation.

  9. Inactivation of Tsc2 in Mesoderm-Derived Cells Causes Polycystic Kidney Lesions and Impairs Lung Alveolarization.

    Science.gov (United States)

    Ren, Siying; Luo, Yongfeng; Chen, Hui; Warburton, David; Lam, Hilaire C; Wang, Larry L; Chen, Ping; Henske, Elizabeth P; Shi, Wei

    2016-12-01

    The tuberous sclerosis complex (TSC) proteins are critical negative regulators of the mammalian/mechanistic target of rapamycin complex 1 pathway. Germline mutations of TSC1 or TSC2 cause TSC, affecting multiple organs, including the kidney and lung, and causing substantial morbidity and mortality. The mechanisms of organ-specific disease in TSC remain incompletely understood, and the impact of TSC inactivation on mesenchymal lineage cells has not been specifically studied. We deleted Tsc2 specifically in mesoderm-derived mesenchymal cells of multiple organs in mice using the Dermo1-Cre driver. The Dermo1-Cre-driven Tsc2 conditional knockout mice had body growth retardation and died approximately 3 weeks after birth. Significant phenotypes were observed in the postnatal kidney and lung. Inactivation of Tsc2 in kidney mesenchyme caused polycystic lesions starting from the second week of age, with increased cell proliferation, tubular epithelial hyperplasia, and epithelial-mesenchymal transition. In contrast, Tsc2 deletion in lung mesenchyme led to decreased cell proliferation, reduced postnatal alveolarization, and decreased differentiation with reduced numbers of alveolar myofibroblast and type II alveolar epithelial cells. Two major findings thus result from this model: inactivation of Tsc2 in mesoderm-derived cells causes increased cell proliferation in the kidneys but reduced proliferation in the lungs, and inactivation of Tsc2 in mesoderm-derived cells causes epithelial-lined renal cysts. Therefore, Tsc2-mTOR signaling in mesenchyme is essential for the maintenance of renal structure and for lung alveolarization. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  10. MicroRNAs: Potential Biomarkers and Therapeutic Targets for Alveolar Bone Loss in Periodontal Disease

    Directory of Open Access Journals (Sweden)

    Tadayoshi Kagiya

    2016-08-01

    Full Text Available Periodontal disease is an inflammatory disease caused by bacterial infection of tooth-supporting structures, which results in the destruction of alveolar bone. Osteoclasts play a central role in bone destruction. Osteoclasts are tartrate-resistant acid phosphatase (TRAP-positive multinucleated giant cells derived from hematopoietic stem cells. Recently, we and other researchers revealed that microRNAs are involved in osteoclast differentiation. MicroRNAs are novel, single-stranded, non-coding, small (20–22 nucleotides RNAs that act in a sequence-specific manner to regulate gene expression at the post-transcriptional level through cleavage or translational repression of their target mRNAs. They regulate various biological activities such as cellular differentiation, apoptosis, cancer development, and inflammatory responses. In this review, the roles of microRNAs in osteoclast differentiation and function during alveolar bone destruction in periodontal disease are described.

  11. Modulating TRAIL-Mediated Apoptosis in Prostate Cancer Using Synthetic Triterpenoids

    National Research Council Canada - National Science Library

    Hyer, Marc L

    2005-01-01

    .... Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, leading to rapid induction of TRAIL-mediated apoptosis, while having no adverse effects on normal human mammary epithelial cells (HMEC...

  12. [Cleft lip, alveolar and palate sequelae. Proposal of new alveolar score by the Alveolar Cleft Score (ACS) classification].

    Science.gov (United States)

    Molé, C; Simon, E

    2015-06-01

    The management of cleft lip, alveolar and palate sequelae remains problematic today. To optimize it, we tried to establish a new clinical index for diagnostic and prognostic purposes. Seven tissue indicators, that we consider to be important in the management of alveolar sequelae, are listed by assigning them individual scores. The final score, obtained by adding together the individual scores, can take a low, high or maximum value. We propose a new classification (ACS: Alveolar Cleft Score) that guides the therapeutic team to a prognosis approach, in terms of the recommended surgical and prosthetic reconstruction, the type of medical care required, and the preventive and supportive therapy to establish. Current studies are often only based on a standard radiological evaluation of the alveolar bone height at the cleft site. However, the gingival, the osseous and the cellular areas bordering the alveolar cleft sequelae induce many clinical parameters, which should be reflected in the morphological diagnosis, to better direct the surgical indications and the future prosthetic requirements, and to best maintain successful long term aesthetic and functional results. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  13. The influence of topic and systemic administration of copaiba oil on the alveolar wound healing after tooth extraction in rats.

    Science.gov (United States)

    Dias-da-Silva, Marco A; Pereira, Andresa C; Marin, Miguel Cc; Salgado, Miguel Ac

    2013-10-01

    The Copaiba oil has been used as an auxiliary treatment of inflammations, skin disorders and stomach ulcers, however, in dentistry, this "alternative" medicine has not been investigated yet. The purpose of this study was to evaluate the influence of topic and systemic administration of copaiba oil on the alveolar wound healing after tooth extraction. Twenty-eight wistar male rats had their lower first molar teeth extracted. Subsequently, they were divided in four groups, according to the treatment performed: (a) alveolar socket irrigation with copaiba oil; (b) alveolar socket irrigation with physiological serum; (c) daily gavage with copaiba oil or (d) daily gavage with physiological serum. After the sacrifice, the mandibles were removed and processed in order to obtain decalcified histological sections. The results demonstrated high level of epithelial migration, small number of inflammatory cells and vascular enhancement in the animals which received systemic administration of copaiba oil. The rats treated with topic administration of copaiba oil presented ulcerations and large number of inflammatory cells. An increased bone neoformation was observed in both groups treated with copaiba oil when compared with placebo group. It could be concluded that topic or systemic administration of copaiba oil leads to a better alveolar bone healing, however the topic application on connective tissue should be carefully considered, regarding the whole socket wound healing. Key words:Alveolar wound healing, oil-resin, copaiba.

  14. Intravenous S-Ketamine Does Not Inhibit Alveolar Fluid Clearance in a Septic Rat Model

    Science.gov (United States)

    Weber, Nina C.; van der Sluijs, Koen; Hackl, Florian; Hotz, Lorenz; Dahan, Albert; Hollmann, Markus W.; Berger, Marc M.

    2014-01-01

    We previously demonstrated that intratracheally administered S-ketamine inhibits alveolar fluid clearance (AFC), whereas an intravenous (IV) bolus injection had no effect. The aim of the present study was to characterize whether continuous IV infusion of S-ketamine, yielding clinically relevant plasma concentrations, inhibits AFC and whether its effect is enhanced in acute lung injury (ALI) which might favor the appearance of IV S-ketamine at the alveolar surface. AFC was measured in fluid-instilled rat lungs. S-ketamine was administered IV over 6 h (loading dose: 20 mg/kg, followed by 20 mg/kg/h), or intratracheally by addition to the instillate (75 µg/ml). ALI was induced by IV lipopolysaccharide (LPS; 7 mg/kg). Interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-3 were measured by ELISA in plasma and bronchoalveolar lavage fluid. Isolated rat alveolar type-II cells were exposed to S-ketamine (75 µg/ml) and/or LPS (1 mg/ml) for 6 h, and transepithelial ion transport was measured as short circuit current (ISC). AFC was 27±5% (mean±SD) over 60 min in control rats and was unaffected by IV S-ketamine. Tracheal S-ketamine reduced AFC to 18±9%. In LPS-treated rats, AFC decreased to 16±6%. This effect was not enhanced by IV S-ketamine. LPS increased IL-6 and CINC-3 in plasma and bronchoalveolar lavage fluid. In alveolar type-II cells, S-ketamine reduced ISC by 37% via a decrease in amiloride-inhibitable sodium transport. Continuous administration of IV S-ketamine does not affect rat AFC even in endotoxin-induced ALI. Tracheal application with direct exposure of alveolar epithelial cells to S-ketamine decreases AFC by inhibition of amiloride-inhibitable sodium transport. PMID:25386677

  15. When Is an Alveolar Type 2 Cell an Alveolar Type 2 Cell? A Conundrum for Lung Stem Cell Biology and Regenerative Medicine.

    Science.gov (United States)

    Beers, Michael F; Moodley, Yuben

    2017-07-01

    Generating mature, differentiated, adult lung cells from pluripotent cells, such as induced pluripotent stem cells and embryonic stem cells, offers the hope of both generating disease-specific in vitro models and creating definitive and personalized therapies for a host of debilitating lung parenchymal and airway diseases. With the goal of advancing lung-regenerative medicine, several groups have developed and reported on protocols using defined media, coculture with mesenchymal components, or sequential treatments mimicking lung development, to obtain distal lung epithelial cells from stem cell precursors. However, there remains significant controversy about the degree of differentiation of these cells compared with their primary counterparts, coupled with a lack of consistency or uniformity in assessing the resultant phenotypes. Given the inevitable, exponential expansion of these approaches and the probable, but yet-to-emerge second and higher generation techniques to create such assets, we were prompted to pose the question, what makes a lung epithelial cell a lung epithelial cell? More specifically for this Perspective, we also posed the question, what are the minimum features that constitute an alveolar type (AT) 2 epithelial cell? In addressing this, we summarize a body of work spanning nearly five decades, amassed by a series of "lung epithelial cell biology pioneers," which carefully describes well characterized molecular, functional, and morphological features critical for discriminately assessing an AT2 phenotype. Armed with this, we propose a series of core criteria to assist the field in confirming that cells obtained following a differentiation protocol are indeed mature and functional AT2 epithelial cells.

  16. Apoptosis in the human periodontal membrane evaluated in primary and permanent teeth

    DEFF Research Database (Denmark)

    Bille, Marie-Louise Bastholm; Thomsen, Bjarke; Kjær, Inger

    2011-01-01

    that resorption is connected to apoptosis of the epithelial cells of Malassez. The purpose of this study is to localize cells undergoing apoptosis in the periodontal membrane of human primary and permanent teeth. Materials and methods. Human primary and permanent teeth were examined immunohistochemically...... for apoptosis and epithelial cells of Malassez in the periodontal membrane. All teeth examined were extracted in connection with treatment. Results. Apoptosis was seen in close proximity to the root surface and within the epithelial cells of Malassez. This pattern of apoptotis is similar in the periodontal...... membrane in primary and permanent teeth. Conclusions. The inter-relationship between apoptotis and root resorption cannot be concluded from the present study. Apoptosis seen in close proximity to the root surface presumably corresponds to the highly innervated layer of the periodontal membrane...

  17. Stem Cell Factor Expression after Renal Ischemia Promotes Tubular Epithelial Survival

    NARCIS (Netherlands)

    Stokman, Geurt; Stroo, Ingrid; Claessen, Nike; Teske, Gwendoline J. D.; Weening, Jan J.; Leemans, Jaklien C.; Florquin, Sandrine

    2010-01-01

    Background: Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration

  18. Airway epithelial cell response to human metapneumovirus infection

    International Nuclear Information System (INIS)

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

    2007-01-01

    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)-κB, 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

  19. Pulmonary epithelial permeability in rats with bleomycin-induced pneumonitis

    Energy Technology Data Exchange (ETDEWEB)

    Anazawa, Yoshiki; Isawa, Toyoharu; Teshima, Takeo; Miki, Makoto; Motomiya, Masakichi (Tohoku Univ., Sendai (Japan). Research Inst. for Tuberculosis and Cancer)

    1992-07-01

    This study was performed to investigate the mechanism by which [sup 99m]Tc-DTPA molecules pass through the pulmonary epithelium following inhalation of [sup 99m]Tc-DTPA aerosol. Interstitial pneumonitis was induced in 6-week-old male rats by instilling 1 mg/kg of bleomycin into the trachea. Disappearance of radioactivity from the lungs was measured with a gamma camera every 2 weeks to estimate pulmonary epithelial permeability, and light- and electron-microscopic histopathologic examinations were performed at the same intervals. There was a statistically significant increase in the pulmonary epithelial permeability at 2 weeks after the instillation of bleomycin. However, subsecquent changes in pulmonary epithelial permeability were not uniform; some animals showed recovery and some showed further increase and/or partial recovery. Microscopically, increase in the capillary bed, round cell infiltration, and widening of the interstitial space were observed in addition to the presence of macrophages in the alveolar spaces at 2 weeks. Electron microscopic examination revealed vacuolization, thinning and detachment of the alveolar epithelium, and denudation of the basement membrane. Prominent fibrosis, honeycombing, thinning of the pulmonary epithelium, and increase in collagen fibers were observed after 18 weeks. We consider that vacuolization, thinning, and detachment of the pulmonary epithelium and denudation of the basement membrane are related to the increase in pulmonary epithelial permeability in bleomycin-induced interstitial pneumonitis. (author).

  20. Perawatan Ortodontik Gigi Anterior Berjejal dengan Tulang Alveolar yang Tipis

    Directory of Open Access Journals (Sweden)

    Miesje K. Purwanegara

    2015-09-01

    Full Text Available Anterior teeth movement in orthodontic treatment is limited to labiolingual direction by very thin alveolar bone. An uncontrolled anterior tooth movement to labiolingual direction can cause alveolar bone perforation at its root segment. This case report is to remind us that alveolar bone thickness limits orthodontc tooth movement. A case of crowded anterior teeth with thin alveolar bone in malocclusion I is reported. This case is treated using adgewise orthodontic appliance. Protraction of anterior teeth is anticipated due to thin alveolar bone on the anterior surface. The conclusion is although the alveolar bone surrounding the crowded anterior teeth is thin, by controlling the movement the teeth reposition is allowed.

  1. Crossroads of Wnt and Hippo in epithelial tissues.

    Science.gov (United States)

    Bernascone, Ilenia; Martin-Belmonte, Fernando

    2013-08-01

    Epithelial tissues undergo constant growth and differentiation during embryonic development and to replace damaged tissue in adult organs. These processes are governed by different signaling pathways that ultimately control the expression of genes associated with cell proliferation, patterning, and death. One essential pathway is Wnt, which controls tubulogenesis in several epithelial organs. Recently, Wnt has been closely linked to other signaling pathways, such as Hippo, that orchestrate proliferation and apoptosis to control organ size. There is evidence that epithelial cell junctions may sequester the transcription factors that act downstream of these signaling pathways, which would represent an important aspect of their functional regulation and their influence on cell behavior. Here, we review the transcriptional control exerted by the Wnt and Hippo signaling pathways during epithelial growth, patterning, and differentiation and recent advances in understanding of the regulation and crosstalk of these pathways in epithelial tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Distribution of BMP6 in the alveolar bone during mouse mandibular molar eruption.

    Science.gov (United States)

    Oralová, Veronika; Chlastáková, Ivana; Radlanski, Ralf Johannes; Matalová, Eva

    2014-01-01

    Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth-bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.

  3. Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Labovsky Vivian

    2012-06-01

    Full Text Available Abstract Background While breast cancer (BC is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG, receptor activator of nuclear factor kappa B ligand (RANKL, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, stromal cell-derived factor-1 (SDF-1, and their receptors (R in 2 human BC cell lines, MDA-MB-231 and MCF-7. Methods OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, immunocytochemistry and ELISA analyses. Results MCF-7 cells released higher levels of OPG in conditioned media (CM than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. Conclusions MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.

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

    Science.gov (United States)

    Sakai, Norihiko; Tager, Andrew M.

    2013-01-01

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

  5. Alveolar soft-part sarcoma in paranasal sinuses

    Directory of Open Access Journals (Sweden)

    Jie ZHANG

    2015-07-01

    Full Text Available Objective To investigate clinicopathological features, immune phenotype, diagnosis and differential diagnosis of alveolar soft-part sarcoma (ASPS in paranasal sinuses. Methods Retrospective study of clinical manifestations, histopathological features and immunohistochemical features was conducted in one case of ASPS in paranasal sinuses.  Results A 28-year-old female presented with bulging forehead for 2 months. MRI revealed a well-circumscribed lesion in left frontal and ethmoid sinuses extending to anterior skull base that showed slightly hyperintense signal on T1WI and hypointense signal on T2WI without obvious enhancement after contrast administration. The patient subsequently underwent endoscopic open surgery on left ethmoid and bilateral frontal sinuses and performed partial resection of the lesion. Three months after the initial surgery, the patient received reoperation for total removal of residual lesion and reconstructive surgery of anterior skull base. Adjuvant chemotherapy and radiotherapy were not administered. Histologically, the tumor was composed of epithelioid cells arranged in organoid nests and/or alveolar structures varying in size and shape, which were separated by connective tissue richly containing sinusoidal vascular channels. The tumor cells were generally large-sized, round, oval or polygonal with abundant eosinophilic granular or translucent vacuolated cytoplasm. The nuclei showed round or oval shape containing centrally placed and obvious nucleoli. The presence a lot of mono- or multi-nuclear giant cells served as another striking feature. Mitotic activities were rare. Reticular fiber staining indicated that reticular fibers surrounded the nest of tumor cells, and diastase-resistant periodic acid-Schiff (PAS-positive crystalline inclusions were identified within the cytoplasm of tumor cells. Immunohistochemically, the tumor cells were reactive for TFE3, while were negative for glial fibrillary acidic protein (GFAP

  6. Lateralization Technique and Inferior Alveolar Nerve Transposition

    Directory of Open Access Journals (Sweden)

    Angélica Castro Pimentel

    2016-01-01

    Full Text Available Bone resorption of the posterior mandible can result in diminished bone edge and, therefore, the installation of implants in these regions becomes a challenge, especially in the presence of the mandibular canal and its contents, the inferior alveolar nerve. Several treatment alternatives are suggested: the use of short implants, guided bone regeneration, appositional bone grafting, distraction osteogenesis, inclined implants tangential to the mandibular canal, and the lateralization of the inferior alveolar nerve. The aim was to elucidate the success rate of implants in the lateralization technique and in inferior alveolar nerve transposition and to determine the most effective sensory test. We conclude that the success rate is linked to the possibility of installing implants with long bicortical anchor which favors primary stability and biomechanics.

  7. Nintedanib reduces ventilation-augmented bleomycin-induced epithelial-mesenchymal transition and lung fibrosis through suppression of the Src pathway.

    Science.gov (United States)

    Li, Li-Fu; Kao, Kuo-Chin; Liu, Yung-Yang; Lin, Chang-Wei; Chen, Ning-Hung; Lee, Chung-Shu; Wang, Chih-Wei; Yang, Cheng-Ta

    2017-11-01

    Mechanical ventilation (MV) used in patients with acute respiratory distress syndrome (ARDS) can increase lung inflammation and pulmonary fibrogenesis. Src is crucial in mediating the transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) during the fibroproliferative phase of ARDS. Nintedanib, a multitargeted tyrosine kinase inhibitor that directly blocks Src, has been approved for the treatment of idiopathic pulmonary fibrosis. The mechanisms regulating interactions among MV, EMT and Src remain unclear. In this study, we suggested hypothesized that nintedanib can suppress MV-augmented bleomycin-induced EMT and pulmonary fibrosis by inhibiting the Src pathway. Five days after administrating bleomycin to mimic acute lung injury (ALI), C57BL/6 mice, either wild-type or Src-deficient were exposed to low tidal volume (V T ) (6 ml/kg) or high V T (30 ml/kg) MV with room air for 5 hrs. Oral nintedanib was administered once daily in doses of 30, 60 and 100 mg/kg for 5 days before MV. Non-ventilated mice were used as control groups. Following bleomycin exposure in wild-type mice, high V T MV induced substantial increases in microvascular permeability, TGF-β1, malondialdehyde, Masson's trichrome staining, collagen 1a1 gene expression, EMT (identified by colocalization of increased staining of α-smooth muscle actin and decreased staining of E-cadherin) and alveolar epithelial apoptosis (P Src signalling using Src-deficient mice, dampened the MV-augmented profibrotic mediators, EMT profile, epithelial apoptotic cell death and pathologic fibrotic scores (P Src pathway. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  8. Bone graft healing in alveolar osteoplasty in patients with unilateral lip, alveolar process, and palate clefts.

    Science.gov (United States)

    Rychlik, Dariusz; Wójcicki, Piotr

    2012-01-01

    Secondary osteoplasty by means of autogenic spongy bone grafting is the most common procedure used in the reconstruction of the continuity of the maxillary alveolar process. The aim of the study was to analyze retrospectively the effect of certain factors on the course of the bone graft healing process in patients with unilateral complete clefts of the lip, alveolar process, and palate. The investigations involved 62 children aged 8 to 14 years (mean age, 11 years) with unilateral complete cleft of the lip, alveolar process, and palate operated on at the Clinic of Plastic Surgery in Polanica Zdrój from November 2007 to April 2009. All the procedures consisted in the reconstruction of the maxillary alveolar process by means of autogenic spongy bone grafting from the iliac bone. The analysis was performed on the basis of computed tomography scans presenting maxillary alveolar processes in the horizontal cross-sectional planes performed on the second or third postoperative day and after 6 months. They were used as the basis for the measurement of the volume and density (condensation) of the bone graft, the surface of its adhesion to the maxillary alveolar bone, and the volume and density of the healed bone. The following correlation coefficients were determined: between the adhesion surface of the bone to the alveolar bone and the volume of the healed bone, between the adhesion surface of the bone to the alveolar bone and the density of the healed bone, and between the density of the graft and the volume of the healed bone. Increasing the surface of the graft adhesion to the bone ridges of the alveolar cleft contributes to increased volume of the healed bone and slows down the increase in its density (on 6-month follow-up). Crushing of the bone graft increases its resorption and reduces volume of the healed bone.

  9. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion

    NARCIS (Netherlands)

    Grieve, Adam G; Rabouille, Catherine

    2014-01-01

    Epithelial cell extrusion and subsequent apoptosis is a key mechanism to prevent the accumulation of excess cells. By contrast, when driven by oncogene expression, apical cell extrusion is followed by proliferation and represents an initial step of tumorigenesis. E-cadherin (E-cad), the main

  10. Alveolar Ridge Carcinoma. Two Cases Report

    International Nuclear Information System (INIS)

    Pupo Triguero, Raul J; Vivar Bauza, Miriam; Alvarez Infante, Elisa

    2008-01-01

    Two cases with alveolar ridge carcinoma due to prosthetist traumatism are discussed in this paper, after 9 and 10 years of using dental prosthesis. Both patients began with disturbance in the alveolar ridge. The clinical examination and biopsy showed a well differenced carcinoma. The treatment was radical surgery and radiotherapy in the first patient, and conservative surgery with radiotherapy in the second case .The patients had xerostomia after radiotherapy and the woman had difficulties with mastication. The advantages and disadvantages of the treatment were discussed, focused on the prevention and treatment for oral

  11. Arachidonic acid promotes epithelial-to-mesenchymal-like transition in mammary epithelial cells MCF10A.

    Science.gov (United States)

    Martinez-Orozco, Raul; Navarro-Tito, Napoleon; Soto-Guzman, Adriana; Castro-Sanchez, Luis; Perez Salazar, Eduardo

    2010-06-01

    Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. Cancer progression requires the development of metastasis, which is characterized by an increase in cell motility and invasion. Epithelial-to-mesenchymal transition (EMT) is a process, by which epithelial cells are transdifferentiated to a more mesenchymal state. A similar process takes place during tumor progression, when carcinoma cells stably or transiently lose epithelial polarities and acquire a mesenchymal phenotype. Arachidonic acid (AA) is a fatty acid that mediates cellular processes, such as cell survival, angiogenesis, chemotaxis, mitogenesis, migration and apoptosis. However, the role of AA on the EMT process in human mammary epithelial cells remains to be studied. We demonstrate here that AA promotes an increase in vimentin and N-cadherin expression, MMP-9 secretion, a decrease in E-cadherin junctional levels, and the activation of FAK, Src and NF-kappaB in MCF10A cells. Furthermore, AA also promotes cell migration in an Src kinase activity-dependent fashion. In conclusion, our results demonstrate, for the first time, that AA promotes an epithelial-to-mesenchymal-like transition in MCF10A human mammary non-tumorigenic epithelial cells. 2010 Elsevier GmbH. All rights reserved.

  12. The role of death-associated protein 3 in apoptosis, anoikis and human cancer.

    Science.gov (United States)

    Wazir, Umar; Orakzai, Mona Maw; Khanzada, Zubair S; Jiang, Wen G; Sharma, Anup K; Kasem, Abdul; Mokbel, Kefah

    2015-01-01

    Death-associated protein 3 (DAP3) is a molecule with a significant role in the control of both apoptosis and anoikis. Apoptosis is the predominant type of programmed cell death (PCD) which may occur in response to irreparable damage to DNA, or in response to induction by inflammatory cells. Anoikis is subset of apoptosis which occurs in epithelial cells in response to detachment from the surrounding matrix. Both apoptosis and anoikis are of interest in the context of carcinogenesis. In this review, we shall discuss apoptosis and anoikis, and the recent literature regarding the role of DAP3 in both these pathways.

  13. Reduced Frizzled Receptor 4 Expression Prevents WNT/β-Catenin-driven Alveolar Lung Repair in Chronic Obstructive Pulmonary Disease.

    Science.gov (United States)

    Skronska-Wasek, Wioletta; Mutze, Kathrin; Baarsma, Hoeke A; Bracke, Ken R; Alsafadi, Hani N; Lehmann, Mareike; Costa, Rita; Stornaiuolo, Mariano; Novellino, Ettore; Brusselle, Guy G; Wagner, Darcy E; Yildirim, Ali Ö; Königshoff, Melanie

    2017-07-15

    Chronic obstructive pulmonary disease (COPD), in particular emphysema, is characterized by loss of parenchymal alveolar tissue and impaired tissue repair. Wingless and INT-1 (WNT)/β-catenin signaling is reduced in COPD; however, the mechanisms thereof, specifically the role of the frizzled (FZD) family of WNT receptors, remain unexplored. To identify and functionally characterize specific FZD receptors that control downstream WNT signaling in impaired lung repair in COPD. FZD expression was analyzed in lung homogenates and alveolar epithelial type II (ATII) cells of never-smokers, smokers, patients with COPD, and two experimental COPD models by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence. The functional effects of cigarette smoke on FZD4, WNT/β-catenin signaling, and elastogenic components were investigated in primary ATII cells in vitro and in three-dimensional lung tissue cultures ex vivo. Gain- and loss-of-function approaches were applied to determine the effects of FZD4 signaling on alveolar epithelial cell wound healing and repair, as well as on expression of elastogenic components. FZD4 expression was reduced in human and experimental COPD lung tissues as well as in primary human ATII cells from patients with COPD. Cigarette smoke exposure down-regulated FZD4 expression in vitro and in vivo, along with reduced WNT/β-catenin activity. Inhibition of FZD4 decreased WNT/β-catenin-driven epithelial cell proliferation and wound closure, and it interfered with ATII-to-ATI cell transdifferentiation and organoid formation, which were augmented by FZD4 overexpression. Moreover, FZD4 restoration by overexpression or pharmacological induction led to induction of WNT/β-catenin signaling and expression of elastogenic components in three-dimensional lung tissue cultures ex vivo. Reduced FZD4 expression in COPD contributes to impaired alveolar repair capacity.

  14. Alveolar Bone Fracture: Pathognomonic Sign for Clinical Diagnosis

    OpenAIRE

    Gutmacher, Zvi; Peled, Eli; Norman, Doron; Lin, Shaul

    2017-01-01

    Aim: Dental injuries, especially luxation and avulsion, are common. Dental trauma can cause alveolar bone fracture that can lead to tooth loss and malocclusion. Single tooth alveolar bone fractures are difficult to identify unless it protrudes through the overlying mucosa and can be visualized. Pain, malocclusion, and tooth mobility provide signs of suspected alveolar bone fractures. Integrity of the proximate alveolar bone should be examined for fractures where avulsion, luxation, or other t...

  15. Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis.

    Science.gov (United States)

    Cao, Zhongwei; Lis, Raphael; Ginsberg, Michael; Chavez, Deebly; Shido, Koji; Rabbany, Sina Y; Fong, Guo-Hua; Sakmar, Thomas P; Rafii, Shahin; Ding, Bi-Sen

    2016-02-01

    Although the lung can undergo self-repair after injury, fibrosis in chronically injured or diseased lungs can occur at the expense of regeneration. Here we study how a hematopoietic-vascular niche regulates alveolar repair and lung fibrosis. Using intratracheal injection of bleomycin or hydrochloric acid in mice, we show that repetitive lung injury activates pulmonary capillary endothelial cells (PCECs) and perivascular macrophages, impeding alveolar repair and promoting fibrosis. Whereas the chemokine receptor CXCR7, expressed on PCECs, acts to prevent epithelial damage and ameliorate fibrosis after a single round of treatment with bleomycin or hydrochloric acid, repeated injury leads to suppression of CXCR7 expression and recruitment of vascular endothelial growth factor receptor 1 (VEGFR1)-expressing perivascular macrophages. This recruitment stimulates Wnt/β-catenin-dependent persistent upregulation of the Notch ligand Jagged1 (encoded by Jag1) in PCECs, which in turn stimulates exuberant Notch signaling in perivascular fibroblasts and enhances fibrosis. Administration of a CXCR7 agonist or PCEC-targeted Jag1 shRNA after lung injury promotes alveolar repair and reduces fibrosis. Thus, targeting of a maladapted hematopoietic-vascular niche, in which macrophages, PCECs and perivascular fibroblasts interact, may help to develop therapy to spur lung regeneration and alleviate fibrosis.

  16. Azithromycin ameliorates airway remodeling via inhibiting airway epithelium apoptosis.

    Science.gov (United States)

    Liu, Yuanqi; Pu, Yue; Li, Diandian; Zhou, Liming; Wan, Lihong

    2017-02-01

    Azithromycin can benefit treating allergic airway inflammation and remodeling. In the present study, we hypothesized that azithromycin alleviated airway epithelium injury through inhibiting airway epithelium apoptosis via down regulation of caspase-3 and Bax/Bcl2 ratio in vivo and in vitro. Ovalbumin induced rat asthma model and TGF-β1-induced BEAS-2B cell apoptosis model were established, respectively. In vivo experiments, airway epithelium was stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) to histologically evaluate the airway inflammation and remodeling. Airway epithelium apoptotic index (AI) was further analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), while expression of apoptosis related gene (Bax, Bcl2, Caspase-3) in lungs were measured by qRT-PCR and western blotting, respectively. In vitro experiments, apoptosis were evaluated by Flow cytometry (FCM) and TUNEL. Above apoptosis related gene were also measured by qRT-PCR and western blotting. Compared with the OVA group, azithromycin significantly reduced the inflammation score, peribronchial smooth muscle layer thickness, epithelial thickening and goblet cell metaplasia (Pazithromycin-treated rats (Pazithromycin significantly suppressed TGF-β1-induced BEAS-2B cells apoptosis (PAzithromycin is an attractive treatment option for reducing airway epithelial cell apoptosis by improving the imbalance of Bax/Bcl-2 ratio and inhibiting Caspase-3 level in airway epithelium. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Teaching Alveolar Ventilation with Simple, Inexpensive Models

    Science.gov (United States)

    DiCarlo, Stephen E.

    2008-01-01

    When teaching and learning about alveolar ventilation with our class of 300 first-year medical students, we use four simple, inexpensive "models." The models, which encourage research-oriented learning and help our students to understand complex ideas, are distributed to the students before class. The students anticipate something new every day,…

  18. Alveolar ridge augmentation by osteoinduction in rats

    DEFF Research Database (Denmark)

    Pinholt, E M; Bang, G; Haanaes, H R

    1990-01-01

    The purpose of this study was to evaluate bone substitutes for alveolar ridge augmentation by osteoinduction. Allogenic, demineralized, and lyophilized dentin and bone was tested for osteoinductive properties in order to establish an experimental model for further studies. Implantations were perf...

  19. Alveolar proteinosis associated with aluminium dust inhalation.

    Science.gov (United States)

    Chew, R; Nigam, S; Sivakumaran, P

    2016-08-01

    Secondary alveolar proteinosis is a rare lung disease which may be triggered by a variety of inhaled particles. The diagnosis is made by detection of anti-granulocyte-macrophage colony-stimulating factor antibodies in bronchoalveolar lavage fluid, which appears milky white and contains lamellar bodies. Aluminium has been suggested as a possible cause, but there is little evidence in the literature to support this assertion. We report the case of a 46-year-old former boilermaker and boat builder who developed secondary alveolar proteinosis following sustained heavy aluminium exposure. The presence of aluminium was confirmed both by histological examination and metallurgical analysis of a mediastinal lymph node. Despite cessation of exposure to aluminium and treatment with whole-lung lavage which normally results in improvements in both symptoms and lung function, the outcome was poor and novel therapies are now being used for this patient. It may be that the natural history in aluminium-related alveolar proteinosis is different, with the metal playing a mediating role in the disease process. Our case further supports the link between aluminium and secondary alveolar proteinosis and highlights the need for measures to prevent excessive aluminium inhalation in relevant industries. © The Author 2016. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Exogenous hydrogen sulfide (H2S protects alveolar growth in experimental O2-induced neonatal lung injury.

    Directory of Open Access Journals (Sweden)

    Arul Vadivel

    Full Text Available Bronchopulmonary dysplasia (BPD, the chronic lung disease of prematurity, remains a major health problem. BPD is characterized by impaired alveolar development and complicated by pulmonary hypertension (PHT. Currently there is no specific treatment for BPD. Hydrogen sulfide (H2S, carbon monoxide and nitric oxide (NO, belong to a class of endogenously synthesized gaseous molecules referred to as gasotransmitters. While inhaled NO is already used for the treatment of neonatal PHT and currently tested for the prevention of BPD, H2S has until recently been regarded exclusively as a toxic gas. Recent evidence suggests that endogenous H2S exerts beneficial biological effects, including cytoprotection and vasodilatation. We hypothesized that H2S preserves normal alveolar development and prevents PHT in experimental BPD.We took advantage of a recently described slow-releasing H2S donor, GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino phosphinodithioate to study its lung protective potential in vitro and in vivo.In vitro, GYY4137 promoted capillary-like network formation, viability and reduced reactive oxygen species in hyperoxia-exposed human pulmonary artery endothelial cells. GYY4137 also protected mitochondrial function in alveolar epithelial cells. In vivo, GYY4137 preserved and restored normal alveolar growth in rat pups exposed from birth for 2 weeks to hyperoxia. GYY4137 also attenuated PHT as determined by improved pulmonary arterial acceleration time on echo-Doppler, pulmonary artery remodeling and right ventricular hypertrophy. GYY4137 also prevented pulmonary artery smooth muscle cell proliferation.H2S protects from impaired alveolar growth and PHT in experimental O2-induced lung injury. H2S warrants further investigation as a new therapeutic target for alveolar damage and PHT.

  1. Alveolar Bone Fracture: Pathognomonic Sign for Clinical Diagnosis.

    Science.gov (United States)

    Gutmacher, Zvi; Peled, Eli; Norman, Doron; Lin, Shaul

    2017-01-01

    Dental injuries, especially luxation and avulsion, are common. Dental trauma can cause alveolar bone fracture that can lead to tooth loss and malocclusion. Single tooth alveolar bone fractures are difficult to identify unless it protrudes through the overlying mucosa and can be visualized. Pain, malocclusion, and tooth mobility provide signs of suspected alveolar bone fractures. Integrity of the proximate alveolar bone should be examined for fractures where avulsion, luxation, or other tooth trauma is detected. Any suggestion of alveolar fractures should be further investigated with an appropriate radiograph. This case report shows a pathognomonic sign that detects and diagnosis single tooth alveolar bone fractures, i.e. , a localized hematoma crossing the attached gingiva from the free gingival margin to the vestibular mucosa. This should serve as a warning for localized alveolar bone fracture. A visualized hematoma and gentle, careful palpation may help detect covered fractures when the overlying mucosa is not perforated.

  2. Foxp3+Regulatory T Cell Expression of Keratinocyte Growth Factor Enhances Lung Epithelial Proliferation.

    Science.gov (United States)

    Dial, Catherine F; Tune, Miriya K; Doerschuk, Claire M; Mock, Jason R

    2017-08-01

    Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3 + regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.

  3. Evaluation of the alveolar macrophage role in the pulmonary distribution of actinide oxides

    International Nuclear Information System (INIS)

    Guezingar-Liebard, Florence

    1999-01-01

    Actinide oxide inhalation is potentially a risk during the fuel fabrication process in the electronuclear industry. These particles can induce pulmonary lesions. The alveolar macrophage play an important role in the particle sequestration and transport but the actinide toxicity towards these cells is not well known. The aim of this work was to characterize the evolution of particle localisation in lungs after inhalation and to evaluate the role of macrophages in the lesion histo-genesis. We have used of a solid track detector to visualise alpha dose distribution within lung tissue. After 237 NpO 2 , MOX or PuO 2 inhalation by rats, different kinetics of clearance were observed for the sub-pleural and peri-bronchial areas compared to the others alveolar areas. For initial lung burdens that alter the lung clearance, particle aggregates were observed. Their kinetic and localisation vary depending on the aerosol, for a same global dose delivered to the lungs. This could be due to the different specific alpha activities of the particles and to the particle number deposited in the lung to obtain a similar burden but it could be also due to a chemical toxicity of neptunium higher than that of the others actinides. The flow cytometry methods developed allow us to measure apoptosis, phagocytosis and free radicals generation. After addition of soluble uranium to the culture medium, similar results were obtained using either alveolar macrophages extracted from rats or a macrophage cell line. This work confirms that alveolar macrophages are involved in the aggregate formation which induces heterogeneous dose distribution within the different lung tissues. (author) [fr

  4. Rare Lung Diseases II: Pulmonary Alveolar Proteinosis

    Directory of Open Access Journals (Sweden)

    Stephen C Juvet

    2008-01-01

    Full Text Available The present article is the second in a series on rare lung diseases. It focuses on pulmonary alveolar proteinosis (PAP, a disorder in which lipoproteinaceous material accumulates in the alveolar space. PAP was first described in 1958, and for many years the nature of the material accumulating in the lungs was unknown. Major insights into PAP have been made in the past decade, and these have led to the notion that PAP is an autoimmume disorder in which autoantibodies interfere with signalling through the granulocyte-macrophage colony-stimulating factor receptor, leading to macrophage and neutrophil dysfunction. This has spurred new therapeutic approaches to this disorder. The discussion of PAP will begin with a case report, then will highlight the classification of PAP and review recent insights into the pathogenesis of PAP. The approach to therapy and the prognosis of PAP will also be discussed.

  5. Silver Nanoparticles in Alveolar Bone Surgery Devices

    Directory of Open Access Journals (Sweden)

    Stefano Sivolella

    2012-01-01

    Full Text Available Silver (Ag ions have well-known antimicrobial properties and have been applied as nanostrategies in many medical and surgical fields, including dentistry. The use of silver nanoparticles (Ag NPs may be an option for reducing bacterial adhesion to dental implant surfaces and preventing biofilm formation, containing the risk of peri-implant infections. Modifying the structure or surface of bone grafts and membranes with Ag NPs may also prevent the risk of contamination and infection that are common when alveolar bone augmentation techniques are used. On the other hand, Ag NPs have revealed some toxic effects on cells in vitro and in vivo in animal studies. In this setting, the aim of the present paper is to summarize the principle behind Ag NP-based devices and their clinical applications in alveolar bone and dental implant surgery.

  6. Alveolar ridge preservation immediately after tooth extraction.

    Science.gov (United States)

    Feller, L; Khammissa, R A G; Bouckaert, M; Lemmer, J

    2013-10-01

    Ridge preservation procedures immediately after tooth extraction, are commonly used with a view to minimising remodelling and shrinkage of the alveolar ridge, associated with socket healing. These procedures may sometimes be effective, but they cannot completely prevent reduction in dimension of the ridge. Certain biomater als used may actually hamper normal deposition of bone within the healing socket, reducing bone trabeculae that can integrate with the implant surface. However, in extraction sockets in alveolar ridges of low bone density, particles of implanted bone substitute incorporated in the healing bone, may enhance the mechanical support for the implant, provided by normal healed bone of low trabecular density alone. This paper reviews biological rationales and procedures for ridge preservation immediately after extraction and comments on their clinical use.

  7. A co-culture system with an organotypic lung slice and an immortal alveolar macrophage cell line to quantify silica-induced inflammation.

    Directory of Open Access Journals (Sweden)

    Falk Hofmann

    Full Text Available There is growing evidence that amorphous silica nanoparticles cause toxic effects on lung cells in vivo as well as in vitro and induce inflammatory processes. The phagocytosis of silica by alveolar macrophages potentiates these effects. To understand the underlying molecular mechanisms of silica toxicity, we applied a co-culture system including the immortal alveolar epithelial mouse cell line E10 and the macrophage cell line AMJ2-C11. In parallel we exposed precision-cut lung slices (lacking any blood cells as well as residual alveolar macrophages of wild type and P2rx7-/- mice with or without AMJ2-C11 cells to silica nanoparticles. Exposure of E10 cells as well as slices of wild type mice resulted in an increase of typical alveolar epithelial type 1 cell proteins like T1α, caveolin-1 and -2 and PKC-β1, whereas the co-culture with AMJ2-C11 showed mostly a slightly lesser increase of these proteins. In P2rx7-/- mice most of these proteins were slightly decreased. ELISA analysis of the supernatant of wild type and P2rx7-/- mice precision-cut lung slices showed decreased amounts of IL-6 and TNF-α when incubated with nano-silica. Our findings indicate that alveolar macrophages influence the early inflammation of the lung and also that cell damaging reagents e.g. silica have a smaller impact on P2rx7-/- mice than on wild type mice. The co-culture system with an organotypic lung slice is a useful tool to study the role of alveolar macrophages during lung injury at the organoid level.

  8. Effect of butyrate and fermentation products on epithelial integrity in a mucus-secreting human colon cell line

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Jensen, Bent Borg; Theil, Peter Kappel

    2018-01-01

    . This was associated with regulation of different genes involved in epithelial integrity, mucus secretion, apoptosis, oxidative stress, and butyrate transport. In conclusion, butyrate in concentrations that can be achieved by dietary intervention in vivo enhanced the epithelial barrier function in vitro. B...

  9. Stem cell factor expression after renal ischemia promotes tubular epithelial survival.

    Directory of Open Access Journals (Sweden)

    Geurt Stokman

    Full Text Available BACKGROUND: Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration of renal tissue. The cytokine stem cell factor (SCF has been shown to protect the tubular epithelium against apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse model for renal ischemia/reperfusion injury, we studied how expression of c-KIT on tubular epithelium and its ligand SCF protect cells against apoptosis. Administration of SCF specific antisense oligonucleotides significantly decreased specific staining of SCF following ischemia. Reduced SCF expression resulted in impaired renal function, increased tubular damage and increased tubular epithelial apoptosis, independent of inflammation. In an in vitro hypoxia model, stimulation of tubular epithelial cells with SCF activated survival signaling and decreased apoptosis. CONCLUSIONS/SIGNIFICANCE: Our data indicate an important role for c-KIT and SCF in mediating tubular epithelial cell survival via an autocrine pathway.

  10. [Clinical research on repairing alveolar cleft with osteoinduction active material].

    Science.gov (United States)

    She, Xiao-ming; Zhang, Qian; Tian, Kun; Yang, Li; Xiong, Gui-fa

    2010-08-01

    To study the feasibility and authenticity of repairing alveolar defects in alveolar cleft patients with osteoinduction active material (OAM) in clinic. Twenty-seven cases of alveolar defect chosen from clinic were divided into two groups (test group and control group). For test group (12 cases), OAM was transplanted to repair the alveolar cleft. For control group (15 cases), autogenous ilium cancellous bone were transplanted into the defect region to repair alveolar cleft. At 6 months after operation, CT and three-dimensional reconstruction were used to observe alveolar appearance, and the effect and clinical success rate of recover alveolar cleft by using different repair material were compared. In the 27 cases, all the maxillary continuity was restored except two of test group and two of control group. There was no significant difference between test group and control group regarding the clinical success rate of the alveolar cleft repair (P = 1.000). OAM was used to repair the alveolar cleft that can result in new bone formations and the burgeon of canines from the bone grafted areas. There is no significant difference between OAM and autogenous ilium cancellous bone regarding the effect of the alveolar cleft repair.

  11. Alveolar ridge preservation in the esthetic zone.

    Science.gov (United States)

    Jung, Ronald E; Ioannidis, Alexis; Hämmerle, Christoph H F; Thoma, Daniel S

    2018-02-27

    In the esthetic zone, in the case of tooth extraction, the clinician is often confronted with a challenge regarding the optimal decision-making process for providing a solution using dental implants. This is because, after tooth extraction, alveolar bone loss and structural and compositional changes of the covering soft tissues, as well as morphological alterations, can be expected. Ideally, the therapeutic plan starts before tooth extraction and it offers three options: spontaneous healing of the extraction socket; immediate implant placement; and techniques for preserving the alveolar ridge at the site of tooth removal. The decision-making process mainly depends on: (i) the chosen time-point for implant placement and the ability to place a dental implant; (ii) the quality and quantity of soft tissue in the region of the extraction socket; (iii) the remaining height of the buccal bone plate; and (iv) the expected rates of implant survival and success. Based on scientific evidence, three time-periods for alveolar ridge preservation are described in the literature: (i) soft-tissue preservation with 6-8 weeks of healing after tooth extraction (for optimization of the soft tissues); (ii) hard- and soft-tissue preservation with 4-6 months of healing after tooth extraction (for optimization of the hard and soft tissues); and (iii) hard-tissue preservation with > 6 months of healing after tooth extraction (for optimization of the hard tissues). © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Oral epithelial dysplasia classification systems

    DEFF Research Database (Denmark)

    Warnakulasuriya, S; Reibel, J; Bouquot, J

    2008-01-01

    . In this report, we review the oral epithelial dysplasia classification systems. The three classification schemes [oral epithelial dysplasia scoring system, squamous intraepithelial neoplasia and Ljubljana classification] were presented and the Working Group recommended epithelial dysplasia grading for routine...

  13. Effects of topical vitamin E on keratocyte apoptosis after traditional photorefractive keratectomy.

    Science.gov (United States)

    Bilgihan, K; Adiguzel, U; Sezer, C; Akyol, G; Hasanreisoglu, B

    2001-01-01

    To evaluate the keratocyte apoptosis and effects of topical vitamin E on keratocyte apoptosis after photorefractive surgery. Rabbits were divided into 7 groups, and all groups were compared with controls after epithelial scraping, epithelial scrape and photorefractive keratectomy (PRK) (traditional PRK), transepithelial PRK, production of a corneal flap with microkeratome and laser-assisted in situ keratomileusis (LASIK). The effects of topical Vitamin E treatment were investigated in the traditional PRK group. The terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay (to detect DNA fragmentation in situ) and light microscopy have been used to detect apoptosis in rabbit cornea. Transepithelial PRK induced minimal keratocyte apoptosis, less than in all other refractive surgical procedures. The greatest amount of keratocyte apoptosis was observed after traditional PRK (p = 0.001), therefore we tested the effects of topical vitamin E in this group. The number of apoptotic keratocytes significantly reduced after vitamin E therapy (p < 0.005). Keratocytes undergo apoptosis after refractive surgery in response to mechanical epithelial removal, preparing of corneal flap and excimer laser stromal photoablation. The topical application of vitamin E immediately after surgery can prevent keratocyte apoptosis, and this result suggests that free radicals may be partly responsible for keratocyte apoptosis after excimer laser keratectomy. Copyright 2001 S. Karger AG, Basel

  14. Apoptosis – is it good or bad?

    Directory of Open Access Journals (Sweden)

    Bakir Mehić

    2012-08-01

    induced by TNF-α. The over expression of antiapoptotic proteins may allow injured cells to survive, and autophagy may assist by providing critical metabolites. Apoptotic cells induce anergy or an immunosuppressive phenotype, whereas necrotic cells augment inflammation, in part by binding the receptor C-type lectin domain family 9 on dendritic cells.Clinical implications of apoptosis are consist in more than 50% of neoplasm’s that have defects in the apoptotic machinery mutations in the tumor-suppressor gene TP53, that is called the “guardian of the genome,” initiates apoptosis in response to DNA damage induced by radiation, chemical agents, oxidative stress, and other agents.[3] Abnormalities in apoptosis can increase susceptibility to autoimmune diseases.[4] There is growing evidence that neuronal apoptosis plays a key role in neonatal brain disorders.[5] Hepatocytes are particularly prone to apoptosis in response to various types of stress, including infections.[6] Necrosis predominates in ischemic injury, but often there are apoptotic cells in the hypoxic penumbra in myocardial infarction and stroke and in globally hypoxic zones after reperfusion injury. Sepsis is perhaps the most remarkable clinical setting in which apoptosis occurs. Massive apoptosis of immune effectors’ cells and gastrointestinal epithelial cells develops in patients with sepsis.[7] The profound loss of immune effectors’ cells in sepsis inhibits the ability of the immune system to eradicate the primary infection and renders the patient susceptible to nosocomial infections. Finally, why apoptosis is good? Because without apoptosis, 2 tons of bone marrow and lymph nodes and a 16-km intestine would probably accumulate in a human by the age of 8o.[8

  15. Ghrelin inhibits ovarian epithelial carcinoma cell proliferation in vitro.

    Science.gov (United States)

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

    2013-11-01

    The only orexigenic peptide, ghrelin, which is primarily produced by the gastrointestinal tract, has been implicated in malignant cell proliferation and invasion. Ghrelin is a natural ligand of the growth hormone secretagogue receptor 1a (GHSR1a). However, the role of ghrelin in ovarian epithelial carcinoma remains unknown since the expression of GHSR1a in ovary is not confirmed. The aim of the present study was to assess expression of ghrelin and its receptor in human ovarian epithelial carcinoma and to examine the effect of ghrelin on carcinoma cell proliferation. Frozen sections of ovarian samples and the human ovarian epithelial carcinoma cell line, HO-8910, were used to characterize the expression of ghrelin/GHSR1a axis and the effect of ghrelin on proliferation. We found that ghrelin and GHSR1a are expressed in ovarian epithelial carcinoma in vivo and in vitro. Ghrelin inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, and this inhibition may be abolished by the ghrelin receptor antagonist D-Lys-3-GH-releasing peptide-6 and ghrelin neutralizing antibody. Ghrelin enhances HO-8910 cell apoptosis and autophagy. The activation of mammalian target of rapamycin (mTOR) signaling pathway blocks the effects of ghrelin-induced autophagy and apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation induced by ghrelin. In conclusion, the present study demonstrates that ghrelin inhibits the proliferation of human HO-8910 ovarian epithelial carcinoma cells by inducing apoptosis and autophagy via the mTOR signaling pathway. This study provides a novel regulatory signaling pathway of ghrelin-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy.

  16. Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

    International Nuclear Information System (INIS)

    Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

    2015-01-01

    To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis

  17. ING1 induces apoptosis through direct effects at the mitochondria

    DEFF Research Database (Denmark)

    Bose, P; Thakur, S; Thalappilly, S

    2013-01-01

    The ING family of tumor suppressors acts as readers and writers of the histone epigenetic code, affecting DNA repair, chromatin remodeling, cellular senescence, cell cycle regulation and apoptosis. The best characterized member of the ING family, ING1,interacts with the proliferating cell nuclear...... translocates to the mitochondria of primary fibroblasts and established epithelial cell lines in response to apoptosis inducing stimuli, independent of the cellular p53 status. The ability of ING1 to induce apoptosis in various breast cancer cell lines correlates well with its degree of translocation...... to the mitochondria after UV treatment. Endogenous ING1 protein specifically interacts with the pro-apoptotic BCL2 family member BAX, and colocalizes with BAX in a UV-inducible manner. Ectopic expression of a mitochondria-targeted ING1 construct is more proficient in inducing apoptosis than the wild type ING1 protein...

  18. Intranasal Fentanyl Intoxication Leading to Diffuse Alveolar Hemorrhage.

    Science.gov (United States)

    Ruzycki, Shannon; Yarema, Mark; Dunham, Michael; Sadrzadeh, Hossein; Tremblay, Alain

    2016-06-01

    Increasing rates of opioid abuse, particularly fentanyl, may lead to more presentations of unusual effects of opioid toxicity. Diffuse alveolar hemorrhage is a rare complication of fentanyl overdose. A 45-year-old male presented in hypoxic respiratory failure secondary to diffuse alveolar hemorrhage requiring intubation. Comprehensive drug screening detected fentanyl without exposure to cocaine. Further history upon the patient's recovery revealed exposure to snorted fentanyl powder immediately prior to presentation. Diffuse alveolar hemorrhage is a potential, though rare, presentation of opioid intoxication. Recognition of less common complications of opioid abuse such as diffuse alveolar hemorrhage is important in proper management of overdoses.

  19. Pulmonary epithelial permeability after inhaling saline, distilled water ''fog'' and cold air

    International Nuclear Information System (INIS)

    Borland, C.; Chamberlain, A.; Barber, B.; Higenbottam, T.

    1985-01-01

    It is recognized that hyperventilation of cold air and the inhalation of fine mists of distilled water provoke significant bronchoconstriction in the asthmatic individual, yet little is known as to how these provocations affect the structural integrity of the alveolar epithelial membrane. In 11 normal subjects, the following effects have been studied: cold air hyperventilation for three minutes, inhalation of 80 L of ultrasonically nebulized distilled water ''fog,'' and 80 L of isotonic saline ''fog'' on the half time clearance (T1/2) from the alveoli of technetium 99m diethylene triamine pentaacetate (DTPA), inhaled as an aerosol. The DTPA T1/2 provided a measurement of pulmonary epithelial permeability

  20. PERFORATION OF INFERIOR ALVEOLAR NERVE BY MAXILLARY ARTERY. LA PERFORACION DEL NERVIO ALVEOLAR INFERIOR POR LA ARTERIA MAXILAR

    OpenAIRE

    Vanishree S Nayak; Ramachandra Bhat K; Prakash Billakanti Babu

    2011-01-01

    Infratemporal fossa is clinically important anatomical area for the delivery of local anesthetic agents in dentistry and maxillofacial surgery. Variations in the anatomy of the inferior alveolar nerve and maxillary artery were studied in infratemporal dissection. During routine dissection of the head in an adult male cadaver an unusual variation in the origin of the inferior alveolar nerve and its relationship with the surrounding structures was observed. The inferior alveolar nerve originate...

  1. Segment distraction to reduce a wide alveolar cleft before alveolar bone grafting.

    NARCIS (Netherlands)

    Binger, T.; Katsaros, C.; Rucker, M.; Spitzer, W.J.

    2003-01-01

    OBJECTIVE: To demonstrate a method for reduction of wide alveolar clefts prior to bone grafting. This method aims to facilitate bone grafting and achieve adequate soft tissue coverage of the graft with attached gingiva. CASE REPORT: Treatment of a patient with bilateral cleft lip and palate with a

  2. Alveolar soft part sarcoma: A rare diagnosis

    Directory of Open Access Journals (Sweden)

    Priyanka Sarkar

    2013-01-01

    Full Text Available Alveolar soft-part sarcoma (ASPS is an extremely rare disease arising from connective tissues with a propensity for recurrence and metastasis. Clinically, it can be confused with hemangioma or arterio-venous malformations. Thus, a high index of suspicion and histopathological examination are required to make a definitive diagnosis. We report a case of recurrent ASPS in a young female with multiple sites involvement without any features of metastasis who has been treated with excision of the symptomatic lesions followed by chemotherapy.

  3. The Inhibitor of Apoptosis Protein-Binding Domain of Smac Is Not Essential for Its Proapoptotic Activity

    OpenAIRE

    Roberts, Darren L.; Merrison, Wendy; MacFarlane, Marion; Cohen, Gerald M.

    2001-01-01

    Smac/DIABLO, a recently identified inhibitor of apoptosis protein (IAP)-binding protein, is released from the mitochondria during apoptosis and reportedly potentiates apoptosis by relieving the inhibition of IAPs on caspases. We now describe the molecular characterization of Smac β, an alternatively spliced form of Smac, which lacks the mitochondrial-targeting sequence found in Smac and has a cortical distribution in both human embryonic kidney 293 and breast epithelial tumor MCF-7 cells. Sma...

  4. Nostril Base Augmentation Effect of Alveolar Bone Graft

    Directory of Open Access Journals (Sweden)

    Woojin Lee

    2013-09-01

    Full Text Available Background The aims of alveolar bone grafting are closure of the fistula, stabilization ofthe maxillary arch, support for the roots of the teeth adjacent to the cleft on each side.We observed nostril base augmentation in patients with alveolar clefts after alveolar bonegrafting. The purpose of this study was to evaluate the nostril base augmentation effect ofsecondary alveolar bone grafting in patients with unilateral alveolar cleft.Methods Records of 15 children with alveolar clefts who underwent secondary alveolar bonegrafting with autogenous iliac cancellous bone between March of 2011 and May of 2012 werereviewed. Preoperative and postoperative worm’s-eye view photographs and reconstructedthree-dimensional computed tomography (CT scans were used for photogrammetry. Thedepression of the nostril base and thickness of the philtrum on the cleft side were measuredin comparison to the normal side. The depression of the cleft side pyriform aperture wasmeasured in comparison to the normal side on reconstructed three-dimensional CT.Results Significant changes were seen in the nostril base (P=0.005, the philtrum length(P=0.013, and the angle (P=0.006. The CT measurements showed significant changes in thepyriform aperture (P<0.001 and the angle (P<0.001.Conclusions An alveolar bone graft not only fills the gap in the alveolar process but alsoaugments the nostril base after surgery. In this study, only an alveolar bone graft was performedto prevent bias from other procedures. Nostril base augmentation can be achieved byperforming alveolar bone grafts in children, in whom invasive methods are not advised.

  5. ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

    Science.gov (United States)

    Yan, Hongchao; Qiu, Linglin; Xie, Xiaolei; Yang, He; Liu, Yongli; Lin, Xiaoman; Huang, Hongxiang

    2017-03-01

    The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1‑ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (Povarian epithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (Povarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

  6. Specific forms of BAFF favor BAFF receptor-mediated epithelial cell survival.

    Science.gov (United States)

    Lahiri, Ayan; Varin, Marie-Michèle; Le Pottier, Laëtitia; Pochard, Pierre; Bendaoud, Boutahar; Youinou, Pierre; Pers, Jacques-Olivier

    2014-06-01

    Although B cell activating factor (BAFF) and its receptor BR3 are produced and expressed by many cells, their role has been restricted to the lymphocyte lineage. Using various techniques (RT-PCR, indirect immunofluorescence, flow cytometry analysis), we observed the expression of BR3 and the production of BAFF by the human salivary gland cell line, by epithelial cells from biopsies of Sjögren's syndrome patients and their controls, but also by salivary gland epithelial cells in culture. To decipher the role of BAFF and BR3 on epithelial cells, BAFF and BR3 were neutralized by blocking antibodies or RNA specific inhibitor (siBR3) and epithelial cell survival was analyzed. Blocking BR3 promotes epithelial cell apoptosis in vitro. This apoptosis resulted in the nuclear translocation of PKCδ. BAFF neutralization by various anti-BAFF antibodies leads to different effects depending on the antibody used suggesting that only some forms of BAFF are required for epithelial cell survival. Our study demonstrates that BR3 is involved in the survival of cultured epithelial cells due to an autocrine effect of BAFF. It also suggests that epithelial cells produce different forms of BAFF and that only some of them are responsible for this effect. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Mechanisms of Apoptosis on Human Lens Epithelium after Ultraviolet Light Exposure

    Science.gov (United States)

    Kim, Seong-Taeck

    2011-01-01

    Purpose The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes. Methods Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway. Results Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased. Conclusions Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells. PMID:21655046

  8. Molecular basis of potassium channels in pancreatic duct epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Novak, Ivana

    2013-01-01

    Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K channels...... other cell types, preferably in epithelia, and, where known, their identification and functions in pancreatic ducts and in adenocarcinoma cells. We conclude by pointing out some outstanding questions and future directions in pancreatic K channel research with respect to the physiology of secretion...

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    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.

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

    International Nuclear Information System (INIS)

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

    2013-01-01

    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

  11. Transcriptional networks in epithelial-mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Christo Venkov

    Full Text Available Epithelial-mesenchymal transition (EMT changes polarized epithelial cells into migratory phenotypes associated with loss of cell-cell adhesion molecules and cytoskeletal rearrangements. This form of plasticity is seen in mesodermal development, fibroblast formation, and cancer metastasis.Here we identify prominent transcriptional networks active during three time points of this transitional process, as epithelial cells become fibroblasts. DNA microarray in cultured epithelia undergoing EMT, validated in vivo, were used to detect various patterns of gene expression. In particular, the promoter sequences of differentially expressed genes and their transcription factors were analyzed to identify potential binding sites and partners. The four most frequent cis-regulatory elements (CREs in up-regulated genes were SRY, FTS-1, Evi-1, and GC-Box, and RNA inhibition of the four transcription factors, Atf2, Klf10, Sox11, and SP1, most frequently binding these CREs, establish their importance in the initiation and propagation of EMT. Oligonucleotides that block the most frequent CREs restrain EMT at early and intermediate stages through apoptosis of the cells.Our results identify new transcriptional interactions with high frequency CREs that modulate the stability of cellular plasticity, and may serve as targets for modulating these transitional states in fibroblasts.

  12. Alveolar bone loss in obese subjects.

    Science.gov (United States)

    Alabdulkarim, Maher; Bissada, Nabil; Al-Zahrani, Mohammad; Ficara, Anthony; Siegel, Burton

    2005-04-01

    Obesity was found to be significantly associated with periodontal disease prevalence as measured by probing depth and clinical attachment loss. The aim of this study was to examine if obesity correlates with chronic periodontitis as diagnosed by radiographic alveolar bone loss. Four hundred subjects > or =18 years old were included; 200 with body mass index (BMI) > or =30 kg/m2 (obese) and 200 with BMI periodontitis. Obesity was found to be significantly associated with periodontitis in the uni-variate regression analysis (OR = 2.37, 95% CI, 1.55-3.63). After adjusting for age, gender, smoking, employment, diabetes, marital status, and number of teeth present, obese subjects were found to be 1.86 times more likely to have periodontitis (95% CI, 0.99-3.51) than non-obese ones. When the sample was stratified based on age, the multivariate association was statistically significant among individuals or = 40 years of age the association was statistically insignificant (OR = 1.06, 95% CI, 0.57-1.95). Stratifying the sample based on gender and smoking status revealed a stronger association among females than males (OR = 3.14 vs. 1.95) and among non-smokers than smokers (OR = 3.36 vs. 2.22). Obesity is associated with increased prevalence of periodontitis as measured by radiographic alveolar bone loss, especially among younger individuals. Prevention and management of obesity may be considered to promote better systemic and periodontal health.

  13. Prominent Intracytoplasmic Crystals in Alveolar Soft Part Sarcoma (ASPS): An Aid in Cytological Diagnosis.

    Science.gov (United States)

    Mannan, Rahul; Bhasin, Tejinder Singh; Kaur, Parampreet; Manjari, Mridu; Gill, Karamjit Singh

    2014-02-01

    Alveolar soft part sarcoma (ASPS) is a rare neoplasm of unknown histogenesis with poor prognosis. Due to the epithelioid appearance of the neoplastic cells, ASPS may resemble many neoplastic conditions, such as metastatic epithelial cell tumours with clear cell change, metastatic renal cell carcinoma, granular cell tumour, epithelioid sarcoma, malignant melanoma and even paragangliomas. Presence of abundant, rod like crystals in the cytoplasm of tumour cells is an important finding characteristic of this tumour, which helps in differentiating it from the other entities. The case study highlights the importance of correlating cytological features that help in reaching the diagnosis such as the background, cell morphology and presence of characteristic rod shaped crystals as immunohistochemical studies are often non-conclusive. The case also is unique as it demonstrates presence of intra-cytoplamic crystals in such abundance.

  14. Celiac Disease: Role of the Epithelial BarrierSummary

    Directory of Open Access Journals (Sweden)

    Michael Schumann

    2017-03-01

    Full Text Available In celiac disease (CD a T-cell–mediated response to gluten is mounted in genetically predisposed individuals, resulting in a malabsorptive enteropathy histologically highlighted by villous atrophy and crypt hyperplasia. Recent data point to the epithelial layer as an under-rated hot spot in celiac pathophysiology to date. This overview summarizes current functional and genetic evidence on the role of the epithelial barrier in CD, consisting of the cell membranes and the apical junctional complex comprising sealing as well as ion and water channel-forming tight junction proteins and the adherens junction. Moreover, the underlying mechanisms are discussed, including apoptosis of intestinal epithelial cells, biology of intestinal stem cells, alterations in the apical junctional complex, transcytotic uptake of gluten peptides, and possible implications of a defective epithelial polarity. Current research is directed toward new treatment options for CD that are alternatives or complementary therapeutics to a gluten-free diet. Thus, strategies to target an altered epithelial barrier therapeutically also are discussed. Keywords: Celiac Sprue, Gluten-Sensitive Enteropathy, Tight Junction, Epithelial Polarity, Partitioning-Defective Proteins, α-Gliadin 33mer

  15. Prevention of Alveolar Osteitis After Third Molar Surgery ...

    African Journals Online (AJOL)

    2017-05-16

    May 16, 2017 ... development of alveolar osteitis were obtained and analyzed. Comparative statistics were done using ... KEY WORDS: Alveolar osteitis, chlorhexidine, prevention, warm saline. Department of Dental. Surgery .... healing by inducing vasodilatation of the vasculature of oral cavity, and thus enhances migration ...

  16. Alveolar ridge augmentation by osteoinductive materials in goats

    DEFF Research Database (Denmark)

    Pinholt, E M; Haanaes, H R; Roervik, M

    1992-01-01

    The purpose of the present study was to determine whether alveolar ridge augmentation could be induced in goats. In 12 male goats allogenic, demineralized, and lyophilized dentin or bone was implanted subperiosteally on the buccal sides of the natural edentulous regions of the alveolar process...

  17. Tongue-Palate Contact of Perceptually Acceptable Alveolar Stops

    Science.gov (United States)

    Lee, Alice; Gibbon, Fiona E.; O'Donovan, Cliona

    2013-01-01

    Increased tongue-palate contact for perceptually acceptable alveolar stops has been observed in children with speech sound disorders (SSD). This is a retrospective study that further investigated this issue by using quantitative measures to compare the target alveolar stops /t/, /d/ and /n/ produced in words by nine children with SSD (20 tokens of…

  18. Classification of alveolar bone destruction patterns on maxillary ...

    African Journals Online (AJOL)

    Objective: The defective diagnosis of alveolar structures is one of most serious handicaps when assessing available periodontal treatment options for the prevention of tooth loss. The aim of this research was to classify alveolar bone defects in the maxillary molar region which is a challenging area for dental implant ...

  19. Pulmonary alveolar proteinosis in a child from an informal settlement

    African Journals Online (AJOL)

    clinical, histopathological, biochemical and genetic data.[3]. Surfactant homeostasis is critical for lung function and is tightly regulated, in part by pulmonary granulocyte-macrophage colony- stimulating factor (GM-CSF), which is required for surfactant clearance by alveolar macrophages and alveolar macrophage maturation.

  20. Alveolar ridge preservation and biologic width management for ...

    African Journals Online (AJOL)

    Alveolar bone atrophy is a chronically progressive, irreversible process which results in bone loss in both the buccal, lingual and apico-coronal region. Without bone preservation measures, bone resorption is experienced and continues for life. Preservation of alveolar ridge is indicated when a tooth-supported fixed partial ...

  1. Diffuse alveolar hemorrhage in a young woman with systemic lupus ...

    African Journals Online (AJOL)

    Diffuse Alveolar Hemorrhage (DAH) is rarely reported complication of Systemic Lupus Erythematosus (SLE). A young woman diagnosed SLE, with a previously normal plain chest radiograph, developed acute onset cough, dyspnoea and hemoptysis. The repeat urgent chest radiograph revealed alveolar opacities. The triad ...

  2. Cultured fibroblasts from alveolar and gingival mucosae are biologically and biochemically different

    International Nuclear Information System (INIS)

    Lanz, J.; Banes, A.

    1986-01-01

    Tissues removed from the alveolar or gingival mucosa of 5 patients were separated into cell populations to assess the relative contributions each might make in wound healing intraorally. Growth curves and protein synthetic patterns of fibroblasts, free of epithelial cells, were obtained at pass 5. The morphologies of the two cell types were not grossly different. However, the AM cells (alveolar mucosa) had a generation time (gt) of 18.7 hrs. whereas the gt for KG cells (keratinized gingiva) was 49.6 hrs. Cells labeled in vitro with 35 S-methionine had distinct patterns of protein synthesis. The AM cells had more of the 275, 220, 92, 80, 50 and 46 kd bands on the autoradiogram of a 7.5% PAGE slab gel than did the KG cells. The KG cells contained more of the 165, 84, 68, 60, 54, 51, 43, 36, and 32a kd bands. In a wound healing situation, the AM cells may be the first fibroblasts to rapidly divide to fill a defect, whereas the KG cells may require a longer time period to divide. This is the first report of biochemical and biological differences in these two fibroblast populations from cultured, human tissues

  3. Electron-microscopic studies of alveolar macrophages from gamma-ray irradiated guinea pigs

    International Nuclear Information System (INIS)

    Krystev, Kh.; Najdenski, Kh.M.; Velyanov, D.K.; Radoevska, S.A.

    1988-01-01

    The alveolar macrophages (AM) were obtained from whole body gamma-irradiated guinea pigs (0.5 Gy and 2 Gy; 92.5 rad/min). The cell suspension contained granulocytes, lymphocytes and disintegrating epithelial and white blood cells, as well as two types of microphages: large (possessing nuclei of saddle bag-like or highly folded form) and small (with spherical or eggshaped nuclei). Eleven electronograms were presented showing all ultrastuctural changes of both small and large AM. The morphological differences between the small and large alveolar macrophages were slight. Marked changes were observed in the large AM on day 1 following 0.5 Gy irradiation: a considerable increase in dimensions of phagosomes turning in digestive vacuoles, lamellarly limited and containing osmiophilic, irregularly formed, densely or lamellarly arranged matter; folded nuclei with slightly vacuolized cytoplasm. The ultrastructural changes in the AM of sublethal dose (2 Gy) irradiated animals were stronger and regenerative processes in them were less possible. On day 30 after irradiation several damaged AM were observed with large digestive vacuoles of fine-grain content, vacuolized endoplasmic reticulum, entirely lyzed nuclear chromatin and free nuclei without cytoplasm. All observations were a convincing indication that guinea pigs AM were more radiosensitive than that obtained from rats and mice

  4. Mannose-functionalized solid lipid nanoparticles are effective in targeting alveolar macrophages.

    Science.gov (United States)

    Costa, Ana; Sarmento, Bruno; Seabra, Vítor

    2018-03-01

    Mannose receptor is highly expressed on alveolar macrophages, being a potential target to promote the specific local drug delivery of anti-tuberculosis agents through the use of functionalized nanocarriers. In this work, isoniazid (Isn)-loaded solid lipid nanoparticles (SLN), reinforced with stearylamine (SA) were produced by double emulsion technique and further surface-functionalized with mannose in a straightforward chemical approach. Upon pre-formulation assessment, SLN close to 500 nm average size, positively charged and with association efficiency of ISN close to 50% were obtained. Functionalization with mannose was performed after SLN production and confirmed by Fourier transform infrared spectroscopy (FTIR). Both functionalized and non-functionalized SLN demonstrated to devoid of toxicity when tested in human lung epithelial cell line (NCI-H441) and differentiated THP-1 (dTHP-1), reducing the intrinsic cytotoxicity of Isn when incorporated into SLN. Uptake studies were conducted on same macrophage-like cells and the results showed that fluorescent mannosylated SLN (M-SLN) were more efficient in be internalized comparatively to SLN. Moreover, the uptake of M-SLN was reduced when cells were pre-incubated with mannose, demonstrating the receptor-dependence internalization of functionalized SLN. These functionalized nanocarriers may represent a useful platform to target alveolar macrophages for delivering anti-infective drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Serum YKL-40 is a reliable biomarker for pulmonary alveolar proteinosis.

    Science.gov (United States)

    Bonella, Francesco; Long, Xiaoping; He, Xuan; Ohshimo, Shinichiro; Griese, Matthias; Guzman, Josune; Costabel, Ulrich

    2017-10-01

    Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by alveolar filling. YKL-40, a chitinase-like protein produced by macrophages and epithelial cells, is increased in patients with interstitial lung diseases. We aimed to evaluate the role of YKL-40 as a biomarker for PAP. A total of 34 patients with autoimmune PAP and 50 healthy controls were studied. YKL-40 was measured by ELISA in serum and bronchoalveolar lavage fluid (BALF). Chitinase coding gene polymorphisms (CHI3L1-329 and -131) were detected by PCR and pyrosequencing. Correlations between serum YKL-40 levels and disease outcome were analysed. Baseline serum and BALF levels of YKL-40 were higher in PAP patients than in controls (286 ± 27 ng/mL vs 42 ± 4 ng/mL, P 40 levels correlated with diffusing capacity of the lung for carbon monoxide (DL CO ) at baseline (P = 0.002) and over time (P 40 levels than those who remained stable or improved (P 40. YKL-40 is elevated in serum and BALF of PAP patients, and may be of clinical utility to predict outcome in PAP. © 2017 Asian Pacific Society of Respirology.

  6. Arachidonate metabolism increases as rat alveolar type II cells differentiate in vitro

    International Nuclear Information System (INIS)

    Lipchik, R.J.; Chauncey, J.B.; Paine, R.; Simon, R.H.; Peters-Golden, M.

    1990-01-01

    Rat type II alveolar epithelial cells are known to undergo morphological and functional changes when maintained in culture for several days. Having previously demonstrated that these cells can deacylate free arachidonic acid (AA) and metabolize it to products of the cyclooxygenase pathway, the present study was undertaken to determine whether in vitro differentiation was accompanied by alterations in the availability and metabolism of AA. We assessed the constitutive and ionophore A23187-induced deacylation and metabolism of endogenous AA, as well as the metabolism of exogenously supplied AA, in primary cultures of rat type II cells at days 2, 4, and 7 after isolation. Levels of free endogenous AA were increased at day 4, whereas eicosanoid synthesis, predominantly prostaglandin E2 and prostacyclin, increased markedly only at day 7. A similar time course of augmentation of prostanoid release was seen in response to exogenous AA. Type II cells cultured on fibronectin, intended to hasten cell flattening and spreading, demonstrated accelerated increases in available free AA in response to A23187; cells cultured on basement membrane derived from Engelbreth-Holm-Swarm mouse sarcoma, known to maintain the type II phenotype, exhibited diminished levels of available free AA. From these findings, we conclude that alterations in arachidonate metabolism are linked to alterations in cellular phenotype. The potentiation of eicosanoid synthesis accompanying in vitro differentiation suggests a possible role for the alveolar epithelium in the modulation of inflammation and fibrosis in the distal lung

  7. Decreased IGF Type 1 Receptor Signaling in Mammary Epithelium during Pregnancy Leads to Reduced Proliferation, Alveolar Differentiation, and Expression of Insulin Receptor Substrate (IRS)-1 and IRS-2

    Science.gov (United States)

    Sun, Zhaoyu; Shushanov, Sain; LeRoith, Derek

    2011-01-01

    The IGFs and the IGF type 1 receptor (IGF-1R) are essential mediators of normal mammary gland development in mice. IGF-I and the IGF-1R have demonstrated functions in formation and proliferation of terminal end buds and in ductal outgrowth and branching during puberty. To study the functions of IGF-1R during pregnancy and lactation, we established transgenic mouse lines expressing a human dominant-negative kinase dead IGF-1R (dnhIGF-1R) under the control of the whey acidic protein promoter. We provide evidence that the IGF-1R pathway is necessary for normal epithelial proliferation and alveolar formation during pregnancy. Furthermore, we demonstrate that the whey acidic protein-dnhIGF-1R transgene causes a delay in alveolar differentiation including lipid droplet formation, lumen expansion, and β-casein protein expression. Analysis of IGF-1R signaling pathways showed a decrease in P-IGF-1R and P-Akt resulting from expression of the dnhIGF-1R. We further demonstrate that disruption of the IGF-1R decreases mammary epithelial cell expression of the signaling intermediates insulin receptor substrate (IRS)-1 and IRS-2. No alterations were observed in downstream signaling targets of prolactin and progesterone, suggesting that activation of the IGF-1R may directly regulate expression of IRS-1/2 during alveolar development and differentiation. These data show that IGF-1R signaling is necessary for normal alveolar proliferation and differentiation, in part, through induction of signaling intermediates that mediate alveolar development. PMID:21628386

  8. Contemporary Approaches in the Repair of Alveolar Clefts

    Directory of Open Access Journals (Sweden)

    Ufuk Tatli

    2014-08-01

    Full Text Available Cleft lip and palate is one of the most common craniofacial anomalies. The repair of the alveolar clefts is an important part of the treatment for patients with cleft lip and palate. The treatment concepts of alveolar bone grafting are still controversial. The corresponding controversial issues are; timing of alveolar bone grafting, graft materials, and timing of the orthodontic expansion. In the present article, aforementioned controversial issues and contemporary treatment modalities of the maxillary alveolar clefts were reviewed in the light of current literature. In conclusion, the most suitable time for alveolar bone grafting is mixed dentition period. Grafting procedure may be performed in the early or late phases of this period depending on some clinical features. Adjunct orthodontic expansion procedures should be performed before and/or after grafting depending on the patient's current features. [Archives Medical Review Journal 2014; 23(4.000: 563-574

  9. Epithelial Myeloid-Differentiation Factor 88 Is Dispensable during Klebsiella Pneumonia.

    Science.gov (United States)

    Anas, Adam A; Claushuis, Theodora A M; Mohan, Rajiv A; Christoffels, Vincent M; Aidinis, Vassilis; Florquin, Sandrine; Van't Veer, Cornelis; Hou, Baidong; de Vos, Alex F; van der Poll, Tom

    2017-05-01

    Klebsiella pneumoniae is a common cause of pneumonia. Previous studies have documented an important role for Toll-like receptors (TLRs) expressed by myeloid cells in the recognition of K. pneumoniae and the initiation of a protective immune response. Lung epithelial cells also express TLRs and can participate in innate immune defense. The aim of this study was to examine the role of the common TLR adaptor protein myeloid-differentiation factor (MyD) 88 in lung epithelium during host defense against K. pneumoniae-induced pneumonia. To this end, we first crossed mice expressing cre recombinase under the control of the surfactant protein C (SftpCcre) or the club cell 10 kD (CC10cre) promoter with reporter mice to show that SftpCcre mice mainly express cre in type II alveolar cells, whereas CC10cre mice express cre almost exclusively in bronchiolar epithelial cells. We then generated mice with cell-targeted deletion of MyD88 in type II alveolar (SftpCcre-MyD88-lox) and bronchiolar epithelial (CC10cre-MyD88-lox) cells, and infected them with K. pneumoniae via the airways. Bacterial growth and dissemination were not affected by the loss of MyD88 in SftpCcre-MyD88-lox or CC10cre-MyD88-lox mice compared with control littermates. Furthermore, inflammatory responses induced by K. pneumoniae in the lung were not dependent on MyD88 expression in type II alveolar or bronchiolar epithelial cells. These results indicate that MyD88 expression in two distinct lung epithelial cell types does not contribute to host defense during pneumonia caused by a common human gram-negative pathogen.

  10. Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

    Science.gov (United States)

    Jeppsson, Sabrina; Srinivasan, Shanthi; Chandrasekharan, Bindu

    2017-02-01

    We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However, the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSS) were used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY -/- ) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 µM) on epithelial proliferation and apoptosis in vitro. DSS-WT mice exhibited enhanced intestinal inflammation, polyp size, and polyp number (7.5 ± 0.8) compared with DSS-NPY -/- mice (4 ± 0.5, P inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and downregulation of miR-375. Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

  11. Hydroxyapatite paste Ostim, without elevation of full-thickness flaps, improves alveolar healing stimulating BMP- and VEGF-mediated signal pathways: an experimental study in humans.

    Science.gov (United States)

    Canuto, R A; Pol, R; Martinasso, G; Muzio, G; Gallesio, G; Mozzati, M

    2013-08-01

    Tooth extraction is considered as the starting point of jaw atrophy via osteoclast activity stimulation. The maintenance of dental alveolar bone depends on surgery procedure and use of materials to maintain prior space favoring bone regeneration. Among substitutes used in dentistry to fill bone defects, Ostim-Pastes (Ostim) is a nanocrystalline paste tested for treatment of severe clinical conditions. This research first investigated the effect of Ostim on alveolar healing, comparing in the same healthy subjects, an Ostim-filled socket with a not-filled one. Moreover, it also proposed a new surgical protocol for the post-extractive socket treatment using the graft materials without elevation of full-thickness flaps. Fourteen patients were enrolled to bilateral maxillary or mandibular extraction that was performed without elevation of full-thickness flaps. In each patient, one socket was filled using Ostim, and the other one was allowed to undergo natural healing. No suture was carried out. Clinical and biologic parameters were screened at 1, 7, and 14 days. Obtained results evidenced that nanocrystalline hydroxyapatite supports bone regeneration, increasing the synthesis of pro-osteogenic factors as bone morphogenetics protein (BMP)-4, BMP-7, alkaline phosphatase, and osteocalcin. Moreover, filling post-extractive socket with nanocrystalline hydroxyapatite paste leads to a complete epithelialization already at 7 days after extraction, despite the fact that the teeth were extracted without elevation of full-thickness flaps . The improved epithelialization is mediated by increased vascular endothelial growth factor (VEGF) expression. No significant change was observed in inflammatory parameters, with exception of an early and transient IL-1β induction, that could trigger and improve alveolar healing. Clinical and biomolecular observations of this explorative study evidenced that nanocrystalline hydroxyapatite improves alveolar socket healing, increasing angiogenesis

  12. Apoptosis of neutrophils

    NARCIS (Netherlands)

    Maianski, N. A.; Maianski, A. N.; Kuijpers, T. W.; Roos, D.

    2004-01-01

    Regulation of the neutrophil life span by apoptosis provides a fine balance between their function as effector cells of host defense and a safe turnover of these potentially harmful cells. Alterations of neutrophil apoptosis are associated with a number of diseases. As do other cell types,

  13. Ubiquitination in apoptosis signaling

    NARCIS (Netherlands)

    van de Kooij, L.W.

    2014-01-01

    The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

  14. Hyperthermia-induced apoptosis

    NARCIS (Netherlands)

    Nijhuis, E.H.A.

    2008-01-01

    This thesis describes a number of studies that investigated several aspects of heat-induced apoptosis in human lymphoid malignancies. Cells harbour both pro- and anti-apoptotic proteins and the balance between these proteins determines whether a cell is susceptible to undergo apoptosis. In this

  15. Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Nir eOsherov

    2012-09-01

    Full Text Available Aspergillus fumigatus is an opportunistic environmental mold that can cause severe allergic responses in atopic individuals and poses a life-threatening risk for severely immunocompromised patients. Infection is caused by inhalation of fungal spores (conidia into the lungs. The initial point of contact between the fungus and the host is a monolayer of lung epithelial cells. Understanding how these cells react to fungal contact is crucial to elucidating the pathobiology of Aspergillus-related disease states. The experimental systems, both in vitro and in vivo, used to study these interactions, are described. Distinction is made between bronchial and alveolar epithelial cells. The experimental findings suggest that lung epithelial cells are more than just innocent bystanders or a purely physical barrier against infection. They can be better described as an active extension of our innate immune system, operating as a surveillance mechanism that can specifically identify fungal spores and activate an offensive response to block infection. This response includes the internalization of adherent conidia and the release of cytokines, antimicrobial peptides and reactive oxygen species. In the case of allergy, lung epithelial cells can dampen an over-reactive immune response by releasing anti-inflammatory compounds such as kinurenine. This review summarizes our current knowledge regarding the interaction of A. fumigatus with lung epithelial cells. A better understanding of the interactions between A. fumigatus and lung epithelial cells has therapeutic implications, as stimulation or inhibition of the epithelial response may alter disease outcome.

  16. Control of ductal vs. alveolar differentiation of mammary clonogens and susceptibility to radiation-induced mammary cancer

    Energy Technology Data Exchange (ETDEWEB)

    Kamiya, Kenji; Yokoro, Kenjiro (Hiroshima Univ. (Japan). Research Inst. for Nuclear Medicine and Biology); Clifton, K.H.; Gould, M.N.

    1991-12-01

    We have developed an in vitro-in vivo transplantation assay for measuring the concentration of clonogenic epithelial cells in cell suspensions of rat mammary tissue. Rat mammary clonogens from organoid cultures are capable of the same degree of PLDR as clonogens in vivo. The growth and differentiation of mammary clonogens to alveolar colonies or ductal colonies is regulated as follows: (a) in the presence of E{sub 2} and high prolactin (Prl), cortisol induces mammary clonogens to proliferate and differentiate to form alveolar colonies which secrete milk and begin losing clonogenic potential, (b) in cortisol deficient rats, Prl and E{sub 2} synergistically stimulate non-secretory ductal colonies, formation of which retain clonogenic potential, (c) E{sub 2} without progesterone stimulates alveolar colony formation in the presence of cortical and high Prl, (d) progesterone inhibits mammary clonogen differentiation to milk-producing cells and induces ductogenesis in a dose responsive fashion in the presence of E{sub 2}, cortisol and high Prl. High prolactin levels coupled with glucocorticoid deficiency increases the susceptibility to mammary carcinogenesis following low dose radiation exposure by increasing the number of total mammary clonogens which are the presumptive target cells and by stimulating their proliferation after exposure. (author).

  17. Control of ductal vs. alveolar differentiation of mammary clonogens and susceptibility to radiation-induced mammary cancer

    International Nuclear Information System (INIS)

    Kamiya, Kenji; Yokoro, Kenjiro; Clifton, K.H.; Gould, M.N.

    1991-01-01

    We have developed an in vitro-in vivo transplantation assay for measuring the concentration of clonogenic epithelial cells in cell suspensions of rat mammary tissue. Rat mammary clonogens from organoid cultures are capable of the same degree of PLDR as clonogens in vivo. The growth and differentiation of mammary clonogens to alveolar colonies or ductal colonies is regulated as follows: a) in the presence of E 2 and high prolactin (Prl), cortisol induces mammary clonogens to proliferate and differentiate to form alveolar colonies which secrete milk and begin losing clonogenic potential, b) in cortisol deficient rats, Prl and E 2 synergistically stimulate non-secretory ductal colonies, formation of which retain clonogenic potential, c) E 2 without progesterone stimulates alveolar colony formation in the presence of cortical and high Prl, d) progesterone inhibits mammary clonogen differentiation to milk-producing cells and induces ductogenesis in a dose responsive fashion in the presence of E 2 , cortisol and high Prl. High prolactin levels coupled with glucocorticoid deficiency increases the susceptibility to mammary carcinogenesis following low dose radiation exposure by increasing the number of total mammary clonogens which are the presumptive target cells and by stimulating their proliferation after exposure. (author)

  18. Molecular genetic and biochemical responses in human airway epithelial cell cultures exposed to titanium nanoparticles in vitro.

    Science.gov (United States)

    Aydın, Elanur; Türkez, Hasan; Hacımüftüoğlu, Fazıl; Tatar, Abdulgani; Geyikoğlu, Fatime

    2017-07-01

    Titanium nanoparticles (NPs) have very wide application areas such as paint, cosmetics, pharmaceuticals, and biomedical applications. And, to translate these nanomaterials to the clinic and industrial domains, their safety needs to be verified, particularly in terms of genotoxicity and cytotoxicity. Therefore, in this study, we aimed to investigate of cytotoxicity and changes in gene expression profiles influenced by commonly titanium (as titanium carbide, titanium carbo-nitride, titanium (II) oxide, titanium (III) oxide, titanium (IV) oxide, titanium nitride, titanium silicon oxide) NPs in human alveolar epithelial (HPAEpiC) and pharynx (HPPC) cell lines in vitro since inhalation is an important pathway for exposure to these NPs. HPAEpiC and HPPC cells were treated with titanium (0-100 µg/mL), NPs for 24 and 48 h, and then cytotoxicity was detected by, [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT), uptake of neutral red (NR) and lactate dehydrogenase (LDH) release assays, while genotoxicity was also analyzed by cDNA array - RT-PCR assay. According to the results of MTT, NR and LDH assays, all tested NPs induced cytotoxicity on both HPAEpiC and HPPC cells in a time- and dose-dependent manner. Determining and analyzing the gene expression profiles of HPAEpiC and HPPC cells, titanium NPs showed more changes in genes related to DNA damage or repair, oxidative stress, and apoptosis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2056-2064, 2017. © 2017 Wiley Periodicals, Inc.

  19. PERFORATION OF INFERIOR ALVEOLAR NERVE BY MAXILLARY ARTERY. Perforation of inferior alveolar nerve by maxillary artery

    OpenAIRE

    Prakash B Billakanti

    2016-01-01

    La fosa infratemporal es un área anatómica clínicamente importante para la administración de agentes anestésicos locales en odontología y cirugía maxilofacial. Fueron estudiadas variaciones en la anatomía del nervio alveolar inferior y la arteria maxilar en la disección infratemporal. Durante la disección rutinaria de la cabeza en el cadáver de un varón adulto, fue observada una variación excepcional en el origen del nervio alveolar inferior y su relación con las estructuras circundantes. El ...

  20. Modulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by Helicobacter pylori in immune pathogenesis of gastric mucosal damage.

    Science.gov (United States)

    Tsai, Hwei-Fang; Hsu, Ping-Ning

    2017-02-01

    Helicobacter pylori infection is associated with chronic gastritis, peptic ulcer, gastric carcinoma, and gastric mucosa-associated lymphoid tissue lymphomas. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Enhanced gastric epithelial cell apoptosis during H. pylori infection was suggested to play an important role in the pathogenesis of chronic gastritis and gastric pathology. In addition to directly triggering apoptosis, H. pylori induces sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in gastric epithelial cells. Human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death-receptor signaling. The induction of TRAIL sensitivity by H. pylori is dependent upon the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex through downregulation of cellular FLICE-inhibitory protein. Moreover, H. pylori infection induces infiltration of T lymphocytes and triggers inflammation to augment apoptosis. In H. pylori infection, significant increases in CCR6 + CD3 + T cell infiltration in the gastric mucosa was observed, and the CCR6 ligand, CCL20 chemokine, was selectively expressed in inflamed gastric tissues. These mechanisms initiate chemokine-mediated T lymphocyte trafficking into inflamed epithelium and induce mucosal injury during Helicobacter infection. This article will review recent findings on the interactions of H. pylori with host-epithelial signaling pathways and events involved in the initiation of gastric pathology, including gastric inflammation and mucosal damage. Copyright © 2016. Published by Elsevier B.V.

  1. PUMA mediates ER stress-induced apoptosis in portal hypertensive gastropathy

    Science.gov (United States)

    Tan, S; Wei, X; Song, M; Tao, J; Yang, Y; Khatoon, S; Liu, H; Jiang, J; Wu, B

    2014-01-01

    Mucosal apoptosis has been demonstrated to be an essential pathological feature in portal hypertensive gastropathy (PHG). p53-upregulated modulator of apoptosis (PUMA) was identified as a BH3-only Bcl-2 family protein that has an essential role in apoptosis induced by a variety of stimuli, including endoplasmic reticulum (ER) stress. However, whether PUMA is involved in mucosal apoptosis in PHG remains unclear, and whether PUMA induces PHG by mediating ER stress remains unknown. The aim of the study is to investigate whether PUMA is involved in PHG by mediating ER stress apoptotic signaling. To identify whether PUMA is involved in PHG by mediating ER stress, gastric mucosal injury and apoptosis were studied in both PHG patients and PHG animal models using PUMA knockout (PUMA-KO) and PUMA wild-type (PUMA-WT) mice. The induction of PUMA expression and ER stress signaling were investigated, and the mechanisms of PUMA-mediated apoptosis were analyzed. GES-1 and SGC7901 cell lines were used to further identify whether PUMA-mediated apoptosis was induced by ER stress in vitro. Epithelial apoptosis and PUMA were markedly induced in the gastric mucosa of PHG patients and mouse PHG models. ER stress had a potent role in the induction of PUMA and apoptosis in PHG models, and the apoptosis was obviously attenuated in PUMA-KO mice. Although the targeted deletion of PUMA did not affect ER stress, mitochondrial apoptotic signaling was downregulated in mice. Meanwhile, PUMA knockdown significantly ameliorated ER stress-induced mitochondria-dependent apoptosis in vitro. These results indicate that PUMA mediates ER stress-induced mucosal epithelial apoptosis through the mitochondrial apoptotic pathway in PHG, and that PUMA is a potentially therapeutic target for PHG. PMID:24625987

  2. Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing*

    OpenAIRE

    Yang, Hee-Young; Kwon, Joseph; Kook, Min-Suk; Kang, Seong Soo; Kim, Se Eun; Sohn, Sungoh; Jung, Seunggon; Kwon, Sang-Oh; Kim, Hyung-Seok; Lee, Jae Hyuk; Lee, Tae-Hoon

    2013-01-01

    Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival sof...

  3. [Massive alveolar haemorrhage in Wegener's granulomatosis].

    Science.gov (United States)

    Valero-Roldán, J; Nuñez-Castillo, D; Fernández-Fígares, C; López-Leiva, I

    2014-01-01

    Wegener's granulomatosis is a systemic vasculitis with involvement of primary granulomatous upper and lower respiratory tract, glomerulonephritis and vasculitis of small vessels. The lung disease ranges from asymptomatic pulmonary nodules to pulmonary infiltrates and fulminant alveolar haemorrhage. The prognosis is poor due to kidney and respiratory failure, although the data are changing due to new treatments with glucocorticoids and cyclophosphamide. We report a case with severe lung disease, which after appropriate anamnesis, multiple tests, and optimal sequential action, the patient was diagnosed with Wegener's granulomatosis. This disease has a low incidence in the Emergency Department, where the patient history supported by the appropriate additional provides a diagnostic suspicion. It is important that the Emergency Department has the skills to manage the stability in these patients in order to resolve their symptoms. Copyright © 2013 Sociedad Española de Médicos de Atención Primaria (SEMERGEN). Publicado por Elsevier España. All rights reserved.

  4. Keratocyte apoptosis and corneal antioxidant enzyme activities after refractive corneal surgery.

    Science.gov (United States)

    Bilgihan, K; Bilgihan, A; Adiguzel, U; Sezer, C; Yis, O; Akyol, G; Hasanreisoglu, B

    2002-01-01

    Refractive corneal surgery induces keratocyte apoptosis and generates reactive oxygen radicals (ROS) in the cornea. The purpose of the present study is to evaluate the correlation between keratocyte apoptosis and corneal antioxidant enzyme activities after different refractive surgical procedures in rabbits. Rabbits were divided into six groups. All groups were compared with the control group (Group 1), after epithelial scraping (Group 2), epithelial scrape and photorefractive keratectomy (PRK) (traditional PRK: Group 3), transepithelial PRK (Group 4), creation of a corneal flap with microkeratome (Group 5) and laser-assisted in situ keratomileusis (LASIK, Group 6). Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay (to detect DNA fragmentation in situ) and light microscopy were used to detect apoptosis in rabbit eyes. Glutathione peroxidase (Gpx) and superoxide dismutase (SOD) activities of the corneal tissues were measured with spectrophotometric methods. Corneal Gpx and SOD activities decreased significantly in all groups when compared with the control group (P<0.05) and groups 2, 3 and 6 showed a significantly higher amount of keratocyte apoptosis (P<0.05). Not only a negative correlation was observed between corneal SOD activity and keratocyte apoptosis (cc: -0.3648) but Gpx activity also showed negative correlation with keratocyte apoptosis (cc: -0.3587). The present study illustrates the negative correlation between keratocyte apoptosis and corneal antioxidant enzyme activities. This finding suggests that ROS may be partly responsible for keratocyte apoptosis after refractive surgery.

  5. Macrophage Akt1 Kinase-Mediated Mitophagy Modulates Apoptosis Resistance and Pulmonary Fibrosis.

    Science.gov (United States)

    Larson-Casey, Jennifer L; Deshane, Jessy S; Ryan, Alan J; Thannickal, Victor J; Carter, A Brent

    2016-03-15

    Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder with increasing incidence. Mitochondrial oxidative stress in alveolar macrophages is directly linked to pulmonary fibrosis. Mitophagy, the selective engulfment of dysfunctional mitochondria by autophagasomes, is important for cellular homeostasis and can be induced by mitochondrial oxidative stress. Here, we show Akt1 induced macrophage mitochondrial reactive oxygen species (ROS) and mitophagy. Mice harboring a conditional deletion of Akt1 in macrophages (Akt1(-/-)Lyz2-cre) and Park2(-/-) mice had impaired mitophagy and reduced active transforming growth factor-β1 (TGF-β1). Although Akt1 increased TGF-β1 expression, mitophagy inhibition in Akt1-overexpressing macrophages abrogated TGF-β1 expression and fibroblast differentiation. Importantly, conditional Akt1(-/-)Lyz2-cre mice and Park2(-/-) mice had increased macrophage apoptosis and were protected from pulmonary fibrosis. Moreover, IPF alveolar macrophages had evidence of increased mitophagy and displayed apoptosis resistance. These observations suggest that Akt1-mediated mitophagy contributes to alveolar macrophage apoptosis resistance and is required for pulmonary fibrosis development. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Caspases: An apoptosis mediator

    Directory of Open Access Journals (Sweden)

    Tapan Kumar Palai

    2015-03-01

    Full Text Available The process of programmed cell death, or apoptosis, is generally characterized by distinct morphological characteristics and energy - dependent biochemical mechanisms. Apoptosis is a widely conserved phenomenon helping many processes, including normal cell turnover, proper development and functioning of the immune system, hormone dependent atrophy etc. Inappropriate apoptosis (either low level or high level leads to many developmental abnormalities like, neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. To use cells for therapeutic purposes through generating cell lines, it is critical to study the cell cycle machinery and signalling pathways that controls cell death and apoptosis. Apoptotic pathways provide a fundamental protective mechanism that decreases cellular sensitivity to damaging events and allow proper developmental process in multi-cellular organisms. Major mediator of apoptosis is a family of proteins known as caspases. There are mainly fourteen types of caspases but out of them only ten caspasese have got essential role in controlling the process of apoptosis. These ten caspases have been categorized into either initiator caspases (caspase 2, 8, 9, 10 or executioner caspases (caspase 3, 6, 7. Although various types of caspases have been identified so far, the exact mechanisms of action of these groups of proteins is still to be fully understood. The aim of this review is to provide a detail overview of role of different caspases in regulating the process of apoptosis.

  7. Accelerated orthodontics with alveolar decortication and augmentation: a case report.

    Science.gov (United States)

    Yezdani, A Arif

    2012-01-01

    This case report reiterates the fact that selective alveolar decortication in conjunction with periodontal alveolar augmentation with a bone graft indubitably and efficaciously produces rapid orthodontic tooth movement. A 29-year-old woman presented with a Class I malocclusion and increased bidentoalveolar protrusion with increased spacing between the maxillary and mandibular incisors. She readily agreed to selective alveolar decortication in conjunction with periodontal alveolar augmentation with a bone graft when presented with the proposal that her malocclusion could be corrected in one-third the treatment time required for conventional orthodontics. A preadjusted edgewise appliance (Roth prescription, 0.022 x 0.028-inch slot) was placed prior to the surgical procedure. One week later, full-thickness labial and lingual flaps were reflected in the maxillary and mandibular arches. The alveolar bone was selectively decorticated and periodontally augmented with a bone graft. Starting 1 week postsurgically, orthodontic adjustments were carried out every 2 weeks. From bracketing to debracketing, the entire orthodontic treatment took 7 months. The rapid orthodontic tooth movement was attributed to the regional acceleratory phenomenon, triggered by selective alveolar decortication. The subsequent periodontal alveolar augmentation with the bone graft repaired the bony dehiscences and enhanced the bone volume and dramatically improved the patient's soft tissue profile.

  8. Beta4 integrin-dependent formation of polarized three-dimensionalarchitecture confers resistance to apoptosis in normal and malignantmammary epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, Valerie M.; Lelievre, Sophie; Lakins, Johnathon N.; Chrenek, Micah A.; Jones, Jonathan C.R.; Giancotti, Filippo; Werb, Zena; Bissell, Mina J.

    2002-08-27

    Tumor cells can evade chemotherapy by acquiring resistanceto apoptosis. We investigated the molecular mechanism whereby malignantand nonmalignant mammary epithelial cells become insensitive toapoptosis. We show that regardless of growth status formation ofpolarized, three-dimensional structures driven by basement membraneconfers protection to apoptosis in both nonmalignant and malignantmammary epithelial cells. By contrast, irrespective of their malignantstatus, nonpolarized structures are sensitive to induction of apoptosis.Resistance to apoptosis requires ligation of beta4 integrins, whichregulates tissue polarity, hemidesmosome formation and NFkB activation.Expression of beta4 integrin that lacks the hemidesmosome targetingdomain interferes with tissue polarity and NFkB activation and permitsapoptosis. These results indicate that integrin-induced polarity maydrive tumor cell resistance to apoptosis-inducing agents via effects onNFkB.

  9. Keratocyte loss in Acanihamoeba Keratitis: Phagocytosis, necrosis or apoptosis?

    Directory of Open Access Journals (Sweden)

    Vemuganti Geeta

    2000-01-01

    Full Text Available Purpose: Pathogenesis of Acanthamoeba keratitis involves breakdown of epithelial barrier, stromal invasion by Acanthamoeba, loss of keratocytes, inflammatory response and finally stromal necrosis. The loss of keratocytes, believed to be due to the phagocytic activity of the parasite, occurs disproportionate to and independent of the parasite load, thereby suggesting additional modes of cell loss. To test our hypothesis that the loss of keratocytes in Acanthamoeba keratitis is due to apoptosis, we did both histology and histochemistry on the corneal tissues. Methods: Routine Haematoxylin and Eosin, Gomori′s Methenamine Silver and Periodic acid Schiff stained sections of five corneal tissues from penetrating keratoplasty and eviscerated eyes were reviewed. TUNEL staining was done for morphological detection of apoptosis in three cases, using formalin-fixed, paraffin-processed tissues. Results: Histological changes were epithelial ulceration, loss of keratocytes in all layers, inflammation in anterior two-thirds of the stroma with necrosis, and deeper quiet stroma. Acanthamoeba trophozoites were found in the anterior stroma while the cysts were more in the deeper stroma, with minimal or no inflammatory response. TUNEL staining was positive in keratocytic nuclei in all layers. Conclusions: This study demonstrates that one of the modes of keratocyte loss in Acanthamoeba keratitis is by apoptosis, possibly in addition to the necrotic process and phagocytic activity of the parasite. The death of inflammatory cells also appears to be mediated by apoptosis.

  10. Physician Education: Apoptosis.

    Science.gov (United States)

    Kataoka; Tsuruo

    1996-01-01

    We have come to understand apoptosis as not merely a single form of cell death, but as a fundamental theme in cell biology that has far-reaching implications in the fields of physiology and pathology. At the present time, however, the mechanism of apoptosis is not clearly understood, as research into apoptosis is still at the initial stages. Nevertheless, the links between apoptosis and a variety of pathological conditions are gradually becoming clearer. In this article, we will provide a simple explanation of apoptosis and its mechanism as a novel concept of cell death and discuss the way in which apoptosis has been linked to a variety of pathological conditions. WHAT IS APOPTOSIS?: In normal tissue, cells that are no longer needed are rapidly eliminated without affecting the overall function of the tissue. In this process cells undergo an active and spontaneous suicide called programmed cell death. In fact, the majority of physiological cell deaths take the form of apoptosis. The word apoptosis is used, in contrast to necrosis, to describe the situation in which a cell actively pursues a course toward death upon receiving certain stimuli [1]. The morphological changes of apoptosis found in most cell types first involve contraction in cell volume and condensation of the nucleus. When this happens the intracellular organelles such as the mitochondria retain their normal morphology. As apoptosis proceeds, blebbing of the plasma membrane occurs, and the nucleus becomes fragmented. Finally, the cell itself fragments to form apoptotic bodies that are engulfed by nearby phagocytes. With respect to biochemical changes, it is known that the chromosomes become fragmented into nucleosome units, and DNA forms characteristic ladder patterns when subjected to agarose gel electrophoresis. MECHANISM OF APOPTOSIS: It has been reported that apoptosis is induced in various cells by many kinds of irritations, but the precise mechanism is still unclear. Cell injuries that induce

  11. Reaper-Induced Apoptosis

    National Research Council Canada - National Science Library

    Perry, Jennifer

    2005-01-01

    Reaper is a central regulator of apoptosis in the fly, Drosophila melanogaster. At the start of this proposal our laboratory identified what was believed to be a pro-apoptotic human homolog of Reaper...

  12. Viruses and apoptosis.

    Science.gov (United States)

    Roulston, A; Marcellus, R C; Branton, P E

    1999-01-01

    Successful viral replication requires not only the efficient production and spread of progeny, but also evasion of host defense mechanisms that limit replication by killing infected cells. In addition to inducing immune and inflammatory responses, infection by most viruses triggers apoptosis or programmed cell death of the infected cell. This cell response often results as a compulsory or unavoidable by-product of the action of critical viral replicative functions. In addition, some viruses seem to use apoptosis as a mechanism of cell killing and virus spread. In both cases, successful replication relies on the ability of certain viral products to block or delay apoptosis until sufficient progeny have been produced. Such proteins target a variety of strategic points in the apoptotic pathway. In this review we summarize the great amount of recent information on viruses and apoptosis and offer insights into how this knowledge may be used for future research and novel therapies.

  13. Expression of matrix metalloproteinase-1 in alveolar macrophages, type II pneumocytes, and airways in smokers: relationship to lung function and emphysema.

    Science.gov (United States)

    Wallace, Alison M; Loy, Leanna B; Abboud, Raja T; D'Armiento, Jeanine M; Coxson, Harvey O; Muller, Nestor L; Kalloger, Steve; Li, Xing; Mark Elliott, W; English, John C; Finley, Richard J; Paré, Peter D

    2014-08-01

    An imbalance between proteolytic enzymes and their inhibitors is thought to be involved in the pathogenesis of chronic obstructive pulmonary disease. Matrix metalloproteinase-1, also known as interstitial collagenase, has been implicated as a potentially important proteinase in the genesis of chronic obstructive pulmonary disease and, more specifically, emphysema. We performed quantitative immunohistochemical assessment of matrix metalloproteinase-1 expression in the resected lung of 20 smokers/ex-smokers who had varying severity of airflow obstruction and emphysema and compared this with the lungs of 5 nonsmokers. Emphysema was measured using a morphometric measure of the lungs' surface area/volume ratio and with qualitative and quantitative computed tomography (CT) measures of emphysema. There were significantly more matrix metalloproteinase-1-expressing alveolar macrophages and type II pneumocytes as well as a greater percentage of small airways that stained positively for matrix metalloproteinase-1 in the lungs of smokers than in those of nonsmokers (p lung surface area/volume ratio and to qualitative estimates of emphysema on CT. These findings suggest that cigarette smoking increases expression of matrix metalloproteinase-1 in alveolar macrophages as well as in alveolar and small airway epithelial cells. Smokers who develop emphysema have increased alveolar macrophage expression of matrix metalloproteinase-1.

  14. ATM promotes apoptosis and suppresses tumorigenesis in response to Myc

    Science.gov (United States)

    Pusapati, Raju V.; Rounbehler, Robert J.; Hong, Sungki; Powers, John T.; Yan, Mingshan; Kiguchi, Kaoru; McArthur, Mark J.; Wong, Paul K.; Johnson, David G.

    2006-01-01

    Overexpression of the c-myc oncogene contributes to the development of a significant number of human cancers. In response to deregulated Myc activity, the p53 tumor suppressor is activated to promote apoptosis and inhibit tumor formation. Here we demonstrate that p53 induction in response to Myc overexpression requires the ataxia-telangiectasia mutated (ATM) kinase, a major regulator of the cellular response to DNA double-strand breaks. In a transgenic mouse model overexpressing Myc in squamous epithelial tissues, inactivation of Atm suppresses apoptosis and accelerates tumorigenesis. Deregulated Myc expression induces DNA damage in primary transgenic keratinocytes and the formation of H2AX and phospho-SMC1 foci in transgenic tissue. These findings suggest that Myc overexpression causes DNA damage in vivo and that the ATM-dependent response to this damage is critical for p53 activation, apoptosis, and the suppression of tumor development. p53 | DNA damage

  15. Nicotine transport in lung and non-lung epithelial cells.

    Science.gov (United States)

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

    2017-11-01

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

  16. Coronaviruses in polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J. W.; Bekker, C. P.; Voorhout, W. F.; Horzinek, M. C.; van der Ende, A.; Strous, G. J.; Rottier, P. J.

    1995-01-01

    Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable

  17. Embryonic epithelial membrane transporters.

    Science.gov (United States)

    Horster, M

    2000-12-01

    Embryonic epithelial membrane transporters are organized into transporter families that are functional in several epithelial organs, namely, in kidney, lung, pancreas, intestine, and salivary gland. Family members (subtypes) are developmentally expressed in plasma membranes in temporospatial patterns that are 1) similar for one subtype within different organs, like aquaporin-1 (AQP1) in lung and kidney; 2) different between subtypes within the same organ, like the amiloride-sensitive epithelial sodium channel (ENaC) in lung; and 3) apparently matched among members of different transporter families, as alpha-ENaC with AQP1 and -4 in lung and with AQP2 in kidney. Finally, comparison of temporal expression patterns in early embryonic development of transporters from different families [e.g., cystic fibrosis transmembrane conductance regulator (CFTR), ENaC, and outer medullary potassium channel] suggests regulatory activating or inactivating interactions in defined morphogenic periods. This review focuses on embryonic patterns, at the mRNA and immunoprotein level, of the following transporter entities expressed in epithelial cell plasma membranes: ENaC; the chloride transporters CFTR, ClC-2, bumetanide-sensitive Na-K-Cl cotransporter, Cl/OH, and Cl/HCO(3); the sodium glucose transporter-glucose transporter; the sodium/hydrogen exchanger; the sodium-phosphate cotransporter; the ATPases; and AQP. The purpose of this article is to relate temporal and spatial expression patterns in embryonic and in early postnatal epithelia to developmental changes in organ structure and function.

  18. Epithelial-mesenchymal transition in tissue repair and fibrosis.

    Science.gov (United States)

    Stone, Rivka C; Pastar, Irena; Ojeh, Nkemcho; Chen, Vivien; Liu, Sophia; Garzon, Karen I; Tomic-Canic, Marjana

    2016-09-01

    The epithelial-mesenchymal transition (EMT) describes the global process by which stationary epithelial cells undergo phenotypic changes, including the loss of cell-cell adhesion and apical-basal polarity, and acquire mesenchymal characteristics that confer migratory capacity. EMT and its converse, MET (mesenchymal-epithelial transition), are integral stages of many physiologic processes and, as such, are tightly coordinated by a host of molecular regulators. Converging lines of evidence have identified EMT as a component of cutaneous wound healing, during which otherwise stationary keratinocytes (the resident skin epithelial cells) migrate across the wound bed to restore the epidermal barrier. Moreover, EMT plays a role in the development of scarring and fibrosis, as the matrix-producing myofibroblasts arise from cells of the epithelial lineage in response to injury but are pathologically sustained instead of undergoing MET or apoptosis. In this review, we summarize the role of EMT in physiologic repair and pathologic fibrosis of tissues and organs. We conclude that further investigation into the contribution of EMT to the faulty repair of fibrotic wounds might identify components of EMT signaling as common therapeutic targets for impaired healing in many tissues. Graphical Abstract Model for injury-triggered EMT activation in physiologic wound repair (left) and fibrotic wound healing (right).

  19. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Tang, J.; Jiang, Y. [Southern Medical University, Nanfang Hospital, Department of Anesthesia, Guangzhou, China, Department of Anesthesia, Nanfang Hospital, Southern Medical University, Guangzhou (China); Tang, Y.; Chen, B. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China); Sun, X. [Laboratory of Traditional Chinese Medicine Syndrome, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou (China); Su, L.; Liu, Z. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China)

    2013-06-25

    Gut-derived endotoxin and pathogenic bacteria have been proposed as important causative factors of morbidity and death during heat stroke. However, it is still unclear what kind of damage is induced by heat stress. In this study, the rat intestinal epithelial cell line (IEC-6) was treated with heat stress or a combination of heat stress and lipopolysaccharide (LPS). In addition, propofol, which plays an important role in anti-inflammation and organ protection, was applied to study its effects on cellular viability and apoptosis. Heat stress, LPS, or heat stress combined with LPS stimulation can all cause intestinal epithelial cell damage, including early apoptosis and subsequent necrosis. However, propofol can alleviate injuries caused by heat stress, LPS, or the combination of heat stress and LPS. Interestingly, propofol can only mitigate LPS-induced intestinal epithelial cell apoptosis, and has no protective role in heat-stress-induced apoptosis. Th