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Sample records for epithelial cells prime

  1. Cell-Type-Specific Chromatin States Differentially Prime Squamous Cell Carcinoma Tumor-Initiating Cells for Epithelial to Mesenchymal Transition.

    PubMed

    Latil, Mathilde; Nassar, Dany; Beck, Benjamin; Boumahdi, Soufiane; Wang, Li; Brisebarre, Audrey; Dubois, Christine; Nkusi, Erwin; Lenglez, Sandrine; Checinska, Agnieszka; Vercauteren Drubbel, Alizée; Devos, Michael; Declercq, Wim; Yi, Rui; Blanpain, Cédric

    2017-02-02

    Epithelial to mesenchymal transition (EMT) in cancer cells has been associated with metastasis, stemness, and resistance to therapy. Some tumors undergo EMT while others do not, which may reflect intrinsic properties of their cell of origin. However, this possibility is largely unexplored. By targeting the same oncogenic mutations to discrete skin compartments, we show that cell-type-specific chromatin and transcriptional states differentially prime tumors to EMT. Squamous cell carcinomas (SCCs) derived from interfollicular epidermis (IFE) are generally well differentiated, while hair follicle (HF) stem cell-derived SCCs frequently exhibit EMT, efficiently form secondary tumors, and possess increased metastatic potential. Transcriptional and epigenomic profiling revealed that IFE and HF tumor-initiating cells possess distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT that correlate with accessibility of key epithelial and EMT transcription factor binding sites. These findings highlight the importance of chromatin states and transcriptional priming in dictating tumor phenotypes and EMT.

  2. Hypoxia primes human normal prostate epithelial cells and cancer cell lines for the NLRP3 and AIM2 inflammasome activation

    PubMed Central

    Panchanathan, Ravichandran; Liu, Hongzhu; Choubey, Divaker

    2016-01-01

    The molecular mechanisms by which hypoxia contributes to prostatic chronic inflammation (PCI) remain largely unknown. Because hypoxia stimulates the transcriptional activity of NF-κB, which “primes” cells for inflammasome activation by inducing the expression of NLRP3 or AIM2 receptor and pro-IL-1β, we investigated whether hypoxia could activate the NLRP3 and AIM2 inflammasome in human normal prostate epithelial cells (PrECs) and cancer cell lines. Here we report that hypoxia (1% O2) treatment of PrECs, prostate cell lines, and a macrophage cell line (THP-1) increased the levels of NLRP3, AIM2, and pro-IL-1β. Further, hypoxia in cells potentiated activation of the NLRP3 and AIM2 inflammasome activity. Notably, hypoxia “primed” cells for NLRP3 and AIM2 inflammasome activation through stimulation of the NF-κB activity. Our observations support the idea that hypoxia in human prostatic tumors contributes to PCI, in part, by priming cells for the activation of NLRP3 and AIM2 inflammasome. PMID:27058421

  3. Polarity Reversal by Centrosome Repositioning Primes Cell Scattering during Epithelial to Mesenchymal Transition

    PubMed Central

    Burute, Mithila; Prioux, Magali; Blin, Guillaume; Truchet, Sandrine; Letort, Gaëlle; Tseng, Qingzong; Bessy, Thomas; Lowell, Sally; Young, Joanne; Filhol-Cochet, Odile; Théry, Manuel

    2017-01-01

    Summary During epithelial to mesenchymal transition (EMT), cells lining the tissue periphery break up their cohesion to migrate within the tissue. This dramatic reorganization involves a poorly characterized reorientation of the apico-basal polarity of static epithelial cells into the front-rear polarity of migrating mesenchymal cells. To investigate the spatial coordination of intracellular reorganization with morphological changes, we monitored centrosome positioning during EMT in vivo, in developing mouse embryos and mammary gland, and in vitro, in cultured 3D cell aggregates and micro-patterned cell doublets. In all conditions, centrosomes moved from their off-centered position next to intercellular junctions toward extra-cellular matrix adhesions on the opposite side of the nucleus, resulting in an effective internal polarity reversal. This move appeared supported by controlled microtubule network disassembly. Sequential release of cell confinement using dynamic micropatterns, and modulation of microtubule dynamics, confirmed that centrosome repositioning was responsible for further cell disengagement and scattering. PMID:28041907

  4. Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells

    PubMed Central

    Haldar, Arun K.; Piro, Anthony S.; Finethy, Ryan; Espenschied, Scott T.; Brown, Hannah E.; Giebel, Amanda M.; Frickel, Eva-Maria; Nelson, David E.

    2016-01-01

    ABSTRACT The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis. The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host. PMID:27965446

  5. Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells.

    PubMed

    Haldar, Arun K; Piro, Anthony S; Finethy, Ryan; Espenschied, Scott T; Brown, Hannah E; Giebel, Amanda M; Frickel, Eva-Maria; Nelson, David E; Coers, Jörn

    2016-12-13

    The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host.

  6. Epithelial-to-mesenchymal transition (EMT) induced by inflammatory priming elicits mesenchymal stromal cell-like immune-modulatory properties in cancer cells.

    PubMed

    Ricciardi, M; Zanotto, M; Malpeli, G; Bassi, G; Perbellini, O; Chilosi, M; Bifari, F; Krampera, M

    2015-03-17

    Epithelial-to-mesenchymal transition (EMT) has a central role in cancer progression and metastatic dissemination and may be induced by local inflammation. We asked whether the inflammation-induced acquisition of mesenchymal phenotype by neoplastic epithelial cells is associated with the onset of mesenchymal stromal cell-like immune-regulatory properties that may enhance tumour immune escape. Cell lines of lung adenocarcinoma (A549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induction by either the supernatant of mixed-lymphocyte reactions or inflammatory cytokines. EMT occurrence following inflammatory priming elicited multiple immune-regulatory effects in cancer cells resulting in NK and T-cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase, but not Fas ligand pathway, was involved at least in part in these effects, as shown by the use of specific inhibitors. EMT induced by inflammatory stimuli confers to cancer cells some mesenchymal stromal cell-like immune-modulatory properties, which could be a cue for cancer progression and metastatic dissemination by favouring immune escape.

  7. P2Y1 Receptor Signaling Contributes to High Salt-Induced Priming of the NLRP3 Inflammasome in Retinal Pigment Epithelial Cells

    PubMed Central

    Prager, Philipp; Hollborn, Margrit; Steffen, Anja; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Systemic hypertension is a risk factor of age-related macular degeneration (AMD), a chronic inflammatory disease. Acute hypertension is caused by increased extracellular osmolarity after intake of dietary salt (NaCl). We determined in cultured human retinal pigment epithelial (RPE) cells whether high extracellular NaCl alters the gene expression of inflammasome-associated proteins, and whether autocrine/paracrine purinergic (P2) receptor signaling contributes to the NaCl-induced NLRP3 gene expression. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Gene and protein expression levels were determined with real-time RT-PCR and Western blot analysis, respectively. IL-1β and IL-18 levels were evaluated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. High extracellular NaCl induced NLRP3 and pro-IL-1β gene expression, while the gene expression of further inflammasome-associated proteins (NLRP1, NLRP2, NLRP6, NLRP7, NLRP12, NLRC4, AIM2, ASC, procaspase-1, pro-IL-18) was not altered or below the detection threshold. The NaCl-induced NLRP3 gene expression was partially dependent on the activities of phospholipase C, IP3 receptors, protein kinase C, the serum and glucocorticoid-regulated kinase, p38 MAPK, ERK1/2, JNK, PI3K, and the transcription factors HIF-1 and NFAT5. Pannexin-dependent ATP release and P2Y1 receptor activation is required for the full induction of NLRP3 gene expression. High NaCl induced a transient increase of the NLRP3 protein level and a moderate NLRP3 inflammasome activation, as indicated by the transient increase of the cytosolic level of mature IL-1β. High NaCl also induced secretion of IL-18. Conclusion High extracellular NaCl induces priming of the NLRP3 inflammasome in RPE cells, in part via P2Y1 receptor signaling. The inflammasome priming effect of NaCl suggests that high intake of dietary salt may promote

  8. Enhanced Ca(2+) response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines.

    PubMed

    Catalioto, Rose-Marie; Valenti, Claudio; Maggi, Carlo Alberto; Giuliani, Sandro

    2015-09-15

    Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1β enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Lung Epithelial Progenitor Cells

    PubMed Central

    Rawlins, Emma L.

    2008-01-01

    The current enthusiasm for stem cell research stems from the hope that damaged or diseased tissues may one day be repaired through the manipulation of endogenous or exogenous stem cells. The postnatal human respiratory system is highly accessible and provides unique opportunities for the application of such techniques. Several putative adult lung epithelial stem cells have been identified in the mouse model system. However, their in vivo capabilities to contribute to different lineages, and their control mechanisms, remain unclear. If stem cell–based therapies are to be successful in the lung, it is vitally important that we understand the normal behavior of adult lung stem cells, and how this is regulated. Lung embryonic progenitor cells are much better defined and characterized than their adult counterparts. Moreover, experiments on a variety of developing tissues are beginning to uncover general mechanisms by which embryonic progenitors influence final organ size and structure. This provides a framework for the study of lung embryonic progenitor cells, facilitating experimental design and interpretation. A similar approach to investigating adult lung stem cells could produce rapid advances in the field. PMID:18684716

  10. Integrins and epithelial cell polarity.

    PubMed

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity.

  11. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

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

  12. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

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

  13. Brain May Prime Metastatic Cell Growth.

    PubMed

    2016-01-01

    Metastasizing tumor cells lose expression of the tumor suppressor PTEN at a much higher rate when they enter the brain compared to other organs, suggesting that the brain's unique microenvironment may prime metastatic cells for aggressive growth, a recent study reports. The findings may have implications for developing targeted therapies for brain metastases. ©2015 American Association for Cancer Research.

  14. Tcf15 Primes Pluripotent Cells for Differentiation

    PubMed Central

    Davies, Owen R.; Lin, Chia-Yi; Radzisheuskaya, Aliaksandra; Zhou, Xinzhi; Taube, Jessica; Blin, Guillaume; Waterhouse, Anna; Smith, Andrew J.H.; Lowell, Sally

    2013-01-01

    Summary The events that prime pluripotent cells for differentiation are not well understood. Inhibitor of DNA binding/differentiation (Id) proteins, which are inhibitors of basic helix-loop-helix (bHLH) transcription factor activity, contribute to pluripotency by blocking sequential transitions toward differentiation. Using yeast-two-hybrid screens, we have identified Id-regulated transcription factors that are expressed in embryonic stem cells (ESCs). One of these, Tcf15, is also expressed in the embryonic day 4.5 embryo and is specifically associated with a novel subpopulation of primed ESCs. An Id-resistant form of Tcf15 rapidly downregulates Nanog and accelerates somatic lineage commitment. We propose that because Tcf15 can be held in an inactive state through Id activity, it may prime pluripotent cells for entry to somatic lineages upon downregulation of Id. We also find that Tcf15 expression is dependent on fibroblast growth factor (FGF) signaling, providing an explanation for how FGF can prime for differentiation without driving cells out of the pluripotent state. PMID:23395635

  15. Progress Towards Drosophila Epithelial Cell Culture

    PubMed Central

    Simcox, Amanda

    2015-01-01

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

  16. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation

    PubMed Central

    Juncadella, Ignacio J.; Kadl, Alexandra; Sharma, Ashish K.; Shim, Yun M.; Hochreiter-Hufford, Amelia; Borish, Larry; Ravichandran, Kodi S.

    2013-01-01

    Lung epithelial cells can influence immune responses to airway allergens1,2. Airway epithelial cells also undergo apoptosis after encountering environmental allergens3; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells1,4,5. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens. PMID:23235830

  17. Eosinophils promote epithelial to mesenchymal transition of bronchial epithelial cells.

    PubMed

    Yasukawa, Atsushi; Hosoki, Koa; Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C

    2013-01-01

    Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

  18. Patterning Bacterial Communities on Epithelial Cells

    PubMed Central

    Dwidar, Mohammed; Leung, Brendan M.; Yaguchi, Toshiyuki; Takayama, Shuichi; Mitchell, Robert J.

    2013-01-01

    Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv) gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions. PMID:23785519

  19. Epithelial TRPV1 Signaling Accelerates Gingival Epithelial Cell Proliferation

    PubMed Central

    Takahashi, N.; Matsuda, Y.; Yamada, H.; Tabeta, K.; Nakajima, T.; Murakami, S.; Yamazaki, K.

    2014-01-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca2+ levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  20. Airway epithelial cell responses to ozone injury

    SciTech Connect

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

    1995-03-01

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

  1. Bone marrow-derived lung epithelial cells.

    PubMed

    Krause, Diane S

    2008-08-15

    Bone marrow-derived cells can take on the phenotype of epithelial cells and express epithelial-specific genes in multiple organs. Here, we focus on recent data on the appearance of marrow-derived epithelial cells in the adult lung. These findings have garnered significant skepticism because in most cases marrow-derived epithelial cells are very rare, the marrow cell of origin is not known, the techniques for detection have needed improvement, and there seem to be multiple mechanisms by which this occurs. Recent studies have focused on these concerns. Once these important concerns are addressed, further studies on the function(s) of these cells will need to be performed to determine whether this engraftment has any clinical significance-either beneficial or detrimental.

  2. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2007-10-01

    Epithelial Stem Cells PRINCIPAL INVESTIGATOR: Peter D. Eirew CONTRACTING ORGANIZATION: British Columbia Cancer Agency...NUMBER Characterization of Human Mammary Epithelial Stem Cells 5b. GRANT NUMBER W81XWH-06-1-0702 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Abstract The mammary epithelium in normal adult female mice contains undifferentiated stem cells with extensive in vivo regenerative and self-renewal

  3. Myb permits multilineage airway epithelial cell differentiation

    PubMed Central

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

    2014-01-01

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

  4. Purification of kidney epithelial cell growth inhibitors.

    PubMed Central

    Holley, R W; Böhlen, P; Fava, R; Baldwin, J H; Kleeman, G; Armour, R

    1980-01-01

    Two high molecular weight growth inhibitors have been isolated from the culture medium of BSC-1 cells, epithelial cells of African green monkey kidney. The purified kidney epithelial cell growth inhibitors, at ng/ml concentrations, reversibly arrest the growth of BSC-1 cells in the G1 phase of the cell cycle. Their action is selective; they are most active on BSC-1 cells, are less active as inhibitors of the growth of rat lung and human breast epithelial cells, and do not inhibit the growth of 3T3 mouse embryo fibroblasts ad human skin fibroblasts in culture. Their growth inhibitory action on BSC-1 cell cultures is counteracted by epidermal growth factor or calf serum. PMID:6969400

  5. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-08

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes.

  6. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed.

  7. Control of lens epithelial cell survival

    PubMed Central

    1993-01-01

    We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell- cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture. PMID:8491781

  8. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  9. Evidence for epithelial-mesenchymal transitions in adult liver cells.

    PubMed

    Sicklick, Jason K; Choi, Steve S; Bustamante, Marcia; McCall, Shannon J; Pérez, Elizabeth Hernández; Huang, Jiawen; Li, Yin-Xiong; Rojkind, Marcos; Diehl, Anna Mae

    2006-10-01

    Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.

  10. Epithelial stem cells and intestinal cancer.

    PubMed

    Tan, Shawna; Barker, Nick

    2015-06-01

    The mammalian intestine is comprised of an epithelial layer that serves multiple functions in order to maintain digestive activity as well as intestinal homeostasis. This epithelial layer contains highly proliferative stem cells which facilitate its characteristic rapid regeneration. How these stem cells contribute to tissue repair and normal homeostasis are actively studied, and while we have a greater understanding of the molecular mechanisms and cellular locations that underlie stem cell regulation in this tissue, much still remains undiscovered. This review describes epithelial stem cells in both intestinal and non-intestinal tissues, as well as the strategies that have been used to further characterize the cells. Through a discussion of the current understanding of intestinal self-renewal and tissue regeneration in response to injury, we focus on how dysregulation of critical signaling pathways results in potentially oncogenic aberrations, and highlight issues that should be addressed in order for effective intestinal cancer therapies to be devised.

  11. Epithelial cell extrusion: Pathways and pathologies.

    PubMed

    Gudipaty, Swapna Aravind; Rosenblatt, Jody

    2016-05-19

    To remove dying or unwanted cells from an epithelium while preserving the barrier function of the layer, epithelia use a unique process called cell extrusion. To extrude, the cell fated to die emits the lipid Sphingosine 1 Phosphate (S1P), which binds the G-protein-coupled receptor Sphingosine 1 Phosphate receptor 2 (S1P2) in the neighboring cells that activates Rho-mediated contraction of an actomyosin ring circumferentially and basally. This contraction acts to squeeze the cell out apically while drawing together neighboring cells and preventing any gaps to the epithelial barrier. Epithelia can extrude out cells targeted to die by apoptotic stimuli to repair the barrier in the face of death or extrude live cells to promote cell death when epithelial cells become too crowded. Indeed, because epithelial cells naturally turn over by cell death and division at some of the highest rates in the body, epithelia depend on crowding-induced live cell extrusion to preserve constant cell numbers. If extrusion is defective, epithelial cells rapidly lose contact inhibition and form masses. Additionally, because epithelia act as the first line of defense in innate immunity, preservation of this barrier is critical for preventing pathogens from invading the body. Given its role in controlling constant cell numbers and maintaining barrier function, a number of different pathologies can result when extrusion is disrupted. Here, we review mechanisms and signaling pathways that control epithelial extrusion and discuss how defects in these mechanisms can lead to multiple diseases. We also discuss tactics pathogens have devised to hijack the extrusion process to infect and colonize epithelia.

  12. Interferon-γ Reduces the Proliferation of Primed Human Renal Tubular Cells

    PubMed Central

    García-Sánchez, Omar; López-Novoa, José Miguel; López-Hernández, Francisco J.

    2014-01-01

    Background/Aims Chronic kidney disease (CKD) is a progressive deterioration of the kidney function, which may eventually lead to renal failure and the need for dialysis or kidney transplant. Whether initiated in the glomeruli or the tubuli, CKD is characterized by progressive nephron loss, for which the process of tubular deletion is of key importance. Tubular deletion results from tubular epithelial cell death and defective repair, leading to scarring of the renal parenchyma. Several cytokines and signaling pathways, including transforming growth factor-β (TGF-β) and the Fas pathway, have been shown to participate in vivo in tubular cell death. However, there is some controversy about their mode of action, since a direct effect on normal tubular cells has not been demonstrated. We hypothesized that epithelial cells would require specific priming to become sensitive to TGF-β or Fas stimulation and that this priming would be brought about by specific mediators found in the pathological scenario. Methods Herein we studied whether the combined effect of several stimuli known to take part in CKD progression, namely TGF-β, tumor necrosis factor-α, interferon-γ (IFN-γ), and Fas stimulation, on primed resistant human tubular cells caused cell death or reduced proliferation. Results We demonstrate that these cytokines have no synergistic effect on the proliferation or viability of human kidney (HK2) cells. We also demonstrate that IFN-γ, but not the other stimuli, reduces the proliferation of cycloheximide-primed HK2 cells without affecting their viability. Conclusion Our results point at a potentially important role of IFN-γ in defective repair, leading to nephron loss during CKD. PMID:24575118

  13. Epithelial cell-extracellular matrix interactions and stem cells in airway epithelial regeneration.

    PubMed

    Coraux, Christelle; Roux, Jacqueline; Jolly, Thomas; Birembaut, Philippe

    2008-08-15

    In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

  14. Epithelial: lamina propria lymphocyte interactions promote epithelial cell differentiation

    PubMed Central

    Dahan, Stephanie; Roda, Giulia; Pinn, David; Roth-Walter, Franziska; Kamalu, Okebugwu; Martin, Andrea P.; Mayer, Lloyd

    2010-01-01

    Background & Aims Lymphoepithelial interactions in the gut can occur in the epithelium and the sub-epithelial space. We asked whether Normal, Crohn’s Disease (CD) or Ulcerative colitis (UC) lamina propria lymphocytes (LPL) could promote intestinal epithelial cell (IEC) growth and differentiation. Methods T84 cells were co-cultured with freshly isolated LPL for varying periods. After removal of LPL, IECs were lysed and subjected to i) measurement of intestinal alkaline phosphatase (IAP) activity; ii) Western blot analysis for MAPK and Akt activation; and iii) Real Time-PCR to assess CDX2 mRNA levels. Tissue sections were immunostained for evidence of MAPK and PI3K activation, CDX2 and IAP; and CDX2 mRNA expression was assessed on human colonic biopsies. Results IAP activity was increased in T84 cells co-cultured for 8 days with Normal LPL (p<0.05), and even greater with CD LPL (p<0.001). Crypt IECs in active CD mucosa expressed IAP ex vivo. Phospho-MAPK (ERK1/2, p38, and JNK) and phospho-Akt were seen as early as 30 min after co-culture. MAPK activation was greatest in T84 cells co-cultured with CD LPL. There was a specific increase in P-p38 MAPK and P-Akt staining in the nuclei of crypt IECs in active vs inactive CD, normal mucosa and UC mucosa. CDX2 mRNA expression was increased in CD LPL co-cultured T84 cells which not correlated with the CDX2 protein localization ex vivo. Conclusion Our observations indicate that there is crosstalk between LPL and IECs, which leads to IEC differentiation. Moreover, in CD mucosa, the differentiation of IEC is accelerated. PMID:18045591

  15. Oxygen transfer rate identifies priming compounds in parsley cells.

    PubMed

    Schilling, Jana Viola; Schillheim, Britta; Mahr, Stefan; Reufer, Yannik; Sanjoyo, Sandi; Conrath, Uwe; Büchs, Jochen

    2015-11-25

    In modern agriculture, the call for an alternative crop protection strategy increases because of the desired reduction of fungicide and pesticide use and the continuously evolving resistance of pathogens and pests to agrochemicals. The direct activation of the plant immune system does not provide a promising plant protection measure because of high fitness costs. However, upon treatment with certain natural or synthetic compounds, plant cells can promote to a fitness cost-saving, primed state of enhanced defense. In the primed state, plants respond to biotic and abiotic stress with faster and stronger activation of defense, and this is often associated with immunity and abiotic stress tolerance. Until now, the identification of chemical compounds with priming-inducing activity (so-called plant activators) relied on tedious and invasive approaches, or required the late detection of secreted furanocoumarin phytoalexins in parsley cell cultures. Thus, simple, fast, straightforward, and noninvasive techniques for identifying priming-inducing compounds for plant protection are very welcome. This report demonstrates that a respiration activity-monitoring system (RAMOS) can identify compounds with defense priming-inducing activity in parsley cell suspension in culture. RAMOS relies on the quasi-continuous, noninvasive online determination of the oxygen transfer rate (OTR). Treatment of parsley culture cells with the known plant activator salicylic acid (SA), a natural plant defense signal, resulted in an OTR increase. Addition of the defense elicitor Pep13, a cell wall peptide of Phythophthora sojae, induced two distinctive OTR peaks that were higher in SA-primed cells than in unprimed cells upon Pep13 challenge. Both, the OTR increase after priming with SA and the Pep13 challenge were dose-dependent. Furthermore, there was a close correlation of a compound's activity to enhance the oxygen consumption in parsley cells and its capacity to prime Pep13-induced furanocoumarin

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  18. Epithelial Immunization Induces Polyfunctional CD8+ T Cells and Optimal Mousepox Protection

    PubMed Central

    Siciliano, Nicholas A.; DeHaven, Brian C.; Snook, Adam E.

    2014-01-01

    We assessed several routes of immunization with vaccinia virus (VACV) in protecting mice against ectromelia virus (ECTV). By a wide margin, skin scarification provided the greatest protection. Humoral immunity and resident-memory T cells notwithstanding, several approaches revealed that circulating, memory CD8+ T cells primed via scarification were functionally superior and conferred enhanced virus control. Immunization via the epithelial route warrants further investigation, as it may also provide enhanced defense against other infectious agents. PMID:24899206

  19. Cell fusion between gastric epithelial cells and mesenchymal stem cells results in epithelial-to-mesenchymal transition and malignant transformation.

    PubMed

    He, Xianghui; Li, Baosong; Shao, Yang; Zhao, Na; Hsu, Yiling; Zhang, Zhixiang; Zhu, Liwei

    2015-01-30

    The discovery of cancer stem cells and tumor heterogeneity prompted the exploration of additional mechanisms aside from genetic mutations for carcinogenesis and cancer progression. The aim of the present study was to investigate the effect of cell fusion between mesenchymal stem cells and the gastric epithelial cells in tumorigenesis. Cell fusion between cord blood mesenchymal stem cells and human gastric epithelial cells was performed in vitro. Cell scratch and transwell assays were performed to determine migration and invasion abilities of the hybrids. The expressions of epithelial-mesenchymal transition-related proteins and genes were analyzed by immunocytochemistry and real time quantitative PCR. Tumorigenesis of the hybrids was evaluated through in vivo inoculation in nude mice. Hybrids expressed the phenotypes of both donor cells. Aneuploidy was observed in 84.1% of cells. The hybrids showed increased proliferation, migration and invasion abilities compared with the parental cells. In addition, the expression of N-cadherin and vimentin in the hybrids was significantly higher than that of the epithelial cells, and the mRNA expression of the epithelial-mesenchymal transition-related genes, Twist and Slug, in the hybrids was also increased compared with that of the parental epithelial cells. Furthermore, the hybrids formed masses of epithelial origin with glandular structures in BALB/c nude mice. These findings suggest that cell fusion between gastric epithelial cells and mesenchymal stem cells may result in epithelial to mesenchymal transition and malignant transformation.

  20. Association of mesenchymal cells and immunoglobulins with differentiating epithelial cells

    PubMed Central

    Bukovsky, Antonin; Caudle, Michael R; Keenan, Jeffrey A; Upadhyaya, Nirmala B; Van Meter, Stuart E; Wimalasena, Jay; Elder, Robert F

    2001-01-01

    Background Mesenchymal-epithelial interactions play an important role in the physiology and pathology of epithelial tissues. Mesenchymal cells either associate with epithelium basement membrane [pericytes and perivascular monocyte-derived cells (MDC)] or reside within epithelium (MDC and T cells). Although intraepithelial mesenchymal cells were suggested to contribute to the epithelium physiology, their association with particular steps in differentiation of epithelial cells, interactions among themselves, and their fate remain unclear. We studied epitopes of mesenchymal cells and their products (immunoglobulins) in stratified epithelium of uterine ectocervix, which is one of the prototypes of complete cellular differentiation from stem into the aged cells. Results Perivascular CD14 primitive MDC associated with basal (stem) epithelial cells. Thy-1 pericytes of microvasculature secreted intercellular vesicles, which associated with Ki67 postmitotic epithelial cells expressing MHC class I. Intraepithelial T cells showed an association with veiled type MDC [dendritic cell (DC) precursors] among parabasal cells, and exhibited fragmentation after entering intermediate (mature) epithelial layers. Mature DC secreted CD68 and exhibited fragmentation after reaching mid intermediate layers. Binding of IgM was detected at the top of each layer: in the upper parabasal, upper intermediate, and most surface epithelial cells. IgG was confined to the entire superficial layer. Conclusions These data suggest that the phylogenetically and ontogenetically developed hierarchy of mesenchymal cells (MDC, pericytes, T cells) and immunoglobulins (IgM, IgG) accompanies differentiation of epithelial cells from immature into the mature and aged phenotype. Further studies of an involvement of mesenchymal cells in the regulation of tissue homeostasis may bring novel approaches to the prevention and therapy of tissue dysfunctions characterized by permanent tissue immaturity (muscular dystrophy

  1. Protons sensitize epithelial cells to mesenchymal transition.

    PubMed

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

    2012-01-01

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

  2. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

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

    2012-01-01

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

  3. Airway epithelial cells: current concepts and challenges.

    PubMed

    Crystal, Ronald G; Randell, Scott H; Engelhardt, John F; Voynow, Judith; Sunday, Mary E

    2008-09-15

    The adult human bronchial tree is covered with a continuous layer of epithelial cells that play a critical role in maintaining the conduit for air, and which are central to the defenses of the lung against inhaled environmental concomitants. The epithelial sheet functions as an interdependent unit with the other lung components. Importantly, the structure and/or function of airway epithelium is deranged in major lung disorders, including chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Investigations regarding the airway epithelium have led to many advances over the past few decades, but new developments in genetics and stem cell/progenitor cell biology have opened the door to understanding how the airway epithelium is developed and maintained, and how it responds to environmental stress. This article provides an overview of the current state of knowledge regarding airway epithelial stem/progenitor cells, gene expression, cell-cell interactions, and less frequent cell types, and discusses the challenges for future areas of investigation regarding the airway epithelium in health and disease.

  4. Adherence of skin bacteria to human epithelial cells.

    PubMed Central

    Romero-Steiner, S; Witek, T; Balish, E

    1990-01-01

    Aerobic and anaerobic bacteria isolated from human axillae were tested for their capacity to adhere to buccal epithelial cells, immortalized human epithelial (HEp-2) cells, and undifferentiated and differentiated human epithelial cells. In general, both aerobic and anaerobic diphtheroids adhered better to differentiated human epithelial cells than to HEp-2 and undifferentiated human epithelial cells (P less than 0.05). Mannose, galactose, fucose, N-acetyl-D-glucosamine, and fibronectin were also assayed for their capacity to inhibit the adherence of diphtheroids to human epithelial cells. A great deal of variability was observed in the capacity of the latter compounds to inhibit the attachment of aerobic diphtheroids to undifferentiated and differentiated epithelial cells. Overall, mannose appeared to be best at inhibiting the adherence of the aerobic diphtheroids to undifferentiated human epithelial cells. Galactose, fucose, N-acetyl-D-glucosamine, and fibronectin showed a greater capacity to inhibit attachment of aerobic diphtheroids to differentiated than to undifferentiated human epithelial cells. The inhibition of adherence to differentiated human epithelial cells varied with the microorganism and the compound tested; however, the highest and most consistent inhibition of adherence (76.1 to 88.6%) was observed with a 5% solution of N-acetyl-D-glucosamine. The in vitro adherence and adherence inhibition assays presented here demonstrate that a number of adhesins and receptors are involved in the adherence of skin bacteria to human epithelial cells and receptors on human epithelial cells are apparently altered during differentiation. PMID:2298877

  5. CCN1 induces a reversible epithelial-mesenchymal transition in gastric epithelial cells.

    PubMed

    Chai, Jianyuan; Norng, Manith; Modak, Cristina; Reavis, Kevin M; Mouazzen, Wasim; Pham, Jennifer

    2010-08-01

    CCN1 is a matricellular protein that activates many genes related to wound healing and tissue remodeling in fibroblasts, but its effect on epithelial cells remains unclear. This study examined the role of CCN1 in epithelial wound healing using rat gastric epithelial cells and rat stomach ulcer as in vitro and in vivo models, respectively. We found that CCN1 expression is highly upregulated in the epithelial cells adjacent to a wound and remains high until the wound is healed. Upregulation of CCN1 activates a transient epithelial-mesenchymal transition in the epithelial cells at the migrating front and drives wound closure. Once the wound is healed, these epithelial cells and their progeny can resume their original epithelial phenotype. We also found that CCN1-induced E-cadherin loss is not due to transcriptional regulation but rather protein degradation due to the collapse of adherens junctions, which is contributed by beta-catenin translocation. CCN1-activated integrin-linked kinase mediates this process. Finally, our in vivo study showed that locally neutralizing CCN1 drastically impairs wound closure, whereas local injection of recombinant CCN1 protein induces expression of vimentin and smooth muscle alpha-actin in normal gastric mucosal epithelial cells and accelerates re-epithelialization during ulcer healing. In conclusion, our study indicates that CCN1 can induce reversible epithelial-mesenchymal transition, and this feature may have great value for clinical wound healing.

  6. Isolation of epithelial cells with hepatobiliary phenotype.

    PubMed

    Castorina, Sergio; Luca, Tonia; Torrisi, Antonella; Privitera, Giovanna; Panebianco, Mariangela

    2008-01-01

    The regenerative capacity of the liver after partial hepatectomy or chemical injury is well known. In human liver, the resident progenitor cells are called "hepatic progenitor cells" (HPCs) while the term "oval cells" should be discouraged in order to indicate the stem cell compartment. The aim of our study was first to analyse the cellular aspects of liver regeneration through differentiation in cholangiocytes and hepatocytes, and then to characterise resident progenitor cells, using "primary cultured hepatocytes" derived from healthy adult human livers. Human hepatocytes were isolated from fresh surgical specimens of patients who underwent hepatic resections in our Clinical Centre surgery operating room. Hepatic differentiation and function were analysed by immunocytochemistry techniques and the presence of liver epithelial cell populations within normal adult human liver, was demonstrated by immunohistochemistry analysis. These cells expanded in vitro and showed the capacity for self-renewal and multipotent differentiation. Human liver stem cells expressed several mesenchymal markers, such as CD44, but not haematopoietic stem cell markers. In addition, these cells expressed alpha-fetoprotein, albumin, CK7 and CK19, indicating a partial commitment to hepatic and biliary cells. Interestingly the expression of both hepatocytes and biliary markers in HPCs reflects the bipotential nature of the hepatic stem cells toward both the hepatic and biliary lineage. According to their immature and bipotential phenotype, hepatic epithelial cells might represent a pool of precursors in the healthy human adult liver.

  7. The human airway epithelial basal cell transcriptome.

    PubMed

    Hackett, Neil R; Shaykhiev, Renat; Walters, Matthew S; Wang, Rui; Zwick, Rachel K; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G

    2011-05-04

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

  8. The Human Airway Epithelial Basal Cell Transcriptome

    PubMed Central

    Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

    2011-01-01

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

  9. Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion.

    PubMed

    Lu, Yen-Zhen; Wu, Chi-Chin; Huang, Yi-Chen; Huang, Ching-Ying; Yang, Chung-Yi; Lee, Tsung-Chun; Chen, Chau-Fong; Yu, Linda Chia-Hui

    2012-05-01

    Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47(phox) and p67(phox), as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative

  10. Reversible transdifferentiation of alveolar epithelial cells.

    PubMed

    Danto, S I; Shannon, J M; Borok, Z; Zabski, S M; Crandall, E D

    1995-05-01

    Alveolar epithelial type II (AT2) cells have been thought to be the progenitors of terminally differentiated type I (AT1) cells in the adult animal in vivo. In this study, we used an AT1 cell-specific monoclonal antibody (mAb VIII B2) to investigate expression of the AT1 cell phenotype accompanying reversible changes in expression of the AT2 cell phenotype. AT2 cells were isolated and cultured either on attached collagen gels or on gels detached 1 or 4 days after plating and maintained thereafter as floating gels. Monolayers on both attached and floating gels were harvested on days 4 and 8 and analyzed by electron microscopy for changes in morphology and binding of mAb VIII B2. Results indicate that: (1) alveolar epithelial cells (AEC) on attached gels develop characteristics of the AT1 cell phenotype, (2) AEC on gels detached on day 1 maintain features of the AT2 cell phenotype (and do not react with mAb VIII B2), and (3) the expression of AT1 cell phenotypic traits seen by day 4 on attached gels is reversed after detachment. We conclude that commitment to the AT1 and AT2 cell lineages requires continuous regulatory input to maintain the differentiated states, and that transdifferentiation between AT2 and AT1 cells may be reversible.

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

    SciTech Connect

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

    1999-02-01

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

  12. Force mapping in epithelial cell migration

    PubMed Central

    du Roure, Olivia; Saez, Alexandre; Buguin, Axel; Austin, Robert H.; Chavrier, Philippe; Siberzan, Pascal; Ladoux, Benoit

    2005-01-01

    We measure dynamic traction forces exerted by epithelial cells on a substrate. The force sensor is a high-density array of elastomeric microfabricated pillars that support the cells. Traction forces induced by cell migration are deduced from the measurement of the bending of these pillars and are correlated with actin localization by fluorescence microscopy. We use a multiple-particle tracking method to estimate the mechanical activity of cells in real time with a high-spatial resolution (down to 2 μm) imposed by the periodicity of the post array. For these experiments, we use differentiated Madin-Darby canine kidney (MDCK) epithelial cells. Our data provide definite information on mechanical forces exerted by a cellular assembly. The maximum intensity of the forces is localized on the edge of the epithelia. Hepatocyte growth factor promotes cell motility and induces strong scattering activity of MDCK cells. Thus, we compare forces generated by MDCK cells in subconfluent epithelia versus isolated cells after hepatocyte growth factor treatment. Maximal-traction stresses at the edge of a monolayer correspond to higher values than those measured for a single cell and may be due to a collective behavior. PMID:15695588

  13. Isolation by Size of Epithelial Tumor Cells

    PubMed Central

    Vona, Giovanna; Sabile, Abdelmajid; Louha, Malek; Sitruk, Veronique; Romana, Serge; Schütze, Karin; Capron, Frédérique; Franco, Dominique; Pazzagli, Mario; Vekemans, Michel; Lacour, Bernard; Bréchot, Christian; Paterlini-Bréchot, Patrizia

    2000-01-01

    We have developed a new assay, ISET (isolation by size of epithelial tumor cells), which allows the counting and the immunomorphological and molecular characterization of circulating tumor cells in patients with carcinoma, using peripheral blood sample volumes as small as 1 ml. Using this assay, epithelial tumor cells can be isolated individually by filtration because of their larger size when compared to peripheral blood leukocytes. ISET parameters were defined using peripheral blood spiked with tumor cell lines (HepG2, Hep3B, MCF-7, HeLa, and LNCaP). ISET can detect a single, micropipetted tumor cell, added to 1 ml of blood. We also demonstrate that fluorescence in situ hybridization can be used to perform chromosomal analyses on tumor cells collected using ISET. Polymerase chain reaction-based genetic analyses can be applied to ISET-isolated cells, and, as an example, we demonstrate homozygous p53 deletion in single Hep3B cells after filtration and laser microdissection. Finally, we provide evidence for the in vivo feasibility of ISET in patients with hepatocellular carcinoma undergoing tumor resection. ISET, but not reverse transcriptase-polymerase chain reaction, allowed analysis of cell morphology, counting of tumor cells, and demonstration of tumor microemboli spread into peripheral blood during surgery. Overall, ISET constitutes a novel approach that should open new perpectives in molecular medicine. PMID:10623654

  14. Antigen presentation for priming T cells in central system.

    PubMed

    Dasgupta, Shaoni; Dasgupta, Subhajit

    2017-01-01

    Generation of myelin antigen-specific T cells is a major event in neuroimmune responses that causes demyelination. The antigen-priming of T cells and its location is important in chronic and acute inflammation. In autoimmune multiple sclerosis, the effector T cells are considered to generate in periphery. However, the reasons for chronic relapsing-remitting events are obscure. Considering mechanisms, a feasible aim of research is to investigate the role of antigen-primed T cells in lupus cerebritis. Last thirty years of investigations emphasize the relevance of microglia and infiltrated dendritic cells/macrophages as antigen presenting cells in the central nervous system. The recent approach towards circulating B-lymphocytes is an important area in the context. Here, we analyze the existing findings on antigen presentation in the central nervous system. The aim is to visualize signaling events of myelin antigen presentation to T cells and lead to the strategy of future goals on immunotherapy research.

  15. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells.

    PubMed Central

    Simon, R H; DeHart, P D; Todd, R F

    1986-01-01

    The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing. Images PMID:3771800

  16. Traction forces exerted by epithelial cell sheets

    NASA Astrophysics Data System (ADS)

    Saez, A.; Anon, E.; Ghibaudo, M.; du Roure, O.; Di Meglio, J.-M.; Hersen, P.; Silberzan, P.; Buguin, A.; Ladoux, B.

    2010-05-01

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  17. Traction forces exerted by epithelial cell sheets.

    PubMed

    Saez, A; Anon, E; Ghibaudo, M; du Roure, O; Di Meglio, J-M; Hersen, P; Silberzan, P; Buguin, A; Ladoux, B

    2010-05-19

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  18. Cells of Origin of Epithelial Ovarian Cancers

    DTIC Science & Technology

    2015-09-01

    lethal malignancy of the female reproductive system , largely due to the fact that most EOCs are diagnosed only after the cancer has metastasized into the...Epithelial ovarian cancer (EOC) is the most lethal malignancy of the female reproductive system , largely due to the fact that most EOCs are diagnosed only...ovarian cancer by defined multiple genetic changes in a mouse model system . Cancer Cell 1, 53-62. Quartuccio, S.M., Lantvit, D.D., Bosland, M.C., and

  19. Cell density determines epithelial migration in culture.

    PubMed Central

    Rosen, P; Misfeldt, D S

    1980-01-01

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

  20. Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells

    PubMed Central

    Maloum, Faïza; Allaire, Joannie M.; Gagné-Sansfaçon, Jessica; Roy, Evelyne; Belleville, Karine; Sarret, Philippe; Morisset, Jean; Carrier, Julie C.; Mishina, Yuji; Kaestner, Klaus H.

    2011-01-01

    Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1aΔGEC mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1aΔGEC mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia. PMID:21415412

  1. T cell homing to epithelial barriers in allergic disease

    PubMed Central

    Islam, Sabina A; Luster, Andrew D

    2013-01-01

    Allergic inflammation develops in tissues that have large epithelial surface areas that are exposed to the environment, such as the lung, skin and gut. In the steady state, antigen-experienced memory T cells patrol these peripheral tissues to facilitate swift immune responses against invading pathogens. In at least two allergy-prone organs, the skin and the gut, memory T cells are programmed during the initial antigen priming to express trafficking receptors that enable them to preferentially home to these organs. In this review we propose that tissue-specific memory and inflammation-specific T cell trafficking facilitates the development of allergic disease in these organs. We thus review recent advances in our understanding of tissue-specific T cell trafficking and how regulation of T cell trafficking by the chemokine system contributes to allergic inflammation in mouse models and in human allergic diseases of the skin, lung and gut. Inflammation- and tissue-specific T lymphocyte trafficking pathways are currently being targeted as new treatments for non-allergic inflammatory diseases and may yield effective new therapeutics for allergic diseases. PMID:22561834

  2. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2010-10-01

    breast is highly expressed by luminal epithelial cells and is less expressed by basal cells19,20. In contrast, CD49f (a6 integrin) has an inverse pattern...mouse stretched on its back. The hose and nose cone from the anesthetic vaporizer are securely attached to one side of the plate, and a heated pad is...the mouse by a nose cone. Check that the mouse has reached surgical anesthesia by loss of pedal withdrawal reflex . ! cautIon Institutional review

  3. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  4. Detection of Bone Marrow Derived Lung Epithelial Cells

    PubMed Central

    Kassmer, Susannah H.; Krause, Diane S.

    2010-01-01

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

  5. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans.

  6. Lactobacillus Decelerates Cervical Epithelial Cell Cycle Progression

    PubMed Central

    Vielfort, Katarina; Weyler, Linda; Söderholm, Niklas; Engelbrecht, Mattias; Löfmark, Sonja; Aro, Helena

    2013-01-01

    We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells. PMID:23675492

  7. CD169+ macrophages are sufficient for priming of CTLs with specificities left out by cross-priming dendritic cells.

    PubMed

    Bernhard, Caroline A; Ried, Christine; Kochanek, Stefan; Brocker, Thomas

    2015-04-28

    Dendritic cells (DCs) are considered the most potent antigen-presenting cells (APCs), which directly prime or cross-prime MHC I-restricted cytotoxic T cells (CTLs). However, recent evidence suggests the existence of other, as-yet unidentified APCs also able to prime T cells. To identify those APCs, we used adenoviral (rAd) vectors, which do not infect DCs but selectively accumulate in CD169(+) macrophages (MPs). In mice that lack DCs, infection of CD169(+) MPs was sufficient to prime CTLs specific for all epitopes tested. In contrast, CTL responses relying exclusively on cross-presenting DCs were biased to selected strong MHC I-binding peptides only. When both DCs and MPs were absent, no CTL responses could be elicited. Therefore, CD169(+) MPs can be considered APCs that significantly contribute to CTL responses.

  8. Cell reintegration: Stray epithelial cells make their way home.

    PubMed

    Wilson, Tyler J; Bergstralh, Dan T

    2017-06-01

    Ongoing work shows that misplaced epithelial cells have the capacity to reintegrate back into tissue layers. This movement appears to underlie tissue stability and may also control aspects of tissue structure. A recent study reveals that cell reintegration in at least one tissue, the Drosophila follicular epithelium, is based on adhesion molecules that line lateral cell surfaces. In this article we will review these observations, discuss their implications for epithelial tissue development and maintenance, and identify future directions for study. © 2017 WILEY Periodicals, Inc.

  9. Phenotypic plasticity in normal breast derived epithelial cells

    PubMed Central

    2014-01-01

    Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools. PMID:24915897

  10. Spontaneous Production of Immunoglobulin M in Human Epithelial Cancer Cells

    PubMed Central

    Hu, Fanlei; Zhang, Li; Zheng, Jie; Zhao, Ling; Huang, Jing; Shao, Wenwei; Liao, Qinyuan; Ma, Teng; Geng, Li; Yin, C. Cameron; Qiu, Xiaoyan

    2012-01-01

    It is well known that B-1 B cells are the main cell type that is responsible for the production of natural immunoglobulin M (IgM) and can respond to infection by increasing IgM secretion. However, we unexpectedly found that some epithelial cells also can express rearranged IgM transcript that has natural IgM characteristics, such as germline-encoded and restricted rearrangement patterns. Here we studied IgM expression in human non-B cells and found that IgM was frequently expressed by many human epithelial cancer cells as well as non-cancer epithelial cells. Moreover, CD79A and CD79B, two molecules that are physically linked to membranous IgM on the surface of B cells to form the B cell antigen receptor complex, were also expressed on the cell surface of epithelial cancer cells and co-located with IgM. Like the natural IgM, the epithelial cancer cell-derived IgM recognized a series of microbial antigens, such as single-stranded DNA, double-stranded DNA, lipopolysaccharide, and the HEp-2 cell antigen. More important, stimulation of the toll-like receptor 9 (TLR9), which mimics bacterial infection, substantially increased the secretion of IgM in human epithelial cancer cells. These findings indicate that human epithelial cancer cells as well as non-cancer epithelial cells can spontaneously produce IgM with natural antibody activity. PMID:23251529

  11. Promoter methylation in epithelial-enriched and epithelial-depleted cell populations isolated from breast milk.

    PubMed

    Browne, Eva P; Dinc, Signem E; Punska, Elizabeth C; Agus, Sami; Vitrinel, Ayca; Erdag, Gulay Ciler; Anderton, Douglas L; Arcaro, Kathleen F; Yilmaz, Bayram

    2014-11-01

    Breast cancer is the most frequently diagnosed cancer among Turkish women and both the incidence and associated mortality appear to be increasing. Of particular concern is the percentage of young women diagnosed with breast cancer; roughly 20% of all breast cancer diagnoses in Turkey are in women younger than 40 years. Increased DNA methylation in the promoter region of tumor suppressor genes is a promising molecular biomarker, and human milk provides exfoliated breast epithelial cells appropriate for DNA methylation analyses. Comparisons between DNA methylation patterns in epithelial (epithelial-enriched) and nonepithelial (epithelial-depleted) cell fractions from breast milk have not been reported previously. In the present study, we examined promoter methylation of 3 tumor suppressor genes in epithelial-enriched and epithelial-depleted cell fractions isolated from breast milk of 43 Turkish women. Percentage methylation in the promoter region of Rass association domain family 1 (RASSF1), secreted frizzle related protein 1 (SFRP1), and glutathione-S-transferase class pi 1 was determined by pyrosequencing of the epithelial-enriched and epithelial-depleted cell fractions. Pyrosequencing identified a few subjects with significantly increased methylation in 1 or more genes. There was little correlation between the 2 cell fractions within individuals; only 1 woman had increased methylation for 1 gene (SFRP1) in both her enriched and depleted cell fractions. Methylation was positively associated with age for SFRP1 (epithelial-depleted fraction) and with body mass index for RASSF1 (epithelial-enriched cell fraction), respectively. Overall, results show that the methylation signals vary between different cell types in breast milk and suggest that breast milk can be used to assess DNA methylation patterns associated with increased breast cancer risk. © The Author(s) 2014.

  12. Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

    PubMed

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

    2013-12-01

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

  13. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2009-10-01

    Appendix……………………………………………………………………………… 11 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A method for... Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability...Abstracts Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turshvili, Sam Aparicio , Joanne Emerman and Connie Eaves, “Identification of Human Mammary

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

    PubMed

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

    2015-03-01

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

  15. Quercetin Blocks Airway Epithelial Cell Chemokine Expression

    PubMed Central

    Nanua, Suparna; Zick, Suzanna M.; Andrade, Juan E.; Sajjan, Umadevi S.; Burgess, John R.; Lukacs, Nicholas W.; Hershenson, Marc B.

    2006-01-01

    Quercetin (3,3′,4′,5,7-pentahydroxyflavone), a dietary flavonoid, is an inhibitor of phosphatidylinositol (PI) 3-kinase and potent antioxidant. We hypothesized that quercetin blocks airway epithelial cell chemokine expression via PI 3-kinase–dependent mechanisms. Pretreatment with quercetin and the PI 3–kinase inhibitor LY294002 each reduced TNF-α–induced IL-8 and monocyte chemoattractant protein (MCP)-1 (also called CCL2) expression in cultured human airway epithelial cells. Quercetin also inhibited TNF-α–induced PI 3-kinase activity, Akt phosphorylation, intracellular H2O2 production, NF-κB transactivation, IL-8 promoter activity, and steady-state mRNA levels, consistent with the notion that quercetin inhibits chemokine expression by attenuating NF-κB transactivation via a PI 3-kinase/Akt-dependent pathway. Quercetin also reduced TNF-α–induced chemokine secretion in the presence of the transcriptional inhibitor actinomycin D, while inducing phosphorylation of eukaryotic translation initiation factor (eIF)-2α, suggesting that quercetin attenuates chemokine expression by post-transcriptional as well as transcriptional mechanisms. Finally, we tested the effects of quercetin in cockroach antigen–sensitized and –challenged mice. These mice show MCP-1–dependent airways hyperresponsiveness and inflammation. Quercetin significantly reduced lung MCP-1 and methacholine responsiveness. We conclude that quercetin blocks airway cell chemokine expression via transcriptional and post-transcriptional pathways. PMID:16794257

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

    PubMed

    Qu, J H; Sun, X G

    2016-09-11

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

  17. Interleukin-1 stimulates zinc uptake by human thymic epithelial cells

    SciTech Connect

    Coto, J.A.; Hadden, J.W. )

    1991-03-15

    Thymic epithelial cells (TEC) are known to secrete peptides which influence the differentiation and maturation of T-lymphocytes. These peptides include the thymic hormones thymulin, thymosin-{alpha}1, and thymopoietin. The biological activity of thymulin is dependent on the presence of zinc in an equimolar ratio. The authors have shown that both interleukin-1{alpha}(IL-1{alpha}) and interleukin-1{beta}(IL-1{beta}), which stimulate proliferation of TEC, stimulate the uptake of Zn-65 in-vitro independent of this proliferation. Mitomycin-C was used to inhibit the proliferation of TEC. Two other stimulators of proliferation of TEC, bovine pituitary extract (BPE) and epidermal growth factor (EGF), did not stimulate zinc uptake by the TEC independent of proliferation. They have also shown, utilizing in-situ hybridization, that IL-1 and zinc induce metallothionein(MT) mRNA expression in human thymic epithelial cells. The exact role of metallothionein is not clear, but it is thought to be involved in regulation of trace metal metabolism, especially in maintenance of zinc homeostasis. Their current hypothesis is that IL-1 stimulates uptake of zinc into the TEC, followed by its complexing with metallothionein. Zinc is then thought to be transferred from metallothionein to thymulin. Immunostaining, utilizing an antithymulin antibody and a fluoresceinated goat anti-rabbit second antibody, confirms the presence of thymulin in TEC and its dependence on zinc. Upon stimulation, thymulin is then secreted. Known stimulants for thymulin include progesterone, dexamethasone, estradiol, testosterone, and prolactin. None of these secretagogues increase zinc uptake, suggesting the priming of the zinc-thymulin complex is unrelated to the regulation of its secretion.

  18. Tractable mammalian cell infections with protozoan-primed bacteria.

    PubMed

    Drennan, Samuel L; Lama, Amrita; Doron, Ben; Cambronne, Eric D

    2013-04-02

    Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

  19. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

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

    2013-03-01

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

  20. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

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

    2013-01-01

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

  1. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

    Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2004-07-14

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

  4. Building Epithelial Tissues from Skin Stem Cells

    PubMed Central

    Fuchs, E.; Nowak, J.A.

    2009-01-01

    The skin epidermis and its appendages provide a protective barrier that guards against loss of fluids, physical trauma, and invasion by harmful microbes. To perform these functions while confronting the harsh environs of the outside world, our body surface undergoes constant rejuvenation through homeostasis. In addition, it must be primed to repair wounds in response to injury. The adult skin maintains epidermal homeostasis, hair regeneration, and wound repair through the use of its stem cells. What are the properties of skin stem cells, when do they become established during embryogenesis, and how are they able to build tissues with such remarkably distinct architectures? How do stem cells maintain tissue homeostasis and repair wounds and how do they regulate the delicate balance between proliferation and differentiation? What is the relationship between skin cancer and mutations that perturbs the regulation of stem cells? In the past 5 years, the field of skin stem cells has bloomed as we and others have been able to purify and dissect the molecular properties of these tiny reservoirs of goliath potential. We report here progress on these fronts, with emphasis on our laboratory’s contributions to the fascinating world of skin stem cells. PMID:19022769

  5. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

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

    2011-10-01

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

  6. Epithelial Cell Innate Response to Candida albicans

    PubMed Central

    Naglik, J.R.; Moyes, D.

    2011-01-01

    With the advent of treatments and diseases such as AIDS resulting in increasing numbers of patients with suppressed immune systems, fungal diseases are an escalating problem. Candida albicans is the most common of these fungal pathogens, causing infections in many of these patients. It is therefore important to understand how immunity to this fungus is regulated and how it might be manipulated. Although work has been done to identify the receptors, fungal moieties, and responses involved in anti-Candida immunity, most studies have investigated interactions with myeloid or lymphoid cells. Given that the first site of contact of C. albicans with its host is the mucosal epithelial surface, recent studies have begun to focus on interactions of C. albicans with this site. The results are startling yet in retrospect obvious, indicating that epithelial cells play an important role in these interactions, initiating responses and even providing a level of protection. These findings have obvious implications, not just for fungal pathogens, but also for identifying how host organisms can distinguish between commensal and pathogenic microbes. This review highlights some of these recent findings and discusses their importance in the wider context of infection and immunity. PMID:21441481

  7. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions.

    PubMed

    Van Landeghem, Laurianne; Mahé, Maxime M; Teusan, Raluca; Léger, Jean; Guisle, Isabelle; Houlgatte, Rémi; Neunlist, Michel

    2009-11-02

    Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions.

  8. Dedifferentiation of committed epithelial cells into stem cells in vivo

    PubMed Central

    Tata, Purushothama Rao; Mou, Hongmei; Pardo-Saganta, Ana; Zhao, Rui; Prabhu, Mythili; Prabhu, Mythili; Law, Brandon M.; Vinarsky, Vladimir; Cho, Josalyn L.; Breton, Sylvie; Sahay, Amar; Medoff, Benjamin D.; Rajagopal, Jayaraj

    2014-01-01

    Summary Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes, and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. We now present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. Following the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts to repair epithelial injury. Indeed, single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. In contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate was inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may play a more general role in the regeneration of many tissues and in multiple disease states, notably cancer. PMID:24196716

  9. Silk Film Topography Directs Collective Epithelial Cell Migration

    PubMed Central

    Rosenblatt, Mark I.

    2012-01-01

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

  10. Preparation and characterization of viable epithelial cells from rabbit distal colon

    SciTech Connect

    Kaunitz, J.D. Univ. of California, Los Angeles )

    1988-04-01

    A preparation of viable epithelial cells, suitable for transport studies, was prepared from rabbit distal colon. Enzymatic digestion of scraped mucosa liberated a population of intact colonic glands that were dissociated into cells by gentle agitation in culture medium. Metabolism of ({sup 14}C)glucose was constant over 2 h and was inhibited 70% by 1 mM ouabain. Cells were loaded with the trapped, pH-sensitive fluorescent dye 2{prime}-7{prime}-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxy methyl ester and leaked 0.1% of the dye per minute. Cell pH was 7.21 {+-} 0.03 in pH 7.4. Ringer medium. Intracellular potassium concentration, as measured by a nigericin null-point technique, was 128 {+-} 8 mM. Plasma membrane potential measured with the potential-sensitive fluorescent dye 3,3-dipropylthiadicarboxycanine iodide was -52 {+-} 2 mV. Recovery of intracellular pH in acid-loaded cells occurred after exposure to sodium-containing medium and was inhibited by 5 {times} 10{sup {minus}4} M amiloride. It is concluded that viable epithelial cells can be prepared from rabbit distal colon with relative simplicity and in high yield. These cells are suitable for measurement of intracellular pH and membrane potential and are thus a convenient model system for study of colonic cell physiology.

  11. Ionizing radiation predisposes nonmalignant human mammary epithelial cells to undergo transforming growth factor beta induced epithelial to mesenchymal transition.

    PubMed

    Andarawewa, Kumari L; Erickson, Anna C; Chou, William S; Costes, Sylvain V; Gascard, Philippe; Mott, Joni D; Bissell, Mina J; Barcellos-Hoff, Mary Helen

    2007-09-15

    Transforming growth factor beta1 (TGFbeta) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFbeta activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFbeta-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFbeta (0.4 ng/mL) or double treated. All double-treated (IR + TGFbeta) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFbeta alone elicited EMT, although IR increased chronic TGFbeta signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFbeta-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFbeta, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

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

    PubMed Central

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

    2012-01-01

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

  13. Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

    PubMed Central

    Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

    2017-01-01

    Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1

  14. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  15. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Ouabain modulates epithelial cell tight junction

    PubMed Central

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

    2010-01-01

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

  19. Mechanical stretch triggers rapid epithelial cell division through Piezo1.

    PubMed

    Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

    2017-03-02

    Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

  20. Polycomb enables primitive endoderm lineage priming in embryonic stem cells

    PubMed Central

    Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V; Bickmore, Wendy A; Brickman, Joshua M

    2016-01-01

    Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver of these coordinated changes, known as lineage priming, in a process that exploits novel polycomb activities. We find that intragenic levels of the polycomb mark H3K27me3 anti-correlate with changes in transcription, irrespective of the gene’s developmental trajectory or identity as a polycomb target. In contrast, promoter proximal H3K27me3 is markedly higher for PrEn priming genes. Consequently, depletion of this modification stimulates the degree to which ESCs are primed towards PrEn when challenged to differentiate, but has little effect on gene expression in self-renewing ESC culture. These observations link polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision. DOI: http://dx.doi.org/10.7554/eLife.14926.001 PMID:27723457

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

    PubMed

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

    2016-07-19

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

  2. Corneal epithelial stem cells: deficiency and regulation.

    PubMed

    Secker, Genevieve A; Daniels, Julie T

    2008-09-01

    The corneal epithelium is continuously renewed by a population of stem cells that reside in the corneoscleral junction, otherwise known as the limbus. These limbal epithelial stem cells (LESC) are imperative for corneal maintenance with deficiencies leading to in-growth of conjunctival cells, neovascularisation of the corneal stroma and eventual corneal opacity and visual loss. One such disease that has traditionally been thought to be due to LESC deficiency is aniridia, a pan-ocular congenital eye disease due to mutations in the PAX6 gene. Corneal changes or aniridia related keratopathy (ARK) seen in aniridia are typical of LESC deficiency. However, the pathophysiology behind ARK is still ill defined, with current theories suggesting it may be caused by a deficiency in the stem cell niche and adjacent corneal stroma, with altered wound healing responses also playing a role (Ramaesh et al, International Journal of Biochemistry & Cell Biology 37:547-557, 2005) or abnormal epidermal differentiation of LESC (Li et al., The Journal of Pathology 214:9, 2008). PAX6 is considered the master control gene for the eye and is required for normal eye development with expression continuing in the adult cornea, thus inferring a role for corneal repair and regeneration (Sivak et al., Developments in Biologicals 222:41-54, 2000). Studies of models of Pax6 deficiency, such as the small eyed (sey) mouse, should help to reveal the intrinsic and extrinsic mechanisms involved in normal LESC function.

  3. STUDIES ON AN EPITHELIAL (GLAND) CELL JUNCTION

    PubMed Central

    Loewenstein, Werner R.; Kanno, Yoshinobu

    1964-01-01

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

  4. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

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

    2016-01-01

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

  5. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  7. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis.

    PubMed

    Ewald, Andrew J; Brenot, Audrey; Duong, Myhanh; Chan, Bianca S; Werb, Zena

    2008-04-01

    Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly, suggesting common mechanisms of epithelial growth.

  8. Osteopontin traffic in hypoxic renal epithelial cells.

    PubMed

    Hampel, Dierk J; Sansome, Christine; Romanov, Victor I; Kowalski, Aaron J; Denhardt, David T; Goligorsky, Michael S

    2003-01-01

    Osteopontin (OPN), a secretory RGD-containing phosphoprotein, is induced in acute renal injury where it plays a renoprotective role. To investigate in depth the mode of OPN secretion under stress conditions, we analyzed OPN traffic in human renal proximal tubular epithelial cells (RPTEC). Western blot analysis and fluorescence microscopy revealed trace amounts of OPN in intact cells, whereas cytoplasmic OPN levels were significantly increased after 24-48 h hypoxia. Immunoelectron microscopy of RPTEC showed predominantly apical localization of gold-labeled OPN under normal conditions. Hypoxia (24 h) increased 2.5-fold immunodetectable gold-labeled OPN at the apical plasma membrane; further reoxygenation (2 h) augmented apical and basolateral labeling 2- and 10-fold, respectively. Analysis of apical and basolateral medium conditioned by RPTEC grown on semipermeable membranes using a specially developed ELISA showed a global decrease in secreted OPN after hypoxia, which recovered following 2 h reoxygenation. Agents known to disrupt the function of the Golgi apparatus (brefeldin A, monensin) or actin cytoskeleton (cytochalasin B) significantly inhibited OPN-GFP secretion in normoxic cells. In cells recovering from hypoxia, however, OPN secretion required functional Golgi apparatus, but was not affected by cytochalasin B. These findings demonstrate that stress inhibits OPN secretion by the process dependent on the functional Golgi apparatus and actin cytoskeleton; recovery restores OPN secretion, although its polarity may become perturbed. Copyright 2003 S. Karger AG, Basel

  9. Seeding of recipient-originated epithelial cells attenuates epithelial to mesenchymal transition in rat tracheal allotransplantation.

    PubMed

    Huang, Xun; Yan, Xiaolong; Zhang, Zhipei; Li, Xiaofei

    2015-06-01

    The specific role and mechanism of epithelium in the progression of obliterative airway disease (OAD) after tracheal allotransplantation remain poorly understood. In this study, we used rat heterotopic tracheal transplantation to investigate the mechanism of epithelial cell seeding during the process of OAD. Prospective, basic science. Research laboratory. In total, 120 Sprague Dawley (SD) rats and 90 Wistar rats were used. Tracheas from SD rats were implanted into SD rats (syngeneic, n = 30) or Wistar rats (allogeneic, n = 30), and SD rat tracheas (seeded with Wistar rat-derived epithelial cells 6 days after transplantation) were transplanted into Wistar rats (seeded allogeneic, n = 30). Grafts were harvested at 7, 14, or 30 days after transplantation for histologic, quantitative reverse transcriptional polymerase chain reaction or Western blot analyses. Syngrafts retained normal histologic structures, while the corresponding allografts demonstrated less ciliated epithelia and more lumenal occlusion. Seeding of epithelial cells ameliorated the histologic changes, reduced the expression of epithelial to mesenchymal transition (EMT)-related transcriptional factors and mesenchymal markers, and dampened the expression of transforming growth factor β1 (TGF-β1) and phosphorylation of smad3. Seeding of recipient epithelial cells inhibits the progression of OAD by attenuating EMT via TGF-β-Smad signaling in rat heterotopic tracheal allografts. Clinically, the injection of recipient-originated epithelial cells might provide new insights into the treatment for OAD after tracheal allotransplantation. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

  10. Cytomatrix synthesis in MDCK epithelial cells

    SciTech Connect

    Mitchell, J.J.; Low, R.B.; Woodcock-Mitchell, J.L. )

    1990-06-01

    Detailed information regarding the synthesis rates of individual protein components is important in understanding the assembly and dynamics of the cytoskeletal matrix of eukaryotic cells. As an approach to this topic, the dual isotope technique of Clark and Zak, was employed to measure fractional synthesis rates (FSRs) in growing and quiescent cultures of MDCK epithelial cells. Cell protein was labeled to equilibrium with (14C)leucine over several days and then pulse-labeled for 4 hours with (3H)leucine. FSRs (as percent per hour) were calculated from the 3H/14C ratio of cell extracts or individual proteins separated by two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of free leucine in the medium. Synthesis of total cell protein rose from approximately 1.4%/hour in quiescent cells to 3.5%/hour in the growing cultures. The latter rate was sufficient to account for the rate of protein accumulation and a low level of turnover in the growing cultures. The FSR of the buffered-Triton soluble extract was higher and the cytoskeletal FSR significantly lower than that for total protein in quiescent monolayers. This difference, however, was not observed in growing cultures. A distinct pattern of differences was seen in the FSRs of individual cytoskeletal proteins in the quiescent cultures. Vimentin synthesis was significantly lower than that of the keratins and the keratin FSRs were not obviously matched in pairwise fashion. Unexpectedly, the FSRs of alpha- and beta-tubulin diverged in quiescent cells with alpha-tubulin turnover exceeding beta-tubulin. Likewise, components of the microfilament lattice showed unequal fractional synthesis rates, myosin and alpha-actinin being faster than actin. In addition, the FSR for globular actin exceeded that of the cytoskeletal associated form.

  11. Pulmonary Epithelial Cell-Derived Cytokine TGF-β1 Is a Critical Cofactor for Enhanced Innate Lymphoid Cell Function

    PubMed Central

    Denney, Laura; Byrne, Adam J.; Shea, Thomas J.; Buckley, James S.; Pease, James E.; Herledan, Gaelle M.F.; Walker, Simone A.; Gregory, Lisa G.; Lloyd, Clare M.

    2015-01-01

    Summary Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-β had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-β1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-β by expressing the receptor TGF-βRII and ILC chemoactivity was enhanced by TGF-β. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions. PMID:26588780

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

    PubMed

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

    2015-11-01

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

  13. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  14. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells.

    PubMed

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-07-15

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite.

  15. Cholecystokinin octapeptide antagonizes apoptosis in human retinal pigment epithelial cells

    PubMed Central

    Liu, Yuan; Zhang, Yueling; Gu, Zhaohui; Hao, Lina; Du, Juan; Yang, Qian; Li, Suping; Wang, Liying; Gong, Shilei

    2014-01-01

    Although cholecystokinin octapeptide-8 is important for neurological function, its neuroprotective properties remain unclear. We speculated that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against oxidative injury. In this study, retinal pigment epithelial cells were treated with peroxynitrite to induce oxidative stress. Peroxynitrite triggered apoptosis in these cells, and increased the expression of Fas-associated death domain, Bax, caspa-se-8 and Bcl-2. These changes were suppressed by treatment with cholecystokinin octapeptide-8. These results suggest that cholecystokinin octapeptide-8 can protect human retinal pigment epithelial cells against apoptosis induced by peroxynitrite. PMID:25221599

  16. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  17. Cell death at the intestinal epithelial front line.

    PubMed

    Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

    2016-07-01

    The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

  18. Gremlin Activates the Smad Pathway Linked to Epithelial Mesenchymal Transdifferentiation in Cultured Tubular Epithelial Cells

    PubMed Central

    Rodrigues-Diez, Raquel; Rodrigues-Diez, Raúl R.; Lavoz, Carolina; Carvajal, Gisselle; Droguett, Alejandra; Garcia-Redondo, Ana B.; Rodriguez, Isabel; Ortiz, Alberto; Egido, Jesús; Mezzano, Sergio; Ruiz-Ortega, Marta

    2014-01-01

    Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β. PMID:24949470

  19. Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry

    PubMed Central

    Clerc, Pascaline; Carey, Gregory B.; Mehrabian, Zara; Wei, Michael; Hwang, Hyehyun; Girnun, Geoffrey D.; Chen, Hegang; Martin, Stuart S.; Polster, Brian M.

    2012-01-01

    Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed “primed for death” and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O2 consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O2 consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers

  20. Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry.

    PubMed

    Clerc, Pascaline; Carey, Gregory B; Mehrabian, Zara; Wei, Michael; Hwang, Hyehyun; Girnun, Geoffrey D; Chen, Hegang; Martin, Stuart S; Polster, Brian M

    2012-01-01

    Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers

  1. Epithelial-to-mesenchymal transition in penile squamous cell carcinoma.

    PubMed

    Masferrer, Emili; Ferrándiz-Pulido, Carla; Masferrer-Niubò, Magalí; Rodríguez-Rodríguez, Alfredo; Gil, Inmaculada; Pont, Antoni; Servitje, Octavi; García de Herreros, Antonio; Lloveras, Belen; García-Patos, Vicenç; Pujol, Ramon M; Toll, Agustí; Hernández-Muñoz, Inmaculada

    2015-02-01

    Epithelial-to-mesenchymal transition is a phenomenon in epithelial tumors that involves loss of intercellular adhesion, mesenchymal phenotype acquisition and enhanced migratory potential. While the epithelial-to-mesenchymal transition process has been extensively linked to metastatic progression of squamous cell carcinoma, studies of the role of epithelial-to-mesenchymal transition in squamous cell carcinoma containing high risk human papillomaviruses are scarce. Moreover, to our knowledge epithelial-to-mesenchymal transition involvement in human penile squamous cell carcinoma, which can arise through transforming HPV infections or independently of HPV, has not been investigated. We evaluated the presence of epithelial-to-mesenchymal transition markers and their relationship to HPV in penile squamous cell carcinoma. We assessed the expression of E-cadherin, vimentin and the epithelial-to-mesenchymal transition related transcription factors Twist, Zeb1 and Snail by immunohistochemical staining in 64 penile squamous cell carcinoma cases. HPV was detected by polymerase chain reaction amplification. Simultaneous loss of membranous E-cadherin expression and vimentin over expression were noted in 43.5% of penile squamous cell carcinoma cases. HPV was significantly associated with loss of membranous E-cadherin but not with epithelial-to-mesenchymal transition. Recurrence and mortality rates were significantly higher in cases showing epithelial-to-mesenchymal transition. Our findings indicate that in penile squamous cell carcinoma epithelial-to-mesenchymal transition is associated with poor prognosis but not with the presence of HPV. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  2. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

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

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

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

    PubMed

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

    2014-01-01

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

  4. Clinical implications of epithelial cell plasticity in cancer progression.

    PubMed

    Aparicio, Luis A; Blanco, Moisés; Castosa, Raquel; Concha, Ángel; Valladares, Manuel; Calvo, Lourdes; Figueroa, Angélica

    2015-09-28

    In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients.

  5. Human Growth Hormone Promotes Corneal Epithelial Cell Migration in Vitro

    PubMed Central

    Ding, Juan; Wirostko, Barbara; Sullivan, David A

    2015-01-01

    Purpose Corneal wound healing is a highly regulated process that requires the proliferation and migration of epithelial cells and interactions between epithelial cells and stromal fibroblasts. Compounds that can be applied topically to the ocular surface and that have the capability of activating corneal epithelial cells to proliferate and/or migrate would be useful to promote corneal wound healing. We hypothesize that human growth hormone (HGH) will activate Signal Transducer and Activators of Transcription-5 (STAT5) signaling and promote corneal wound healing by enhancing corneal epithelial cell and fibroblast proliferation and/or migration in vitro. The purpose of this study is to test these hypotheses. Methods We studied cell signaling, proliferation and migration using an immortalized human corneal epithelial cell line and primary human corneal fibroblasts in vitro. We also examined whether insulin-like growth factor-1 (IGF-1), a hormone known to mediate many of HGH’s growth promoting actions, may play a role in this effect. Results We show that HGH activates STAT5 signaling and promotes corneal epithelial cell migration in vitro. The migratory effect requires an intact communication between corneal epithelia and fibroblasts, and is not mediated by IGF-1. Conclusion HGH may represent a topical therapeutic to promote corneal epithelial wound healing. This warrants further investigation. PMID:25782399

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

    PubMed

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

    2015-07-15

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

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  9. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

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

  10. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

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

    PubMed

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

    2013-06-27

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

  12. Cultured epithelial cells response to phototherapy with low intensity laser.

    PubMed

    Eduardo, Fernanda P; Mehnert, Dolores U; Monezi, Telma A; Zezell, Denise M; Schubert, Mark M; Eduardo, Carlos P; Marques, Márcia M

    2007-04-01

    Little is known about the intracellular response of epithelial cells to phototherapy. The aim of this in vitro study was to analyze the effect of phototherapy with low-energy lasers with different wavelengths and powers on cultured epithelial cell growth under different nutritional conditions. Epithelial cell cultures (Vero cell line) grown in nutritional deficit in culture medium supplemented with 2% fetal bovine serum (FBS) were irradiated with low-energy laser from one to three times with a GaAlAs laser (660 nm) and InGaAlP (780 nm), 40 and 70 mW, respectively, with 3 or 5 J/cm2. Cell growth was indirectly assessed by measuring the cell mitochondrial activity. Nonirradiated cell cultures grown in nutritional regular medium supplemented with 10% FBS produced higher cell growth than all cultures grown in nutritional deficit irradiated or not. The overall cell growth of cultures grown under nutritionally deficit conditions was significantly improved especially when irradiated with 780 nm for three times. Phototherapy with the laser parameters tested increases epithelial cell growth rate for cells stressed by growth under nutritionally deficient states. This cell growth improvement is directly proportional to the number of irradiations; however, was not enough to reach the full cell growth potential rate of Vero epithelial cell line observed when growing under nutritional regular condition. (c) 2007 Wiley-Liss, Inc.

  13. Signaling pathway for phagocyte priming upon encounter with apoptotic cells.

    PubMed

    Nonaka, Saori; Ando, Yuki; Kanetani, Takuto; Hoshi, Chiharu; Nakai, Yuji; Nainu, Firzan; Nagaosa, Kaz; Shiratsuchi, Akiko; Nakanishi, Yoshinobu

    2017-05-12

    The phagocytic elimination of cells undergoing apoptosis is an evolutionarily conserved innate immune mechanism for eliminating unnecessary cells. Previous studies showed an increase in the level of engulfment receptors in phagocytes after the phagocytosis of apoptotic cells, which leads to the enhancement of their phagocytic activity. However, precise mechanisms underlying this phenomenon require further clarification. We found that the pre-incubation of a Drosophila phagocyte cell line with the fragments of apoptotic cells enhanced the subsequent phagocytosis of apoptotic cells, accompanied by an augmented expression of the engulfment receptors Draper and integrin αPS3. The DNA-binding activity of the transcription repressor Tailless was transiently raised in those phagocytes, depending on two partially overlapping signal-transduction pathways for the induction of phagocytosis as well as the occurrence of engulfment. The RNAi knockdown of tailless in phagocytes abrogated the enhancement of both phagocytosis and engulfment receptor expression. Furthermore, the hemocyte-specific RNAi of tailless reduced apoptotic cell clearance in Drosophila embryos. Taken together, we propose the following mechanism for the activation of Drosophila phagocytes after an encounter with apoptotic cells: two partially overlapping signal-transduction pathways for phagocytosis are initiated; transcription repressor Tailless is activated; expression of engulfment receptors is stimulated; and phagocytic activity is enhanced. This phenomenon most likely ensures the phagocytic elimination of apoptotic cells by stimulated phagocytes and is thus considered as a mechanism to prime phagocytes in innate immunity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

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

    2014-01-01

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

  15. HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

    PubMed Central

    Tugizov, Sharof M.; Herrera, Rossana; Veluppillai, Piri; Greenspan, Deborah; Soros, Vanessa; Greene, Warner C.; Levy, Jay A.; Palefsky, Joel M.

    2010-01-01

    Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission. PMID:21056450

  16. Inflammatory cytokines prime adipose tissue mesenchymal stem cells to enhance malignancy of MCF-7 breast cancer cells via transforming growth factor-β1.

    PubMed

    Trivanović, Drenka; Jauković, Aleksandra; Krstić, Jelena; Nikolić, Srdjan; Okić Djordjević, Ivana; Kukolj, Tamara; Obradović, Hristina; Mojsilović, Slavko; Ilić, Vesna; Santibanez, Juan Francisco; Bugarski, Diana

    2016-03-01

    Mesenchymal stem cells from human adipose tissue (hASCs) are proposed as suitable tools for soft tissue engineering and reconstruction. Although it is known that hASCs have the ability to home to sites of inflammation and tumor niche, the role of inflammatory cytokines in the hASCs-affected tumor development is not understood. We found that interferon-γ (IFN-γ) and/or tumor necrosis factor-α (TNF-α) prime hASCs to produce soluble factors which enhance MCF-7 cell line malignancy in vitro. IFN-γ and/or TNF-α-primed hASCs produced conditioned media (CM) which induced epithelial to mesenchymal transition (EMT) of MCF-7 cells by reducing E-Cadherin and increasing Vimentin expression. Induced EMT was accompanied by increased invasion, migration, and urokinase type-plasminogen activator (uPA) expression in MCF-7 cells. These effects were mediated by increased expression of transforming growth factor-β1(TGF-β1) in cytokines-primed hASCs, since inhibition of type I TGF-β1 receptor on MCF-7 cells and neutralization of TGF-β1 disabled the CM from primed hASCs to increase EMT, cell migration, and uPA expression in MCF-7 cells. Obtained data suggested that IFN-γ and/or TNF-α primed hASCs might enhance the malignancy of MCF-7 cell line by inducing EMT, cell motility and uPA expression in these cells via TGF-β1-Smad3 signalization, with potentially important implications in breast cancer progression.

  17. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  18. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

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

    SciTech Connect

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

    2009-07-01

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

  20. Culture, Immortalization, and Characterization of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Liu, Shaohui; Hatton, Mark P.; Khandelwal, Payal

    2010-01-01

    Purpose. Meibomian gland epithelial cells are essential in maintaining the health and integrity of the ocular surface. However, very little is known about their physiological regulation. In this study, the cellular control mechanisms were explored, first to establish a defined culture system for the maintenance of primary epithelial cells from human meibomian glands and, second, to immortalize these cells, thereby developing a preclinical model that could be used to identify factors that regulate cell activity. Methods. Human meibomian glands were removed from lid segments after surgery, enzymatically digested, and dissociated. Isolated epithelial cells were cultured in media with or without serum and/or 3T3 feeder layers. To attempt immortalization, the cells were exposed to retroviral human telomerase reverse transcriptase (hTERT) and/or SV40 large T antigen cDNA vectors, and antibiotic-resistant cells were selected, expanded, and subcultured. Analyses for possible biomarkers, cell proliferation and differentiation, lipid-related enzyme gene expression, and the cellular response to androgen were performed with biochemical, histologic, and molecular biological techniques. Results. It was possible to isolate viable human meibomian gland epithelial cells and to culture them in serum-free medium. These cells proliferated, survived through at least the fifth passage, and contained neutral lipids. Infection with hTERT immortalized these cells, which accumulated neutral lipids during differentiation, expressed multiple genes for lipogenic enzymes, responded to androgen, and continued to proliferate. Conclusions. The results show that human meibomian gland epithelial cells may be isolated, cultured, and immortalized. PMID:20335607

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

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

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

  2. Cell volume regulation in epithelial physiology and cancer

    PubMed Central

    Pedersen, Stine F.; Hoffmann, Else K.; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed. PMID:24009588

  3. Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming.

    PubMed Central

    Seder, R A; Gazzinelli, R; Sher, A; Paul, W E

    1993-01-01

    Naive CD4+ T cells produce interleukin 2 (IL-2) but little IL-4 or interferon gamma (IFN-gamma). In vitro, they develop into IL-4 or IFN-gamma producers depending on the conditions of the priming culture. Using T-cell receptor transgenic CD4+ T cells, the role of IL-12 and IL-4 in antigen-specific priming was examined. IL-12 substantially enhanced the ability of naive CD4+ T cells to develop into cells that produced IFN-gamma upon restimulation. However, it was not essential since anti-IL-12 antibodies failed to block the priming for IFN-gamma observed in the absence of exogenous IL-12. When both IL-12 and IL-4 were present in the priming culture, IL-12 did not inhibit priming for IL-4 production. In contrast, IL-4 diminished but did not abolish priming for IFN-gamma production. In an accessory cell-independent priming system, IL-12 strikingly augmented priming for IFN-gamma production, indicating that it acts directly on T cells. IFN-gamma itself did not enhance priming for IFN-gamma production in either accessory cell-dependent or independent systems. In an accessory cell-dependent system, the IL-12-mediated enhancement was not blocked by adding neutralizing anti-IFN-gamma monoclonal antibody. However, in an accessory cell-independent system, anti-IFN-gamma antibody did inhibit priming for IFN-gamma production leaving open a role for IFN-gamma in the priming process. These data indicate that IL-12 has a major effect on the inductive phase of T-cell priming by enhancing commitment to IFN-gamma production and thus can profoundly influence the state of immunity that develops. Images Fig. 4 PMID:7901851

  4. Airway epithelial cell wound repair mediated by alpha-dystroglycan.

    PubMed

    White, S R; Wojcik, K R; Gruenert, D; Sun, S; Dorscheid, D R

    2001-02-01

    Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

  5. Ion pump sorting in polarized renal epithelial cells.

    PubMed

    Caplan, M J

    2001-08-01

    The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

  6. Secretion of alpha 1-antitrypsin by alveolar epithelial cells.

    PubMed

    Venembre, P; Boutten, A; Seta, N; Dehoux, M S; Crestani, B; Aubier, M; Durand, G

    1994-06-13

    We have investigated the ability of alveolar epithelial cells (human A549 cell line and rat type-II pneumocytes) to produce alpha 1-antitrypsin (AAT). Northern blot analysis demonstrated the presence of an AAT-specific mRNA transcript in A549 cells. Unstimulated A549 cells secreted immunoreactive AAT at a rate of 0.51 +/- 0.04 ng/10(6) cells/h, with a modified glycosylation compared to serum AAT. AAT formed a complex with neutrophil elastase. Rat type-II pneumocytes secreted immunoreactive AAT. Our results suggest that alveolar epithelial cells could participate in antiprotease defense within the lung through local AAT production.

  7. Antigen persistence is required for dendritic cell licensing and CD8+ T cell cross-priming.

    PubMed

    Jusforgues-Saklani, Hélène; Uhl, Martin; Blachère, Nathalie; Lemaître, Fabrice; Lantz, Olivier; Bousso, Philippe; Braun, Deborah; Moon, James J; Albert, Matthew L

    2008-09-01

    It has been demonstrated that CD4(+) T cells require Ag persistence to achieve effective priming, whereas CD8(+) T cells are on "autopilot" after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8(+) T cells require CD4(+) T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8(+) T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8(+) T cell activation is not due to a problem with cross-presentation. In fact, by providing "extra help" in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8(+) T cells. Our data suggest that the "licensing" of cross-presenting DCs does not occur during their initial encounter with CD4(+) T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8(+) T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8(+) T cell response is helper-dependent.

  8. Peptidases released by necrotic cells control CD8+ T cell cross-priming

    PubMed Central

    Gamrekelashvili, Jaba; Kapanadze, Tamar; Han, Miaojun; Wissing, Josef; Ma, Chi; Jaensch, Lothar; Manns, Michael P.; Armstrong, Todd; Jaffee, Elizabeth; White, Ayla O.; Citrin, Deborah E.; Korangy, Firouzeh; Greten, Tim F.

    2013-01-01

    Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells. PMID:24216478

  9. Metformin inhibits the proliferation of benign prostatic epithelial cells

    PubMed Central

    Ge, Rongbin; Li, Jijun; Johnson, Cameron W.; Rassoulian, Cyrus; Olumi, Aria F.

    2017-01-01

    Objective Benign prostatic hyperplasia (BPH) is the most common proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies have shown that diabetes significantly increases the risk of developing BPH, although whether anti-diabetic medications preventing the development of BPH remains to be defined. We have previously found that stromally expressed insulin-like growth factor 1 (IGF-1) promotes benign prostatic epithelial cell proliferation through paracrine mechanisms. Here, we seek to understand if metformin, a first line medication for the treatment of type 2 diabetes, inhibits the proliferation of benign prostatic epithelial cells through reducing the expression of IGF-1 receptor (IGF-1R) and regulating cell cycle. Methods BPE cell lines BPH-1 and P69, murine fibroblasts3T3 and primary human prostatic fibroblasts were cultured and tested in this study. Cell proliferation and the cell cycle were analyzed by MTS assay and flow cytometry, respectively. The expression of IGF-1R was determined by western-blot and immunocytochemistry. The level of IGF-1 secretion in culture medium was measured by ELISA. Results Metformin (0.5-10mM, 6-48h) significantly inhibited the proliferation of BPH-1 and P69 cells in a dose-dependent and time-dependent manner. Treatment with metformin for 24 hours lowered the G2/M cell population by 43.24% in P69 cells and 24.22% in BPH-1 cells. On the other hand, IGF-1 (100ng/mL, 24h) stimulated the cell proliferation (increased by 28.81% in P69 cells and 20.95% in BPH-1 cells) and significantly enhanced the expression of IGF-1R in benign prostatic epithelial cells. Metformin (5mM) abrogated the proliferation of benign prostatic epithelial cells induced by IGF-1. In 3T3 cells, the secretion of IGF-1 was significantly inhibited by metformin from 574.31pg/ml to 197.61pg/ml. The conditioned media of 3T3 cells and human prostatic fibroblasts promoted the proliferation of epithelial cells and the

  10. Evidence for the multistep nature of in vitro human epithelial cell carcinogenesis

    SciTech Connect

    Rhim, J.S.; Yoo, J.H.; Park, J.H.; Thraves, P.; Salehi, Z.; Dritschilo, A. )

    1990-09-01

    In keeping with the multistep development of human cancer in vivo, a stepwise approach to neoplastic transformation in vitro presents a reasonable strategy. We have recently developed an in vitro multistep model suitable for the study of human epithelial cell carcinogenesis. Upon infection with the adenovirus 12-simian virus 40 hybrid virus, primary human epidermal keratinocytes acquired an indefinite life span in culture but did not undergo malignant conversion. Subsequent addition of Kirsten murine sarcoma virus and human ras oncogene or chemical carcinogens (N-methyl-N{prime}-nitro-N-nitrosoguanidine or 4-nitroquinoline 1-oxide) to these cells induced morphological alterations and the acquisition of neoplastic properties. Subsequently it was found that this line could be transformed neoplastically by a variety of retrovirus-containing H-ras, bas, fes, fms, erbB, and src oncogenes. In addition, we found that the immortalized human epidermal keratinocyte (RHEK-1) line can be transformed neoplastically by exposure to ionizing radiation. Thus, this in vitro system may be useful in studying the interaction of a variety of carcinogenic agents and human epithelial cells. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of viruses, oncogenes, chemical carcinogens, or X-ray irradiation and support a multistep process for neoplastic conversion.

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

    SciTech Connect

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

    1997-10-13

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

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

    SciTech Connect

    Boice, M.L.

    1988-01-01

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

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

    SciTech Connect

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

    1991-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  15. Differentiation of Club Cells to Alveolar Epithelial Cells In Vitro

    PubMed Central

    Zheng, Dahai; Soh, Boon-Seng; Yin, Lu; Hu, Guangan; Chen, Qingfeng; Choi, Hyungwon; Han, Jongyoon; Chow, Vincent T. K.; Chen, Jianzhu

    2017-01-01

    Club cells are known to function as regional progenitor cells to repair the bronchiolar epithelium in response to lung damage. By lineage tracing in mice, we have shown recently that club cells also give rise to alveolar type 2 cells (AT2s) and alveolar type 1 cells (AT1s) during the repair of the damaged alveolar epithelium. Here, we show that when highly purified, anatomically and phenotypically confirmed club cells are seeded in 3-dimensional culture either in bulk or individually, they proliferate and differentiate into both AT2- and AT1-like cells and form alveolar-like structures. This differentiation was further confirmed by transcriptomic analysis of freshly isolated club cells and their cultured progeny. Freshly isolated club cells express Sca-1 and integrin α6, markers commonly used to characterize lung stem/progenitor cells. Together, current study for the first time isolated highly purified club cells for in vitro study and demonstrated club cells’ capacity to differentiate into alveolar epithelial cells at the single-cell level. PMID:28128362

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

    PubMed

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

    2010-12-01

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

  17. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition

    PubMed Central

    Hong, Deli; Messier, Terri L.; Tye, Coralee E.; Dobson, Jason R.; Fritz, Andrew J.; Sikora, Kenneth R.; Browne, Gillian; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

    2017-01-01

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression. PMID:28407681

  18. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition.

    PubMed

    Hong, Deli; Messier, Terri L; Tye, Coralee E; Dobson, Jason R; Fritz, Andrew J; Sikora, Kenneth R; Browne, Gillian; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2017-03-14

    Runx1 is a well characterized transcription factor essential for hematopoietic differentiation and Runx1 mutations are the cause of leukemias. Runx1 is highly expressed in normal epithelium of most glands and recently has been associated with solid tumors. Notably, the function of Runx1 in the mammary gland and how it is involved in initiation and progression of breast cancer is still unclear. Here we demonstrate the consequences of Runx1 loss in normal mammary epithelial and breast cancer cells. We first observed that Runx1 is decreased in tumorigenic and metastatic breast cancer cells. We also observed loss of Runx1 expression upon induction of epithelial-mesenchymal transition (EMT) in MCF10A (normal-like) cells. Furthermore depletion of Runx1 in MCF10A cells resulted in striking changes in cell shape, leading to mesenchymal cell morphology. The epithelial phenotype could be restored in breast cancer cells by re-expressing Runx1. Analyses of breast tumors and patient data revealed that low Runx1 expression is associated with poor prognosis and decreased survival. We addressed mechanisms for the function of Runx1 in maintaining the epithelial phenotype and find Runx1 directly regulates E-cadherin; and serves as a downstream transcription factor mediating TGFβ signaling. We also observed through global gene expression profiling of growth factor depleted cells that induction of EMT and loss of Runx1 is associated with activation of TGFβ and WNT pathways. Thus these findings have identified a novel function for Runx1 in sustaining normal epithelial morphology and preventing EMT and suggest Runx1 levels could be a prognostic indicator of tumor progression.

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

    PubMed Central

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

    2015-01-01

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

  20. Transcytotic passage of albumin through lens epithelial cells.

    PubMed

    Sabah, Judith R; Schultz, Bruce D; Brown, Zach W; Nguyen, Annelise T; Reddan, John; Takemoto, Larry J

    2007-03-01

    To characterize the transcytotic passage of albumin through lens epithelial cells. N/N 1003A rabbit lens epithelial cells were grown to a confluent monolayer on porous filter supports (Transwell Corning, Inc., Corning, NY). Monolayers were exposed apically to Alexa 488-labeled albumin (Alexa 488-BSA) in the absence and presence of endocytic inhibitors (filipin; dansylcadaverine [DCV]). Transcytotic passage of albumin was monitored for 4 hours by quantitating fluorescence in the basolateral compartment. The mechanism of albumin passage was studied by labeling cell monolayers and cryosections of whole rat lenses for clathrin or caveolin. The monolayer of cells formed a barrier to the passage of albumin, as shown by the 44% reduction in albumin passage in comparison to nonseeded membranes. Treatment with filipin or DCV reduced the passage of Alexa 488-BSA through lens epithelial cells by 73% and 66%, respectively. Confocal microscopy showed that albumin passage was predominantly transcellular and demonstrated colocalization of albumin with caveolin-1 and clathrin in lens epithelial and fiber cells. The Transwell apparatus is an excellent system to monitor transport systems across cell monolayers. In this study, rabbit lens epithelial cells formed a confluent monolayer that acted as a barrier to the passive diffusion of albumin. The kinetics of albumin movement across the monolayer and the inhibitor pharmacology suggests that lens cells actively transport albumin from the apical to the basolateral compartment. The inhibitory profile suggests the involvement of caveolae and clathrin-coated vesicles in the transcytotic process.

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

    PubMed

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

    2014-04-01

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

  2. Anatomical location and culture of equine corneal epithelial stem cells.

    PubMed

    Moriyama, Hidekazu; Kasashima, Yoshinori; Kuwano, Atsutoshi; Wada, Shinya

    2014-03-01

    To identify morphologically the locations of equine corneal epithelial stem cells (CESCs) and to culture these cells. We studied the eyes of 12 adult thoroughbred horses. Eye tissues were immunostained for two positive stem cell markers (p63, CK14) and one negative marker (CK3) to identify the locations of CESCs, so we could compare their immunostaining patterns with those of human stem cells previously reported. We compared the proliferation rates and morphological features of epithelial cells isolated from the corneal limbus and central cornea. Undifferentiated cells expressing the same immunostaining pattern as human CESCs were present in the equine corneal limbus. Cultured epithelial cells isolated from the limbus expressed the same immunostaining pattern that CESCs show histologically, but cells isolated from the central cornea did not proliferate and could not be evaluated. Equine CESCs were localized in the epithelial basal layer of the corneal limbus, where melanocytes reside. They could be cultured without loss of their undifferentiated nature. When collecting such stem cells, it may be useful to harvest and culture corneal epithelial tissues in the limbus where melanocytes serve as an indicator of the collecting area. © 2013 American College of Veterinary Ophthalmologists.

  3. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

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

    2012-09-14

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

  4. Epithelial to Mesenchymal Transition of Mesothelial Cells in Tuberculous Pleurisy

    PubMed Central

    Kim, Changhwan; Park, Sung-Hoon; Hwang, Yong Il; Jang, Seung Hun; Kim, Cheol Hong; Jung, Ki-Suck; Min, Kwangseon; Lee, Jae Woong; Jang, Young Sook

    2011-01-01

    Purpose Tuberculous pleurisy is the most frequent extrapulmonary manifestation of tuberculosis. In spite of adequate treatment, pleural fibrosis is a common complication, but the mechanism has not been elucidated. This study is to determine whether epithelial to mesenchymal transition (EMT) of mesothelial cells occurs in tuberculous pleurisy. Materials and Methods Normal pleural mesothelial cells, isolated from irrigation fluids during operations for primary spontaneous pneumothorax, were characterized by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). These cells were treated in vitro with various cytokines, which were produced in the effluents of tuberculous pleurisy. The isolated cells from the effluents of tuberculous pleurisy were analyzed by immunofluorescence and RT-PCR analysis. Results The isolated cells from the irrigation fluid of primary spontaneous pneumothorax had epithelial characteristics. These cells, with transforming growth factor-β1 and/or interleukin-1β treatment, underwent phenotypic transition from epithelial to mesenchymal cells, with the loss of epithelial morphology and reduction in cytokeratin and E-cadherin expression. Effluent analysis from tuberculous pleurisy using immunofluorescence and RT-PCR demonstrated two phenotypes that showed mesenchymal characteristics and both epithelial & mesencymal characteristics. Conclusion Our results suggest that pleural mesothelial cells in tuberculous pleurisy have been implicated in pleural fibrosis through EMT. PMID:21155035

  5. Epithelial cell guidance by self-generated EGF gradients†

    PubMed Central

    Scherber, Cally; Aranyosi, Alexander J.; Kulemann, Birte; Thayer, Sarah P.; Toner, Mehmet; Iliopoulos, Othon

    2012-01-01

    Cancer epithelial cells often migrate away from the primary tumor to invade into the surrounding tissues. Their migration is commonly assumed to be directed by pre-existent spatial gradients of chemokines and growth factors in the target tissues. Unexpectedly however, we found that the guided migration of epithelial cells is possible in vitro in the absence of pre-existent chemical gradients. We observed that both normal and cancer epithelial cells can migrate persistently and reach the exit along the shortest path from microscopic mazes filled with uniform concentrations of media. Using microscale engineering techniques and biophysical models, we uncovered a self-guidance strategy during which epithelial cells generate their own guiding cues under conditions of biochemical confinement. The self-guidance strategy depends on the balance between three interdependent processes: epidermal growth factor (EGF) uptake by the cells (U), the restricted transport of EGF through the structured microenvironment (T), and cell chemotaxis toward the resultant EGF gradients (C). The UTC self-guidance strategy can be perturbed by inhibition of signalling through EGF-receptors and appears to be independent from chemokine signalling. Better understanding of the UTC self-guidance strategy could eventually help devise new ways for modulating epithelial cell migration and delaying cancer cell invasion or accelerating wound healing. PMID:22314635

  6. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  7. Starved epithelial cells uptake extracellular matrix for survival

    PubMed Central

    Muranen, Taru; Iwanicki, Marcin P.; Curry, Natasha L.; Hwang, Julie; DuBois, Cory D.; Coloff, Jonathan L.; Hitchcock, Daniel S.; Clish, Clary B.; Brugge, Joan S.; Kalaany, Nada Y.

    2017-01-01

    Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition. PMID:28071763

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  10. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

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

    1990-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1994-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets.

  14. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

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

  15. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

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

    SciTech Connect

    Stampfer, M.R.

    1982-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  18. Epithelial cell division in the Xenopus laevis embryo during gastrulation.

    PubMed

    Hatte, Guillaume; Tramier, Marc; Prigent, Claude; Tassan, Jean-Pierre

    2014-01-01

    How vertebrate epithelial cells divide in vivo and how the cellular environment influences cell division is currently poorly understood. A sine qua non condition to study cell division in situ is the ease of observation of cell division. This is fulfilled in the Xenopus embryo at the gastrula stage where polarized epithelial cells divide with a high frequency at the surface of the organism. Recently, using this model system, we have shown that epithelial cells divide by asymmetric furrowing and that the mode of cell division is regulated during development. Here, we further characterize epithelial cell division in situ. To this end, we used confocal microscopy to study epithelial cell division in the ectoderm of the Xenopus laevis gastrula. Cell division was followed either by indirect immunofluorescence in fixed embryos or by live imaging of embryos transiently expressing diverse fluorescent proteins. Here, we show that during cytokinesis, the plasma membranes of the two daughter cells are usually separated by a gap. For most divisions, daughter cells make contacts basally at a distance from the furrow tip which creates an inverted teardrop-like shaped volume tightly associated with the furrow. At the end of cytokinesis, the inverted teardrop is resorbed; thus it is a transient structure. Several proteins involved in cytokinesis are localized at the tip of the inverted teardrop suggesting that the formation of the gap could be an active process. We also show that intercalation of neighboring cells between daughter cells occasionally occurs during cytokinesis. Our results reveal an additional level of complexity in the relationship between dividing cells and also with their neighboring cells during cytokinesis in the Xenopus embryo epithelium.

  19. Epithelial-mesenchymal transitions of bile duct epithelial cells in primary hepatolithiasis.

    PubMed

    Zhao, Lijin; Yang, Rigao; Cheng, Long; Wang, Maijian; Jiang, Yan; Wang, Shuguang

    2010-07-01

    The purpose of this study was to explore the role of epithelial-mesenchymal transition in the pathogenesis of hepatolithiasis. Thirty-one patients with primary hepatolithiasis were enrolled in this study. Expressions of E-cadherin, alpha-catenin, alpha-SMA, vimentin, S100A4, TGF-beta1 and P-smad2/3 in hepatolithiasis bile duct epithelial cells were examined by immunohistochemistry staining. The results showed that the expressions of the epithelial markers E-cadherin and alpha-catenin were frequently lost in hepatolithiasis (32.3% and 25.9% of cases, respectively), while the mesenchymal markers vimentin, alpha-SMA and S100A4 were found to be present in hepatolithiasis (35.5%, 29.0%, and 32.3% of cases, respectively). The increased mesenchymal marker expression was correlated with decreased epithelial marker expression. The expressions of TGF-beta1 and P-smad2/3 in hepatolithiasis were correlated with the expression of S100A4. These data indicate that TGF-beta1-mediated epithelial-mesenchymal transition might be involved in the formation of hepatolithiasis.

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

    SciTech Connect

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

    2008-06-26

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

  1. Fibrin glue inhibits migration of ocular surface epithelial cells.

    PubMed

    Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

    2016-10-01

    PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.

  2. Serratia marcescens internalization and replication in human bladder epithelial cells

    PubMed Central

    Hertle, Ralf; Schwarz, Heinz

    2004-01-01

    Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

  3. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    PubMed

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-03-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices.

  4. Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

    PubMed

    Allahverdian, Sima; Harada, Norihiro; Singhera, Gurpreet K; Knight, Darryl A; Dorscheid, Delbert R

    2008-02-01

    Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.

  5. Biphenotypic surface epithelial cells in the gastrointestinal tube with mixed epithelial-myofibroblastic differentiation: a paradigm.

    PubMed

    Németh, István Balázs; Tiszlavicz, László

    2012-04-01

    Epithelial cells and myofibroblasts are well-characterized histomorphological elements of tissues. They are distinguished from one another on the basis of topography and of differences in cytokeratin (CK) and α-smooth muscle actin (SMA) expression. Certain epithelial cells exhibit CK / SMA co-expression. This study aimed to define the immunophenotypical characteristics of these biphenotypic cells with respect to cytodifferentiation (broad spectrum of CKs, SMA), cell-cell interaction (E-cadherin, adenomatous polyposis coli - APC, β-catenin), and cell survival (cyclooxygenase-2 - Cox-2). At the routine gastrointestinal pathology service of the Department of Pathology, University of Szeged, tissue samples were identified from instances of cervical inlet patch (n = 5), Barrett's esophagus (n=5), gastritis (n=5), fundic gland polyp (n=2), gastric neoplastic polyp (n=1), inflammatory bowel disease (n=5), and colonic neoplastic polyp (n=3). that contained epithelial cells expressing SMA. These biphenotypic cells were further immunophenotyped. Foregut-derived biphenotypic cells expressed CKs 7 and 20, while hindgut-derived biphenotypic cells expressed only CK 20. Subepithelial myofibroblasts adjacent to biphenotypic epithelium expressed Cox-2, SMA, and β-catenin, as did biphenotypic cells. Myofibroblasts, however, did not express CKs. In neoplastic polyps, APC expression weakened as cytologic atypism increased, while intermingled biphenotypic cells in neoplastic glands overexpressed APC, as did myofibroblasts beneath. CK subspecies expression in biphenotypic cells reflects embryonic development of the gastrointestinal tract. The immunophenotyping analysis addresses bidirectional (via transdifferentiation from epithelia into myofibroblasts or vice versa) formation of biphenotypic cells within damaged epithelium, a phenomenon that must be further analysed.

  6. Cell-to-cell transfer of M. tuberculosis antigens optimizes CD4 T cell priming.

    PubMed

    Srivastava, Smita; Ernst, Joel D

    2014-06-11

    During Mycobacterium tuberculosis and other respiratory infections, optimal T cell activation requires pathogen transport from the lung to a local draining lymph node (LN). However, the infected inflammatory monocyte-derived dendritic cells (DCs) that transport M. tuberculosis to the local lymph node are relatively inefficient at activating CD4 T cells, possibly due to bacterial inhibition of antigen presentation. We found that infected migratory DCs release M. tuberculosis antigens as soluble, unprocessed proteins for uptake and presentation by uninfected resident lymph node DCs. This transfer of bacterial proteins from migratory to local DCs results in optimal priming of antigen-specific CD4 T cells, which are essential in controlling tuberculosis. Additionally, this mechanism does not involve transfer of the whole bacterium and is distinct from apoptosis or exosome shedding. These findings reveal a mechanism that bypasses pathogen inhibition of antigen presentation by infected cells and generates CD4 T cell responses that control the infection.

  7. Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

    PubMed

    Hosoki, Koa; Kainuma, Keigo; Toda, Masaaki; Harada, Etsuko; Chelakkot-Govindalayathila, Ayshwarya-Lakshmi; Roeen, Ziaurahman; Nagao, Mizuho; D'Alessandro-Gabazza, Corina N; Fujisawa, Takao; Gabazza, Esteban C

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

  8. Epithelial cell apoptosis facilitates Entamoeba histolytica infection in the gut.

    PubMed

    Becker, Stephen M; Cho, Kyou-Nam; Guo, Xiaoti; Fendig, Kirsten; Oosman, Mohammed N; Whitehead, Robert; Cohn, Steven M; Houpt, Eric R

    2010-03-01

    Entamoeba histolytica is the protozoan parasite that causes amebic colitis. The parasite triggers apoptosis on contact with host cells; however, the biological significance of this event during intestinal infection is unclear. We examined the role of apoptosis in a mouse model of intestinal amebiasis. Histopathology revealed that abundant epithelial cell apoptosis occurred in the vicinity of amoeba in histological specimens. Epithelial cell apoptosis occurred rapidly on co-culture with amoeba in vitro as measured by annexin positivity, DNA degradation, and mitochondrial dysfunction. Administration of the pan caspase inhibitor ZVAD decreased the rate and severity of amebic infection in CBA mice by all measures (cecal culture positivity, parasite enzyme-linked immunosorbent assay, and histological scores). Similarly, caspase 3 knockout mice on the resistant C57BL/6 background exhibited even lower cecal parasite antigen burden and culture positive rates than wild type mice. The permissive effect of apoptosis on infection could be tracked to the epithelium, in that transgenic mice that overexpressed Bcl-2 in epithelial cells were more resistant to infection as measured by cecal parasite enzyme-linked immunosorbent assay and histological scores. We concluded that epithelial cell apoptosis in the intestine facilitates amebic infection in this mouse model. The parasite's strategy for inducing apoptosis may point to key virulence factors, and therapeutic maneuvers to diminish epithelial apoptosis may be useful in amebic colitis.

  9. Priming of the Cells: Hypoxic Preconditioning for Stem Cell Therapy.

    PubMed

    Wei, Zheng Z; Zhu, Yan-Bing; Zhang, James Y; McCrary, Myles R; Wang, Song; Zhang, Yong-Bo; Yu, Shan-Ping; Wei, Ling

    2017-10-05

    Stem cell-based therapies are promising in regenerative medicine for protecting and repairing damaged brain tissues after injury or in the context of chronic diseases. Hypoxia can induce physiological and pathological responses. A hypoxic insult might act as a double-edged sword, it induces cell death and brain damage, but on the other hand, sublethal hypoxia can trigger an adaptation response called hypoxic preconditioning or hypoxic tolerance that is of immense importance for the survival of cells and tissues. This review was based on articles published in PubMed databases up to August 16, 2017, with the following keywords: "stem cells," "hypoxic preconditioning," "ischemic preconditioning," and "cell transplantation." Original articles and critical reviews on the topics were selected. Hypoxic preconditioning has been investigated as a primary endogenous protective mechanism and possible treatment against ischemic injuries. Many cellular and molecular mechanisms underlying the protective effects of hypoxic preconditioning have been identified. In cell transplantation therapy, hypoxic pretreatment of stem cells and neural progenitors markedly increases the survival and regenerative capabilities of these cells in the host environment, leading to enhanced therapeutic effects in various disease models. Regenerative treatments can mobilize endogenous stem cells for neurogenesis and angiogenesis in the adult brain. Furthermore, transplantation of stem cells/neural progenitors achieves therapeutic benefits via cell replacement and/or increased trophic support. Combinatorial approaches of cell-based therapy with additional strategies such as neuroprotective protocols, anti-inflammatory treatment, and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the recent progress regarding cell types and applications in regenerative medicine as well as future applications.

  10. Immunolocalization of epithelial and mesenchymal matrix constituents in association with inner enamel epithelial cells.

    PubMed

    Bosshardt, D D; Nanci, A

    1998-02-01

    After crown formation, the enamel organ reorganizes into Hertwig's epithelial root sheath (HERS). Although it is generally accepted that HERS plays an inductive role during root formation, it also has been suggested that it may contribute enamel-related proteins to cementum matrix. By analogy to the enamel-free area (EFA) in rat molars, in which epithelial cells express not only enamel proteins but also "typical" mesenchymal matrix constituents, it has been proposed that HERS cells may also have the potential to produce cementum proteins. To test this hypothesis, we examined the nature of the first matrix layer deposited along the cervical portion of root dentin and the characteristics of the associated cells. Rat molars were processed for postembedding colloidal gold immunolabeling with antibodies to amelogenin (AMEL), ameloblastin (AMBN), bone sialoprotein (BSP), and osteopontin (OPN). To minimize the possibility of false-negative results, several antibodies to AMEL were used. The labelings were compared with those obtained at the EFA. Initial cementum matrix was consistently observed at a time when epithelial cells from HERS covered most of the forming root surface. Cells with mesenchymal characteristics were rarely seen in proximity to the matrix. Both the EFA matrix and initial cementum exhibited collagen fibrils and were intensely immunoreactive for BSP and OPN. AMEL and AMBN were immunodetected at the EFA but not over the initial cementum proper. These two proteins were, however, present at the cervical-most portion of the root where enamel matrix extends for a short distance between dentin and cementum. These data suggest that epithelial cells along the root surface are likely responsible for the deposition of the initial cementum matrix and therefore, like the cells at the EFA, may be capable of producing mesenchymal proteins.

  11. miR-214 regulates lactoferrin expression and pro-apoptotic function in mammary epithelial cells.

    PubMed

    Liao, Yalin; Du, Xiaogu; Lönnerdal, Bo

    2010-09-01

    Lactoferrin (Lf) is an abundantly expressed protein in human milk. Lactoferrin exhibits several important biological functions, and its expression is regulated by multiple environmental factors. Cellular endogenous factors, however, have not been extensively studied with regard to lactoferrin gene expression. In this study, we showed that lactoferrin gene expression and function are directly targeted by miR-214 in HC11 and MCF7 cells. In the lactoferrin mRNA 3 prime untranslated region (UTR) of human, mouse, rat, pig, bovine, camel, and goat species, there is a conserved region that perfectly matches the seed region of miR-214. Transfection of miR-214 mimic in HEK293 cells dose-dependently inhibited the activity of pGL3-control vector containing lactoferrin mRNA 3 prime UTR downstream of the luciferase gene. In HC11 cells, miR-214 overexpression inhibited the induction of lactoferrin expression by beta -estradiol (E2) and dexamethasone-prolactin-insulin (DPI). Furthermore, in MCF7 cells, overexpression of miR-214 markedly decreased lactoferrin expression (P lt 0.05), and inhibition of endogenous miR-214 expression increased lactoferrin expression and cellular apoptotic activities (P lt 0.05). In summary, our data showed that miR-214 is directly involved in lactoferrin expression and lactoferrin mediated cancer susceptibility (proapoptotic activities) in mammary epithelial cells.

  12. Guadecitabine (SGI-110) priming sensitizes hepatocellular carcinoma cells to oxaliplatin.

    PubMed

    Kuang, Yuting; El-Khoueiry, Anthony; Taverna, Pietro; Ljungman, Mats; Neamati, Nouri

    2015-11-01

    Promoter DNA hypermethylation is an important biomarker of hepatocellular carcinoma (HCC), supporting the potential utility of demethylating agents in this disease. Guadecitabine (SGI-110) is a second-generation hypomethylating agent formulated as a dinucleotide of decitabine and deoxyguanosine that yields longer half-life and more extended decitabine exposure than decitabine IV infusion. Here we performed preclinical evaluation of SGI-110 in HCC models to guide the design of a phase I/II clinical trial. HCC cell lines and xenograft models were used to determine the antitumor activity of SGI-110 as a single agent and in combination with oxaliplatin. Pretreatment with low doses of SGI-110 significantly synergized with oxaliplatin yielding enhanced cytotoxicity. The combination of SGI-110 and oxaliplatin was well tolerated and significantly delayed tumor growth in mice compared to oxaliplatin alone. Bromouridine-labeled RNA sequencing (Bru-seq) was employed to elucidate the effects of SGI-110 and/or oxaliplatin on genome-wide transcription. SGI-110 and the combination treatment inhibited the expression of genes involved in WNT/EGF/IGF signaling. DNMT1 and survivin were identified as novel PD markers to monitor the efficacy of the combination treatment. In conclusion, SGI-110 priming sensitizes HCC cells to oxaliplatin by inhibiting distinct signaling pathways. We expect that this combination treatment will show low toxicity and high efficacy in patients. Our study supports the use of the combination of low doses of SGI-110 and oxaliplatin in HCC patients.

  13. Enhancer repertoires are reshaped independently of early priming and heterochromatin dynamics during B cell differentiation

    PubMed Central

    Choukrallah, Mohamed-Amin; Song, Shuang; Rolink, Antonius G.; Burger, Lukas; Matthias, Patrick

    2015-01-01

    A widely accepted model posits that activation of enhancers during differentiation goes through a priming step prior to lineage commitment. To investigate the chronology of enhancer repertoire establishment during hematopoiesis, we monitored epigenome dynamics during three developmental stages representing hematopoietic stem cells, B-cell progenitors and mature B-cells. We find that only a minority of enhancers primed in stem cells or progenitors become active at later stages. Furthermore, most enhancers active in differentiated cells were not primed in earlier stages. Thus, the enhancer repertoire is reshaped dynamically during B-cell differentiation and enhancer priming in early stages does not appear to be an obligate step for enhancer activation. Furthermore, our data reveal that heterochromatin and Polycomb-mediated silencing have only a minor contribution in shaping enhancer repertoires during cell differentiation. Together, our data revisit the prevalent model about epigenetic reprogramming during hematopoiesis and give insights into the formation of gene regulatory networks. PMID:26477271

  14. Effect of Helicobacter pylori on gastric epithelial cells

    PubMed Central

    Alzahrani, Shatha; Lina, Taslima T; Gonzalez, Jazmin; Pinchuk, Irina V; Beswick, Ellen J; Reyes, Victor E

    2014-01-01

    The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world’s population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori’s avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori. PMID:25278677

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

    EPA Science Inventory

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

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

    PubMed Central

    St Johnston, Daniel

    2016-01-01

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

  17. Cooperative Interactions During Human Mammary Epithelial Cell Immortalization

    DTIC Science & Technology

    2005-07-01

    Immortal Transformation of Cultured Human Mammary Epithelial Cells. Cellular Oncology, 26:248-251, 2004. Rodier , F., Kim, S-H., Nijjar, T., Yaswen, P...Promoter, Mol. Cell Biol.: 25:3923-3933, 2005. Goldstein, J, Rodier , F, Garbe, J, Stampfer, M, Campisi, J, Caspase-independent cytochrome c release is a

  18. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

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

  19. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

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

  20. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

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

    2012-01-01

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

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

    EPA Science Inventory

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

  2. Identification of Phosphorylation Sites on Extracellular Corneal Epithelial Cell Maspin

    PubMed Central

    Narayan, Malathi; Mirza, Shama P.; Twining, Sally S.

    2011-01-01

    Maspin, a 42-kDa non classical serine protease inhibitor (serpin) is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation mass spectrometry was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310, Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues on extracellular maspin was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin. PMID:21365746

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2007-03-01

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

  5. Coronavirus entry and release in polarized epithelial cells: a review.

    PubMed

    Cong, Yingying; Ren, Xiaofeng

    2014-09-01

    Most coronaviruses cause respiratory or intestinal infections in their animal or human host. Hence, their interaction with polarized epithelial cells plays a critical role in the onset and outcome of infection. In this paper, we review the knowledge regarding the entry and release of coronaviruses, with particular emphasis on the severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. As these viruses approach the epithelial surfaces from the apical side, it is not surprising that coronavirus cell receptors are exposed primarily on the apical domain of polarized epithelial cells. With respect to release, all possibilities appear to occur. Thus, most coronaviruses exit through the apical surface, several through the basolateral one, although the Middle East respiratory syndrome coronavirus appears to use both sides. These observations help us understand the local or systematic spread of the infection within its host as well as the spread of the virus within the host population.

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2014-10-01

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

  8. Intestinal epithelial cells and their role in innate mucosal immunity.

    PubMed

    Maldonado-Contreras, A L; McCormick, Beth A

    2011-01-01

    The mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts are covered by a layer of epithelial cells that are responsible for sensing and promoting a host immune response in order to establish the limits not only for commensal microorganisms but also for foreign organisms or particles. This is a remarkable task as the human body represents a composite of about 10 trillion human-self cells plus non-self cells from autochthonous or indigenous microbes that outnumber human cells 10:1. Hence, the homeostasis of epithelial cells that line mucosal surfaces relies on a fine-tuned immune system that patrols the boundaries between human and microbial cells. In the case of the intestine, the epithelial layer is composed of at least six epithelial cell lineages that act as a physiological barrier in addition to aiding digestion and the absorption of nutrients, water and electrolytes. In this review, we highlight the immense role of the intestinal epithelium in coordinating the mucosal innate immune response.

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

    PubMed Central

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

    2014-01-01

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

  10. EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells

    PubMed Central

    Xiao, Jianqiao; Palefsky, Joel M.; Herrera, Rossana; Berline, Jennifer; Tugizov, Sharof M.

    2009-01-01

    We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with β1 and αv family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2low recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells. PMID:19394065

  11. Effects of lipopolysaccharide, multiwalled carbon nanotubes, and the combination on lung alveolar epithelial cells

    PubMed Central

    Pacurari, M; May, I; Tchounwou, PB

    2016-01-01

    Multi-walled carbon nanotubes (MWCNT) have been shown to induce lung fibrosis in animal models, however the underlying molecular factors/mechanisms are still unclear. In this study, we investigated the effects of lipopolysaccharide (LPS), MWCNT, and the combination of LPS and MWCNT on the expression of matrix metalloproteinase-9 and metalloproteinase-12 (MMP-9, MMP-12), collagen 3A1 (Col3A1), and transforming growth factor beta (TGFβ) in alveolar epithelial A549 cells. MMPs are proteinases that degrade extracellular matrix and play a role in lung fibrosis. A549 cells were exposed to LPS (1 ng/ml), MWCNT (20 μg/ml), and the combination and analyzed for paracellular permeability, TGFβ, Col3A1, MMP-9, MMP-12, NF-κB activation, and cell migration by real-time PCR and immunofluorescence. LPS, the combination of LPS and MWCNT, and MWCNT only at the highest tested dose induced blue dextran extravation. LPS and MWCNT increased the expression of TGFβ and its downstream target gene Col3A, and MMP-9 and MMP-12 mRNA. MWCNT potently induced cell migration toward wound healing, whereas LPS slightly induced cell migration. Both, LPS and MWCNT, induced NF-κB nuclear translocation. Our results indicate that MWCNT activated alveolar epithelial cells to promote fibrogenesis, and that LPS differentially primes molecular factors involved in lung remodeling. These findings suggest a role of alveolar epithelial cells in fibrogenesis and also may aid in the design and development of tests for screening of fibrogenic agents. PMID:26880698

  12. MAPK pathway mediates epithelial-mesenchymal transition induced by paraquat in alveolar epithelial cells.

    PubMed

    Huang, Min; Wang, Ya-Peng; Zhu, Ling-Qin; Cai, Qian; Li, Hong-Hui; Yang, Hui-Fang

    2016-11-01

    Epithelial-mesenchymal transition (EMT) is believed to be involved in lung fibrosis process induced by paraquat (PQ); however, the molecular mechanism of this process has not been clearly established. The present study investigated the potential involvement of EMT after PQ poisoning. The expressions of EMT markers, such as E-cadherin and α-smooth muscle actin (α-SMA), at multiple time points after exposure to different concentrations of PQ were evaluated by western blot analysis. Following PQ treatment, EMT induction was observed under microscopy. Related fibrosis genes, including Matrix metalloproteinase 2 (MMP-2), Matrix metalloproteinase 9 (MMP-9), collagens type I (COL I), and type III (COL III), were also evaluated by measuring their mRNA levels using RT-PCR analysis. Signaling pathways were analyzed using selective pharmacological inhibitors for MAPK. Cell migration ability was evaluated by scratch wound and Transwell assays. The data showed that PQ-induced epithelial RLE-6NT cells to develop mesenchymal cell characteristics, as indicated by a significant decrease in the epithelial marker E-cadherin and a significant increase in the extracellular matrix (ECM) marker α-smooth muscle actin in a dose and time-dependent manner. Moreover, PQ-treated RLE-6NT cells had an EMT-like phenotype with elevated expression of MMP-2, MMP-9, and COL I and COL III and enhanced migration ability. Signal pathway analysis revealed that PQ-induced EMT led to ERK-1 and Smad2 phosphorylation through activation of the MAPK pathway. The results of the current study indicate that PQ-induced pulmonary fibrosis occurs via EMT, which is mediated by the MAPK pathway. This implies that the MAPK pathway is a promising therapeutic target in alveolar epithelial cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1407-1414, 2016. © 2015 Wiley Periodicals, Inc.

  13. TRPV channels as thermosensory receptors in epithelial cells.

    PubMed

    Lee, Hyosang; Caterina, Michael J

    2005-10-01

    Temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels are critical contributors to normal pain and temperature sensation and therefore represent attractive targets for pain therapy. When these channels were first discovered, most attention was focused on their potential contributions to direct thermal activation of peripheral sensory neurons. However, recent anatomical, physiological, and behavioral studies have provided evidence that TRPV channels expressed in skin epithelial cells may also contribute to thermosensation in vitro and in vivo. Here, we review these studies and speculate on possible communication mechanisms from cutaneous epithelial cells to sensory neurons.

  14. Collaboration of epithelial cells with organized mucosal lymphoid tissues.

    PubMed

    Neutra, M R; Mantis, N J; Kraehenbuhl, J P

    2001-11-01

    Immune surveillance of mucosal surfaces requires the delivery of intact macromolecules and microorganisms across epithelial barriers to organized mucosal lymphoid tissues. Transport, processing and presentation of foreign antigens, as well as local induction and clonal expansion of antigen-specific effector lymphocytes, involves a close collaboration between organized lymphoid tissues and the specialized follicle-associated epithelium. M cells in the follicle-associated epithelium transport foreign macromolecules and microorganisms to antigen-presenting cells within and under the epithelial barrier. Determination of the earliest cellular interactions that occur in and under the follicle-associated epithelium could greatly facilitate the design of effective mucosal vaccines in the future.

  15. Feature quantification and abnormal detection on cervical squamous epithelial cells.

    PubMed

    Zhao, Mingzhu; Chen, Lei; Bian, Linjie; Zhang, Jianhua; Yao, Chunyan; Zhang, Jianwei

    2015-01-01

    Feature analysis and classification detection of abnormal cells from images for pathological analysis are an important issue for the realization of computer assisted disease diagnosis. This paper studies a method for cervical squamous epithelial cells. Based on cervical cytological classification standard and expert diagnostic experience, expressive descriptors are extracted according to morphology, color, and texture features of cervical scales epithelial cells. Further, quantificational descriptors related to cytopathology are derived as well, including morphological difference degree, cell hyperkeratosis, and deeply stained degree. The relationship between quantified value and pathological feature can be established by these descriptors. Finally, an effective method is proposed for detecting abnormal cells based on feature quantification. Integrated with clinical experience, the method can realize fast abnormal cell detection and preliminary cell classification.

  16. Osmosignaling and volume regulation in intestinal epithelial cells.

    PubMed

    Lim, Christina H; Bot, Alice G M; de Jonge, Hugo R; Tilly, Ben C

    2007-01-01

    Most cells have to perform their physiological functions under a variable osmotic stress, which, because of the relatively high permeability of the plasma membrane for water, may result in frequent alterations in cell size. Intestinal epithelial cells are especially prone to changes in cell volume because of their high capacity of salt and water transport and the high membrane expression of various nutrient transporters. Therefore, to avoid excessive shrinkage or swelling, enterocytes, like most cell types, have developed efficient mechanisms to maintain osmotic balance. This chapter reviews selected model systems that can be used to investigate cell volume regulation in intestinal epithelial cells, with emphasis on the regulatory volume decrease, and the methods available to study the compensatory redistribution of (organic) osmolytes. In addition, a brief summary is presented of the pathways involved in osmosensing and osmosignaling in the intestine.

  17. Nicotine transport in lung and non-lung epithelial cells.

    PubMed

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

    2017-11-01

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

  18. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  19. Cell division and cadherin-mediated adhesion regulate lens epithelial cell movement in zebrafish.

    PubMed

    Mochizuki, Toshiaki; Luo, Yi-Jyun; Tsai, Hsieh-Fu; Hagiwara, Akane; Masai, Ichiro

    2017-02-15

    In vertebrates, lens epithelial cells cover the anterior half of the lens fiber core. During development, lens epithelial cells proliferate, move posteriorly and differentiate into lens fiber cells after passing through the equator. To elucidate the mechanisms underlying lens epithelial cell movement, we conducted time-lapse imaging of zebrafish lens epithelium. Lens epithelial cells do not intermingle but maintain their relative positions during development. Cell division induces epithelial rearrangement, which subsequently promotes cell movement towards the equator. These data suggest that cell division is the major driving force for cell movement. In zebrafish, E-cadherin is expressed in lens epithelium, whereas N-cadherin is required for lens fiber growth. E-cadherin reduced lens epithelial cell movement, whereas N-cadherin enhanced it. Laser ablation experiments revealed that lens epithelium is governed by pulling tension, which is modulated by these cadherins. Thus, cell division and cadherin-mediated adhesion regulate lens epithelial cell movement via modulation of epithelial tension. © 2017. Published by The Company of Biologists Ltd.

  20. Keratins are novel markers of renal epithelial cell injury.

    PubMed

    Djudjaj, Sonja; Papasotiriou, Marios; Bülow, Roman D; Wagnerova, Alexandra; Lindenmeyer, Maja T; Cohen, Clemens D; Strnad, Pavel; Goumenos, Dimitrios S; Floege, Jürgen; Boor, Peter

    2016-04-01

    Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  1. Microarray analysis of human epithelial cell responses to bacterial interaction.

    PubMed

    Mans, Jeffrey J; Lamont, Richard J; Handfield, Martin

    2006-09-01

    Host-pathogen interactions are inherently complex and dynamic. The recent use of human microarrays has been invaluable to monitor the effects of various bacterial and viral pathogens upon host cell gene expression programs. This methodology has allowed the host response transcriptome of several cell lines to be studied on a global scale. To this point, the great majority of reports have focused on the response of immune cells, including macrophages and dendritic cells. These studies revealed that the immune response to microbial pathogens is tailored to different microbial challenges. Conversely, the paradigm for epithelial cells has--until recently--held that the epithelium mostly served as a relatively passive physical barrier to infection. It is now generally accepted that the epithelial barrier contributes more actively to signaling events in the immune response. In light of this shift, this review will compare transcriptional profiling data from studies that involved host-pathogen interactions occurring with epithelial cells. Experiments that defined both a common core response, as well as pathogen-specific host responses will be discussed. This review will also summarize the contributions that transcriptional profiling analysis has made to our understanding of bacterial physio-pathogensis of infection. This will include a discussion of how host transcriptional responses can be used to infer the function of virulence determinants from bacterial pathogens interacting with epithelial mucosa. In particular, we will expand upon the lessons that have been learned from gastro-intestinal and oral pathogens, as well as from members of the commensal flora.

  2. Primed Mesenchymal Stem Cells Alter Healing and Improve Rat Medial Collateral Ligament Healing

    PubMed Central

    Saether, Erin E.; Chamberlain, Connie S.; Aktas, Erdem; Leiferman, Ellen M.; Brickson, Stacey L.; Vanderby, Ray

    2016-01-01

    Cell therapy with mesenchymal stem cells (MSCs) can improve tissue healing. It is possible, however, that priming MSCs prior to implantation can further enhance their therapeutic benefit. This study was then performed to test whether priming MSCs to be more anti-inflammatory would enhance healing in a rat ligament model, i.e. a medial collateral ligament (MCL). MSCs were primed for 48 hours using polyinosinic acid and polycytidylic acid (Poly (I:C)) at a concentration of 1µg/ml. Rat MCLs were surgically transected and administered 1×106 cells in a carrier solution at the time of injury. A series of healing metrics were analyzed at days 4 and 14 post-injury in the ligaments that received primed MSCs, unprimed MSCs, or no cells (controls). Applying primed MSCs beneficially altered healing by affecting endothelialization, type 2 macrophage presence, apoptosis, procollagen 1α, and IL-1Ra levels. When analyzing MSC localization, both primed and unprimed MSCs co-localized with endothelial cells and pericytes suggesting a supportive role in angiogenesis. Priming MSCs prior to implantation altered key ligament healing events, resulted in a more anti-inflammatory environment, and improved healing. PMID:26530282

  3. Valproic acid enforces the priming effect of sphingosine-1 phosphate on human mesenchymal stem cells.

    PubMed

    Lim, Jisun; Lee, Seungun; Ju, Hyein; Kim, Yonghwan; Heo, Jinbeom; Lee, Hye-Yeon; Choi, Kyung-Chul; Son, Jaekyoung; Oh, Yeon-Mok; Kim, In-Gyu; Shin, Dong-Myung

    2017-09-01

    Engraftment and homing of mesenchymal stem cells (MSCs) are modulated by priming factors including the bioactive lipid sphingosine-1-phosphate (S1P), by stimulating CXCR4 receptor signaling cascades. However, limited in vivo efficacy and the remaining priming molecules prior to administration of MSCs can provoke concerns regarding the efficiency and safety of MSC priming. Here, we showed that valproic acid (VPA), a histone deacetylase inhibitor, enforced the priming effect of S1P at a low dosage for human umbilical cord-derived MSCs (UC-MSCs). A DNA-methylation inhibitor, 5-azacytidine (5-Aza), and VPA increased the expression of CXCR4 in UC-MSCs. In particular, UC-MSCs primed with a suboptimal dose (50 nM) of S1P in combination with 0.5 mM VPA (VPA+S1P priming), but not 1 µM 5-Aza, significantly improved the migration activity in response to stromal cell-derived factor 1 (SDF-1) concomitant with the activation of both MAPKp42/44 and AKT signaling cascades. Both epigenetic regulatory compounds had little influence on cell surface marker phenotypes and the multi-potency of UC-MSCs. In contrast, VPA+S1P priming of UC-MSCs potentiated the proliferation, colony forming unit-fibroblast, and anti-inflammatory activities, which were severely inhibited in the case of 5-Aza treatment. Accordingly, the VPA+S1P-primed UC-MSCs exhibited upregulation of a subset of genes related to stem cell migration and anti-inflammation response. Thus, the present study demonstrated that VPA enables MSC priming with S1P at a low dosage by enhancing their migration and other therapeutic beneficial activities. This priming strategy for MSCs may provide a more efficient and safe application of MSCs for treating a variety of intractable disorders.

  4. Aneuploidy, oncogene amplification and epithelial to mesenchymal transition define spontaneous transformation of murine epithelial cells

    PubMed Central

    Padilla-Nash, Hesed M.; McNeil, Nicole E.

    2013-01-01

    Human epithelial cancers are defined by a recurrent distribution of specific chromosomal aneuploidies, a trait less typical for murine cancer models induced by an oncogenic stimulus. After prolonged culture, mouse epithelial cells spontaneously immortalize, transform and become tumorigenic. We assessed genome and transcriptome alterations in cultures derived from bladder and kidney utilizing spectral karyotyping, array CGH, FISH and gene expression profiling. The results show widespread aneuploidy, yet a recurrent and tissue-specific distribution of genomic imbalances, just as in human cancers. Losses of chromosome 4 and gains of chromosome 15 are common and occur early during the transformation process. Global gene expression profiling revealed early and significant transcriptional deregulation. Chromosomal aneuploidy resulted in expression changes of resident genes and consequently in a massive deregulation of the cellular transcriptome. Pathway interrogation of expression changes during the sequential steps of transformation revealed enrichment of genes associated with DNA repair, centrosome regulation, stem cell characteristics and aneuploidy. Genes that modulate the epithelial to mesenchymal transition and genes that define the chromosomal instability phenotype played a dominant role and were changed in a directionality consistent with loss of cell adhesion, invasiveness and proliferation. Comparison with gene expression changes during human bladder and kidney tumorigenesis revealed remarkable overlap with changes observed in the spontaneously transformed murine cultures. Therefore, our novel mouse models faithfully recapitulate the sequence of genomic and transcriptomic events that define human tumorigenesis, hence validating them for both basic and preclinical research. PMID:23619298

  5. Epithelial neoplasia in Drosophila entails switch to primitive cell states

    PubMed Central

    Khan, Sumbul J.; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P.; Harsh, Sneh; Pandey, Ravi K.; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-01-01

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl− clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl− clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl− clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl− clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl− clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of “cells-of-origin” in epithelial cancers, namely their propensity for switch to primitive cell states. PMID:23708122

  6. Epithelial neoplasia in Drosophila entails switch to primitive cell states.

    PubMed

    Khan, Sumbul J; Bajpai, Anjali; Alam, Mohammad Atif; Gupta, Ram P; Harsh, Sneh; Pandey, Ravi K; Goel-Bhattacharya, Surbhi; Nigam, Aditi; Mishra, Arati; Sinha, Pradip

    2013-06-11

    Only select cell types in an organ display neoplasia when targeted oncogenically. How developmental lineage hierarchies of these cells prefigure their neoplastic propensities is not yet well-understood. Here we show that neoplastic Drosophila epithelial cells reverse their developmental commitments and switch to primitive cell states. In a context of alleviated tissue surveillance, for example, loss of Lethal giant larvae (Lgl) tumor suppressor in the wing primordium induced epithelial neoplasia in its Homothorax (Hth)-expressing proximal domain. Transcriptional profile of proximally transformed mosaic wing epithelium and functional tests revealed tumor cooperation by multiple signaling pathways. In contrast, lgl(-) clones in the Vestigial (Vg)-expressing distal wing epithelium were eliminated by cell death. Distal lgl(-) clones, however, could transform when both tissue surveillance and cell death were compromised genetically and, alternatively, when the transcription cofactor of Hippo signaling pathway, Yorkie (Yki), was activated, or when Ras/EGFR signaling was up-regulated. Furthermore, transforming distal lgl(-) clones displayed loss of Vg, suggesting reversal of their terminal cell fate commitment. In contrast, reinforcing a distal (wing) cell fate commitment in lgl(-) clones by gaining Vg arrested their neoplasia and induced cell death. We also show that neoplasia in both distal and proximal lgl(-) clones could progress in the absence of Hth, revealing Hth-independent wing epithelial neoplasia. Likewise, neoplasia in the eye primordium resulted in loss of Elav, a retinal cell marker; these, however, switched to an Hth-dependent primitive cell state. These results suggest a general characteristic of "cells-of-origin" in epithelial cancers, namely their propensity for switch to primitive cell states.

  7. RIPK1 and NF-κB signaling in dying cells determines cross-priming of CD8+ T cells

    PubMed Central

    Yatim, Nader; Jusforgues-Saklani, Hélène; Orozco, Susana; Schulz, Oliver; Barreira da Silva, Rosa; Reis e Sousa, Caetano; Green, Douglas R.; Oberst, Andrew; Albert, Matthew L.

    2015-01-01

    Dying cells initiate adaptive immunity by providing both antigens and inflammatory stimuli for dendritic cells (DCs), which in turn activate CD8+ T cells through a process called antigen cross-priming. To define how different forms of programmed cell death influence immunity, we established models of necroptosis and apoptosis, where dying cells are generated by RIPK3 and CASP8 dimerization, respectively. We found that release of inflammatory mediators such as damage-associated molecular patterns (DAMPs) by dying cells was not sufficient for CD8+ T cell cross-priming. Instead, robust cross-priming required RIPK1 signaling and NF-κB-induced transcription within dying cells. Decoupling NF-κB signaling from necroptosis or inflammatory apoptosis reduced priming efficiency and tumor immunity. Our results reveal that coordinated inflammatory and cell death signaling pathways within dying cells orchestrate adaptive immunity. PMID:26405229

  8. Extracellular cleavage of E-cadherin promotes epithelial cell extrusion.

    PubMed

    Grieve, Adam G; Rabouille, Catherine

    2014-08-01

    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 component of adherens junctions, has been shown to be essential for epithelial cell extrusion, but its mechanistic contribution remains unclear. Here, we provide clear evidence that cell extrusion can be driven by the cleavage of E-cad, both in a wild-type and an oncogenic environment. We first show that CDC42 activation in a single epithelial cell results in its efficient matrix metalloproteinase (MMP)-sensitive extrusion through MEK signalling activation and this is supported by E-cad cleavage. Second, using an engineered cleavable form of E-cad, we demonstrate that, by itself, truncation of extracellular E-cad at the plasma membrane promotes apical extrusion. We propose that extracellular cleavage of E-cad generates a rapid change in cell-cell adhesion that is sufficient to drive apical cell extrusion. Whereas in normal epithelia, extrusion is followed by apoptosis, when combined with active oncogenic signalling, it is coupled to cell proliferation.

  9. TMIGD1 is a novel adhesion molecule that protects epithelial cells from oxidative cell injury.

    PubMed

    Arafa, Emad; Bondzie, Philip A; Rezazadeh, Kobra; Meyer, Rosana D; Hartsough, Edward; Henderson, Joel M; Schwartz, John H; Chitalia, Vipul; Rahimi, Nader

    2015-10-01

    Oxidative damage to renal tubular epithelial cells is a fundamental pathogenic mechanism implicated in both acute kidney injury and chronic kidney diseases. Because epithelial cell survival influences the outcome of acute kidney injury and chronic kidney diseases, identifying its molecular regulators could provide new insight into pathobiology and possible new therapeutic strategies for these diseases. We have identified transmembrane and immunoglobulin domain-containing 1 (TMIGD1) as a novel adhesion molecule, which is highly conserved in humans and other species. TMIGD1 is expressed in renal tubular epithelial cells and promotes cell survival. The extracellular domain of TMIGD1 contains two putative immunoglobulin domains and mediates self-dimerization. Our data suggest that TMIGD1 regulates transepithelial electric resistance and permeability of renal epithelial cells. TMIGD1 controls cell migration, cell morphology, and protects renal epithelial cells from oxidative- and nutrient-deprivation-induced cell injury. Hydrogen peroxide-induced oxidative cell injury downregulates TMIGD1 expression and targets it for ubiquitination. Moreover, TMIGD1 expression is significantly affected in both acute kidney injury and in deoxy-corticosterone acetate and sodium chloride (deoxy-corticosterone acetate salt)-induced chronic hypertensive kidney disease mouse models. Taken together, we have identified TMIGD1 as a novel cell adhesion molecule expressed in kidney epithelial cells that protects kidney epithelial cells from oxidative cell injury to promote cell survival.

  10. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

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

    2007-11-10

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

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

    PubMed

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

    2014-03-01

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

  12. Hyperoxia-induced signal transduction pathways in pulmonary epithelial cells

    PubMed Central

    Zaher, Tahereh E.; Miller, Edmund J.; Morrow, Dympna M. P.; Javdan, Mohammad; Mantell, Lin L.

    2007-01-01

    Mechanical ventilation with hyperoxia is necessary to treat critically ill patients. However, prolonged exposure to hyperoxia leads to the generation of excessive reactive oxygen species (ROS), which can cause acute inflammatory lung injury. One of the major effects of hyperoxia is the injury and death of pulmonary epithelium, which is accompanied by increased levels of pulmonary proinflammatory cytokines and excessive leukocyte infiltration. A thorough understanding of the signaling pathways leading to pulmonary epithelial cell injury/death may provide some insights into the pathogenesis of hyperoxia-induced acute inflammatory lung injury. This review focuses on epithelial responses to hyperoxia and some of the major factors regulating pathways to epithelial cell injury/death, and proinflammatory responses upon exposure to hyperoxia. We discuss in detail some of the most interesting players, such as, NF-κB, that can modulate both proinflammatory responses and cell injury/death of lung epithelial cells. A better appreciation for the functions of these factors will no doubt help us to delineate the pathways to hyperoxic cell death and proinflammatory responses. PMID:17349918

  13. Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells

    PubMed Central

    Sajjan, Umadevi; Wang, Qiong; Zhao, Ying; Gruenert, Dieter C.; Hershenson, Marc B.

    2008-01-01

    Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease. Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection. Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting. Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo. Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function. PMID:18787220

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

    PubMed

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

    2014-11-01

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

  15. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells.

    PubMed

    Yoshinaga, Tomoyo; Uwabe, Kenichiro; Naito, Shoichi; Higashino, Kenichi; Nakano, Toru; Numata, Yoshito; Kihara, Akio

    2016-01-01

    Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis.

  16. AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells

    PubMed Central

    Yoshinaga, Tomoyo; Uwabe, Kenichiro; Naito, Shoichi; Higashino, Kenichi; Nakano, Toru; Numata, Yoshito

    2016-01-01

    Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-β1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-β signaling pathway. Our findings regarding the effects of AM251 on the TGF-β signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis. PMID:27936102

  17. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture

    PubMed Central

    Gidfar, Sanaz; Milani, Farnoud Y.; Milani, Behrad Y.; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J.; Sagha, Hossein; Djalilian, Ali R.

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells. PMID:28054657

  18. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture.

    PubMed

    Gidfar, Sanaz; Milani, Farnoud Y; Milani, Behrad Y; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J; Sagha, Hossein; Djalilian, Ali R

    2017-01-05

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

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

    PubMed

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

    2013-03-01

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

  20. Peyer's patch dendritic cells process viral antigen from apoptotic epithelial cells in the intestine of reovirus-infected mice.

    PubMed

    Fleeton, Marina N; Contractor, Nikhat; Leon, Francisco; Wetzel, J Denise; Dermody, Terence S; Kelsall, Brian L

    2004-07-19

    We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (sigma1) and nonstructural (sigmaNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both sigma1 and sigmaNS, indicating productive viral replication. In contrast, sigma1, but not sigmaNS, was detected in the subepithelial dome (SED) in association with CD11c(+)/CD8alpha(-)/CD11b(lo) DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained sigma1, but not sigmaNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, sigma1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4(+) T cells in vitro. These studies show that CD8alpha(-)/CD11b(lo) DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4(+) T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.

  1. Cell associated urokinase activity and colonic epithelial cells in health and disease.

    PubMed Central

    Gibson, P R; van de Pol, E; Doe, W F

    1991-01-01

    It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA. PMID:1650741

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

    EPA Science Inventory

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

    Nitrotyrosine (NO2Tyr) is a...

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

    EPA Science Inventory

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

    Nitrotyrosine (NO2Tyr) is a...

  4. Metabolic cooperativity between epithelial cells and adipocytes of mice

    SciTech Connect

    Bartley, J.C.; Emerman, J.T.; Bissell, M.J.

    1981-01-01

    We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from (/sup 14/C)glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy. We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations.

  5. Mesenchymal Stromal Cell-Derived Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Acquisition of Epithelial Stem-Like Cell Properties in Ameloblastoma Epithelial Cells.

    PubMed

    Jiang, Chunmiao; Zhang, Qunzhou; Shanti, Rabie M; Shi, Shihong; Chang, Ting-Han; Carrasco, Lee; Alawi, Faizan; Le, Anh D

    2017-09-01

    Epithelial-mesenchymal transition (EMT), a biological process associated with cancer stem-like or cancer-initiating cell formation, contributes to the invasiveness, metastasis, drug resistance, and recurrence of the malignant tumors; it remains to be determined whether similar processes contribute to the pathogenesis and progression of ameloblastoma (AM), a benign but locally invasive odontogenic neoplasm. Here, we demonstrated that EMT- and stem cell-related genes were expressed in the epithelial islands of the most common histologic variant subtype, the follicular AM. Our results revealed elevated interleukin (IL)-6 signals that were differentially expressed in the stromal compartment of the follicular AM. To explore the stromal effect on tumor pathogenesis, we isolated and characterized both mesenchymal stromal cells (AM-MSCs) and epithelial cells (AM-EpiCs) from follicular AM and demonstrated that, in in vitro culture, AM-MSCs secreted a significantly higher level of IL-6 as compared to the counterpart AM-EpiCs. Furthermore, both in vitro and in vivo studies revealed that exogenous and AM-MSC-derived IL-6 induced the expression of EMT- and stem cell-related genes in AM-EpiCs, whereas such effects were significantly abrogated either by a specific inhibitor of STAT3 or ERK1/2, or by knockdown of Slug gene expression. These findings suggest that AM-MSC-derived IL-6 promotes tumor-stem like cell formation by inducing EMT process in AM-EpiCs through STAT3 and ERK1/2-mediated signaling pathways, implying a role in the etiology and progression of the benign but locally invasive neoplasm. Stem Cells 2017;35:2083-2094. © 2017 AlphaMed Press.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    1983-01-01

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

  9. Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells.

    PubMed

    Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J

    2013-04-01

    Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high-resolution whole-slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. "Virtually digested" WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here. Copyright © 2012 American Association for the Study of Liver Diseases.

  10. Structural anomalies of highly malignant respiratory tract epithelial cells

    SciTech Connect

    Manger, R.L.; Heckman, C.A.

    1982-11-01

    These studies were designed to determine whether cytostructural changes were related to malignancy and the loss of growth control in epithelial cells. Three highly malignant cell lines were derived from transplantable carcinomas of the respiratory tract and compared with three respiratory tract epithelial lines of negligible malignancy. Keratin cytoskeletons were visualized by indirect immunofluorescence staining, and sample photomicrographs representing each line were prepared. The criteria used in making the classifications to identify the features common to the highly malignant lines included the nonuniform spacing of cells in the field of view, the cell shape, and the presence of nonfluorescent areas in the lamellar cytoplasm. Since the nonuniformity of keratin distribution in the periphery of the malignant cells suggested a structural anomaly, the cell lines were also examined by scanning electron microscopy. Unlike cells from the lines of negligible malignancy, cells from two of the highly malignant lines showed thickenings in the subterminal portions of the lamellar cytoplasm. The results suggested that specific architectural changes at the cellular level might be linked to the process of epithelial transformation and tumor progression.

  11. Transport Mechanism of Nicotine in Primary Cultured Alveolar Epithelial Cells.

    PubMed

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation.

  12. Keratin cytoskeletons in epithelial cells of internal organs

    PubMed Central

    Sun, Tung-Tien; Shih, Chiaho; Green, Howard

    1979-01-01

    An antiserum against human epidermal keratins was used to detect keratins in frozen sections of various rabbit and human tissues by indirect immunofluorescence. Strong staining was observed in all stratified squamous epithelia (epidermis, cornea, conjunctiva, tongue, esophagus, vagina, and anus), in epidermal appendages (hair follicle, sebaceous gland, ductal and myoepithelial cells of sweat glands), as well as in Hassall's corpuscles of the thymus, indicating that all contain abundant keratins. No staining by the antiserum was observed in fibroblasts, muscle of any type, cartilage, blood vessel, nerve tissue, iris or lens epithelium, or the glomerular or tubular cells of the kidney. In contrast, the antiserum stained the cells of most epithelia of the intestinal tract, urinary tract (urethra, bladder, ureter, collecting ducts of kidney), female genital tract (cervix, cervical glands, uterus, and oviduct), and respiratory tract (trachea and bronchi). Epithelial cells of the fine ductal system in the pancreas and submaxillary gland also stained well. When primary cultures of epithelial cells derived from bladder, intestine, kidney, and trachea were grown on glass coverslips and stained with anti-keratin, fiber networks similar to those of cultured keratinocytes were observed. These results show that keratins constitute a cytoskeleton in epithelial cells of diverse morphology and embryological origin. The stability of keratin filaments probably confers the structural strength necessary for cells covering a free surface. Keratin staining can be used to obtain information about the origin of cell lines. Images PMID:111242

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

    SciTech Connect

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

    1989-03-01

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

  14. Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Sreenivasan, P K; Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells. Images PMID:8454326

  15. G15 sensitizes epithelial breast cancer cells to doxorubicin by preventing epithelial-mesenchymal transition through inhibition of GPR30

    PubMed Central

    Liu, Yu; Du, Fei-Ya; Chen, Wei; Fu, Pei-Fen; Yao, Min-Ya; Zheng, Shu-Sen

    2015-01-01

    Resistance to single or multiple chemotherapeutic drugs is a major obstacle in breast cancer therapy. Recent studies have suggested that GPR30 is implicated in mediating cancer cell proliferation. The aim of this study was to examine the anti-tumor effects of the GPR30 antagonist G15 in breast cancer. We found that low concentrations of G15 had little effect on breast cancer cell viability, but could enhance doxorubicin sensitivity in MDA-MB-231 and MCF-7 cells with epithelial phenotypes. In addition, G15 prevented epithelial breast cancer cells undergoing epithelial-mesenchymal transition (EMT) after doxorubicin induction. Moreover, downregulation of GPR30 suppressed the EMT in breast cancer cells. These results support that G15 enhanced doxorubicin sensitivity and prevented the EMT in epithelial breast cancer cells by inhibiting GPR30 expression. PMID:26175858

  16. Mesenchymal stem cell-mediated endochondral ossification utilising micropellets and brief chondrogenic priming.

    PubMed

    Knuth, C A; Witte-Bouma, J; Ridwan, Y; Wolvius, E B; Farrell, E

    2017-09-22

    With limited autologous and donor bone graft availability, there is an increasing need for alternative graft substitutes. We have previously shown that chondrogenically priming mesenchymal stem cell (MSC) pellets for 28 d in vitro will reproducibly result in endochondral bone formation after in vivo implantation. However, pellet priming time for clinical applications is quite extensive. A micropellet (μpellet)-fibrin construct was developed and coupled, with a shorter priming period, determined by an in vitro time course experiment. In vitro data showed expression of chondrogenic genes and matrix production after 7 d of chondrogenic priming, indicating that briefer priming could possibly be used to induce bone formation in vivo. 7 and 28 d primed pellet, pellet-fibrin and μpellet-fibrin constructs were cultured for in vitro analysis and implanted subcutaneously for 8 weeks into nude mice. μpellet-fibrin constructs, cultured in vitro for 7 or 28 d, produced comparable bone to standard pellets in vivo. MSC-mediated bone formation was achieved following only 7 d of in vitro priming. Bone formation in vivo appeared to be influenced by overall matrix production pre-implantation. Given this short priming time and the injectable nature of the μpellet-fibrin constructs, this approach might be further developed as an injectable bone substitute, leading to a minimally-invasive treatment option, which would allow for tailored filling of bone defects.

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

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

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

  18. Potentiation and tolerance of toll-like receptor priming in human endothelial cells.

    PubMed

    Koch, Stephen R; Lamb, Fred S; Hellman, Judith; Sherwood, Edward R; Stark, Ryan J

    2017-02-01

    Repeated challenge of lipopolysaccharide (LPS) alters the response to subsequent LPS exposures via modulation of toll-like receptor 4 (TLR4). Whether activation of other TLRs can modulate TLR4 responses, and vice versa, remains unclear. Specifically with regards to endothelial cells, a key component of innate immunity, the impact of TLR cross-modulation is unknown. We postulated that TLR2 priming (via Pam3Csk4) would inhibit TLR4-mediated responses while TLR3 priming (via Poly I:C) would enhance subsequent TLR4-inflammatory signaling. We studied human umbilical vein endothelial cells (HUVECs) and neonatal human dermal microvascular endothelial cells (HMVECs). Cells were primed with a combination of Poly I:C (10 μg/ml), Pam3Csk4 (10 μg/ml), or LPS (100 ng/ml), then washed and allowed to rest. They were then rechallenged with either Poly I:C, Pam3Csk4 or LPS. Endothelial cells showed significant tolerance to repeated LPS challenge. Priming with Pam3Csk4 also reduced the response to secondary LPS challenge in both cell types, despite a reduced proinflammatory response to Pam3Csk4 in HMVECs compared to HUVECs. Poly I:C priming enhanced inflammatory and interferon producing signals upon Poly I:C or LPS rechallenge, respectively. Poly I:C priming induced interferon regulatory factor 7, leading to enhancement of interferon production. Finally, both Poly I:C and LPS priming induced significant changes in receptor-interacting serine/threonine-protein kinase 1 activity. Pharmacological inhibition of receptor-interacting serine/threonine-protein kinase 1 or interferon regulatory factor 7 reduced the potentiated phenotype of TLR3 priming on TLR4 rechallenge. These results demonstrate that in human endothelial cells, prior activation of TLRs can have a significant impact on subsequent exposures and may contribute to the severity of the host response.

  19. Activation by histamine of bronchial epithelial cells from nonasthmatic subjects.

    PubMed

    Vignola, A M; Campbell, A M; Chanez, P; Lacoste, P; Michel, F B; Godard, P; Bousquet, J

    1993-10-01

    Histamine is a major mediator of the mast cells that are present between epithelial cells in asthma. In asthma, there is an increased expression of ICAM-1 and HLA-DR and an increased spontaneous release of fibronectin. The effect of histamine was tested on bronchial epithelial cells obtained by bronchial brushing from 22 nonasthmatic subjects. The activation of epithelial cells was assessed by immunocytochemical analysis of the expression of membrane markers (ICAM-1 and HLA-DR) using the alkaline phosphatase-anti-alkaline phosphatase method and the release of fibronectin (enzyme immunoassay). Time-response (three experiments) and dose-response (six experiments) curves showed that the maximal effect was obtained after an incubation time of 24 h and a dose of 1 microM of histamine. For this time course and concentration, there was a highly significant increase in the number of cells expressing ICAM-1 (before histamine: 10 +/- 11%; after histamine: 32 +/- 20%; P < 0.001) and HLA-DR (before histamine: 8 +/- 7%; after histamine: 23 +/- 20%; P < 0.001) and in the release of fibronectin (before histamine: 30 +/- 20 ng/10(5) viable cells; after histamine: 61 +/- 35 ng/10(5) viable cells; P < 0.003). Cycloheximide blocked these effects, suggesting that histamine requires protein synthesis for its action. Pyrilamine (H1-blocker) and ranitidine (H2-blocker) at a concentration of 10 microM decreased the effect of histamine. However, there was no additive effect when both antagonists were added. This study suggests that mast cells present in the airways have a role in the activation of epithelial cells.

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2013-11-01

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

  2. Expression and roles of CCN2 in dental epithelial cells.

    PubMed

    Shimo, Tsuyoshi; Koyama, Eiki; Kurio, Naito; Matsumoto, Kenichi; Okui, Tatsuo; Ibaragi, Soichiro; Yoshioka, Norie; Sasaki, Akira

    2015-01-01

    Connective tissue growth factor (CCN2) regulates diverse cellular functions, including tooth development. In order to delineate the precise role of CCN2 in the epithelium during odontogenesis, we investigated how it is expressed and what roles it may have in primary cultures of epithelial cells derived from developing tooth germ of the bovine fetus. Ccn2 mRNA and protein were strongly expressed in the inner dental epithelium, which is consistent with the expression of transforming growth factor-β2 mRNA and proliferating cell nuclear antigen. Bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) were also expressed in the inner dental epithelium, indicating that CCN2 functionally interacts with these factors in the epithelium. The stimulatory effects of FGF2 on cell proliferation and BMP4 on cell differentiation were additively up-regulated by CCN2 in a newly-established dental epithelium cell culture. Taken together, our data provide clear evidence that CCN2 is synthesized by inner dental epithelial cells, and appears to act as an autocrine factor, which regulates dental epithelial cell proliferation and differentiation in concert with growth factors. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Myosin-X functions in polarized epithelial cells.

    PubMed

    Liu, Katy C; Jacobs, Damon T; Dunn, Brian D; Fanning, Alan S; Cheney, Richard E

    2012-05-01

    Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein-Myo10 localizes to lateral membrane cell-cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.

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

    PubMed Central

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

    2015-01-01

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

  5. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

    PubMed

    Lindemans, Caroline A; Calafiore, Marco; Mertelsmann, Anna M; O'Connor, Margaret H; Dudakov, Jarrod A; Jenq, Robert R; Velardi, Enrico; Young, Lauren F; Smith, Odette M; Lawrence, Gillian; Ivanov, Juliet A; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L; O'Rourke, Kevin P; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas E; Nieuwenhuis, Edward E; Shroyer, Noah F; Liu, Chen; Kolesnick, Richard; van den Brink, Marcel R M; Hanash, Alan M

    2015-12-24

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.

  6. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    PubMed

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  9. Concise review: limbal epithelial stem cell therapy: controversies and challenges.

    PubMed

    O'Callaghan, Anna R; Daniels, Julie T

    2011-12-01

    Limbal epithelial stem cells (LESCs) are a population of stem cells responsible for maintenance and repair of the corneal surface. Injury and disease can result in a deficiency of these stem cells, the vision affecting condition called limbal stem cell deficiency (LSCD) in which the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy was first described in 1997;29:19231932-19231932.and LESCs cultured from either patients or donors have been used to successfully treat LSCD. In this review, some of the challenges and controversies associated with cultured LESC therapy will be discussed including alternative stem cell sources.

  10. Lrig1: a new master regulator of epithelial stem cells

    PubMed Central

    Ordóñez-Morán, Paloma; Huelsken, Joerg

    2012-01-01

    Nat Cell Biol 14 4, 401–408 03042012 The intestine represents the most vigorously renewing, adult epithelial tissue that makes maintenance of its homeostasis a delicate balance between proliferation, cell cycle arrest, migration, differentiation, and cell death. These processes are precisely controlled by a network of developmental signalling cascades, which include Wnt, Notch, BMP/TGFβ, and Hedgehog pathways. A new, elegant study by Wong et al (2012) now adds Lrig1 as a key player in the control of intestinal homeostasis. As for epidermal stem cells, Lrig1 limits the size of the intestinal progenitor compartment by dampening EGF/ErbB-triggered stem cell expansion. PMID:22433838

  11. Crosstalk between intestinal epithelial cell and adaptive immune cell in intestinal mucosal immunity.

    PubMed

    Lu, Jun Tao; Xu, An Tao; Shen, Jun; Ran, Zhi Hua

    2017-05-01

    Constantly challenged by luminal bacteria, intestinal epithelium forms both a physical and biochemical defense against pathogens. Besides, intestinal epithelium senses dynamic and continuous changes in luminal environment and transmits signals to subjacent immune cells accordingly. It has been long accepted that adaptive immune cells fulfill their roles partly by modulating function of intestinal epithelial cells. Recent studies have brought up the proposal that intestinal epithelial cells also actively participate in the regulation of adaptive immunity, especially CD4+ adaptive T cells, which indicates that there is reciprocal crosstalk between intestinal epithelial cells and adaptive immune cells, and the crosstalk may play important role in intestinal mucosal immunity. This Review makes a comprehensive summary about crosstalk between intestinal epithelial cells and CD4+ adaptive T cells in intestinal immunity. Special attention would be given to their implications in inflammatory bowel disease pathogenesis and potential therapeutic targets. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  12. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    PubMed

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

  13. Ultraviolet Irradiation-Induced Volume Alteration of Corneal Epithelial Cells

    PubMed Central

    Wang, Ling; Lu, Luo

    2016-01-01

    Purpose The purpose of the study is to understand how extracellular stresses, such as ultraviolet (UV) irradiation, affect corneal epithelial cells. Cell volume changes, damage to corneal epithelial integrity, and cellular responses were assessed after exposure to UVC stresses. Methods Primary human and rabbit corneal epithelial cells were exposed to UVC light in culture conditions. Ultraviolet C irradiation–induced changes in cell size and volume were measured by real-time microscopy and self-quenching of the fluorescent dye calcein, respectively. The effects of UVC irradiation on Src and focal adhesion kinase (FAK) phosphorylation and FAK-dependent integrin signaling were detected by ELISA, immunoblotting, and immunostaining. Results Ultraviolet C irradiation induced both size and volume shifts in human and rabbit corneal epithelial cells. Ultraviolet C irradiation-induced decrease of cell volume elicited activation of Src and FAK, characterized by increased phosphorylations of SrcY416, FAKY397, and FAKY925. In addition, immunostaining studies showed UVC irradiation–induced increases in phosphorylation of FAK and formation of integrin β5 clustering. Application of Kv channel blockers, including 4-aminopyridine (4-AP), α-DTX, and depressing substance-1 (BDS-1), effectively suppressed UVC irradiation–induced cell volume changes, and subsequently inhibited UVC irradiation–induced phosphorylation of Src/FAK, and formation of integrin β5 clustering, suggesting UVC irradiation–induced volume changes and Src/FAK activation. Hyperosmotic pressure–induced volume decreases were measured in comparison with effects of UVC irradiation on volume and Src/FAK activation. However, Kv channel blocker, 4-AP, had no effect on hyperosmotic pressure–induced responses. Conclusions The present study demonstrates that UVC irradiation–induced decreases in cell volume lead to Src/FAK activation due to a rapid loss of K ions through membrane Kv channels. PMID:27978555

  14. Established thymic epithelial progenitor/stem cell-like cell lines differentiate into mature thymic epithelial cells and support T cell development.

    PubMed

    Chen, Pengfei; Zhang, Jun; Zhan, Yu; Su, Juanjuan; Du, Yarui; Xu, Guoliang; Shi, Yufang; Siebenlist, Ulrich; Zhang, Xiaoren

    2013-01-01

    Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs in vitro. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-κB subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells in vivo. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells in vitro and in vivo. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors.

  15. Priming Mesenchymal Stem Cells with Endothelial Growth Medium Boosts Stem Cell Therapy for Systemic Arterial Hypertension

    PubMed Central

    de Oliveira, Lucas Felipe; Almeida, Thalles Ramos; Ribeiro Machado, Marcus Paulo; Cuba, Marilia Beatriz; Alves, Angélica Cristina; da Silva, Marcos Vinícius; Rodrigues Júnior, Virmondes; Dias da Silva, Valdo José

    2015-01-01

    Systemic arterial hypertension (SAH), a clinical syndrome characterized by persistent elevation of arterial pressure, is often associated with abnormalities such as microvascular rarefaction, defective angiogenesis, and endothelial dysfunction. Mesenchymal stem cells (MSCs), which normally induce angiogenesis and improve endothelial function, are defective in SAH. The central aim of this study was to evaluate whether priming of MSCs with endothelial growth medium (EGM-2) increases their therapeutic effects in spontaneously hypertensive rats (SHRs). Adult female SHRs were administered an intraperitoneal injection of vehicle solution (n = 10), MSCs cultured in conventional medium (DMEM plus 10% FBS, n = 11), or MSCs cultured in conventional medium followed by 72 hours in EGM-2 (pMSC, n = 10). Priming of the MSCs reduced the basal cell death rate in vitro. The administration of pMSCs significantly induced a prolonged reduction (10 days) in arterial pressure, a decrease in cardiac hypertrophy, an improvement in endothelium-dependent vasodilation response to acetylcholine, and an increase in skeletal muscle microvascular density compared to the vehicle and MSC groups. The transplanted cells were rarely found in the hearts and kidneys. Taken together, our findings indicate that priming of MSCs boosts stem cell therapy for the treatment of SAH. PMID:26300922

  16. In vitro methods to culture primary human breast epithelial cells.

    PubMed

    Raouf, Afshin; Sun, Yu Jia

    2013-01-01

    Current evidence suggests that much like leukemia, breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore, knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro.

  17. Microtubule organization is determined by the shape of epithelial cells

    PubMed Central

    Gomez, Juan Manuel; Chumakova, Lyubov; Bulgakova, Natalia A.; Brown, Nicholas H.

    2016-01-01

    Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia. PMID:27779189

  18. In vitro co-culture of epithelial cells and smooth muscle cells on aligned nanofibrous scaffolds.

    PubMed

    Kuppan, Purushothaman; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2017-12-01

    Esophagus is a complex, hollow organ consisting of epithelial cells in the inner mucosal layer and smooth muscle cells in the outer muscle layer. In the present study, we have evaluated the in vitro co-culture of epithelial cells and smooth muscle cells on the aligned nanofibrous scaffold made of PHBV, PHBV-gelatin, PCL and PCL-gelatin developed through electrospinning using rotating drum collector. Epithelial cells were labeled with cell tracker green while the smooth muscle cells were labeled with cell tracker red. Labeled cells were seeded on the aligned nanofibers matrices and tracked using laser scanning confocal microscopy. The results demonstrate that both epithelial and smooth muscle cells attach, extend, and proliferate over these nanofibrous matrices. Confocal z-sectioning shows that epithelial and smooth muscle cells tend to separate into two distinct layers on a single nanofiber system mimicking the in vivo anatomy. Cell viability assay showed that both types of cells are viable and also interact with each other. The functional gene expression of respective cell types demonstrates that both epithelial and smooth muscle cells are phenotypically as well as functionally active when they were co-cultured. Thus the study highlighted that aligned nanofibrous scaffolds could be potential alternative graft for esophageal tissue regeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Phorbal esters and calcium ionophore can prime murine peritoneal macrophages for tumor cell destruction

    SciTech Connect

    Somers, S.D.; Weiel, J.E.; Hamilton, T.A.; Adams, D.O.

    1986-06-01

    Murine macrophages from sites of inflammation develop toward tumoricidal competence by exposure to a macrophage-activating factor such as interferon-..gamma.. (IFN-..gamma..). To explore the biochemical transductional events initiated by IFN-..gamma.., peritoneal macrophages from C57BL/6J mice elicited by various sterile irritants were treated in vitro with two pharmacologic agents that mimic the action of certain second messengers. Phorbol myristate acetate (PMA) and the ionophore A23187 cooperatively reproduced the ability of IFN-..gamma.. to prime macrophages for tumoricidal function. Neither agent alone was able to prime macrophages. The two agents acted on the macrophages, and target susceptibility to kill was not altered by PMA and A23187. Only active phorbol esters, which are known to bind and stimulate protein kinase C, were able to cooperate with A23187 to induce priming. A cell-permeable synthetic diacylglycerol (sn-1,2-dioctanoyl glycerol) could also prime for cytolysis. In the presence of PMA, A23187, and EGTA, addition of Ca/sup + +/ was sufficient for priming, whereas the addition of Mg/sup + +/ was much less efficient. Priming by IFN-..gamma.., however, was not blocked by EGTA. Efflux of /sup 45/Ca/sup + +/ from preloaded cells was significantly increased by A23187 and by IFN-..gamma... Quin-2/AM, an intracellular chelator of Ca/sup + +/, blocked priming by IFN-..gamma...

  20. Secretory component mediates Candida albicans binding to epithelial cells.

    PubMed

    van der Wielen, P A; Holmes, A R; Cannon, R D

    2016-01-01

    Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Bacterial Exposure Induces and Activates Matrilysin in Mucosal Epithelial Cells

    PubMed Central

    López-Boado, Yolanda S.; Wilson, Carole L.; Hooper, Lora V.; Gordon, Jeffrey I.; Hultgren, Scott J.; Parks, William C.

    2000-01-01

    Matrilysin, a matrix metalloproteinase, is expressed and secreted lumenally by intact mucosal and glandular epithelia throughout the body, suggesting that its regulation and function are shared among tissues. Because matrilysin is produced in Paneth cells of the murine small intestine, where it participates in innate host defense by activation of prodefensins, we speculated that its expression would be influenced by bacterial exposure. Indeed, acute infection (10–90 min) of human colon, bladder, and lung carcinoma cells, primary human tracheal epithelial cells, and human tracheal explants with type 1–piliated Escherichia coli mediated a marked (25–50-fold) and sustained (>24 h) induction of matrilysin production. In addition, bacterial infection resulted in activation of the zymogen form of the enzyme, which was selectively released at the apical surface. Induction of matrilysin was mediated by a soluble, non-LPS bacterial factor and correlated with the release of defensin-like bacteriocidal activity. Bacteria did not induce matrilysin in other cell types, and expression of other metalloproteinases by epithelial cells was not affected by bacteria. Matrilysin was not detected in germ-free mice, but the enzyme was induced after colonization with Bacteroides thetaiotaomicron. These findings indicate that bacterial exposure is a potent and physiologically relevant signal regulating matrilysin expression in epithelial cells. PMID:10725342

  2. Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

    PubMed Central

    Tam, Anthony; Wadsworth, Samuel; Dorscheid, Delbert; Man, Shu-Fan Paul; Sin, Don D.

    2014-01-01

    Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium. PMID:24964096

  3. Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

    PubMed

    Ortega, Fabian E; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S; Lauer, Peter; Nelson, W James; Theriot, Julie A

    2017-09-06

    An intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. L. monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here, we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required. © 2017 by The American Society for Cell Biology.

  4. Characterization and genetic manipulation of primed stem cells into a functional naïve state with ESRRB

    PubMed Central

    Rossello, Ricardo Antonio; Pfenning, Andreas; Howard, Jason T; Hochgeschwender, Ute

    2016-01-01

    AIM To identify differences between primed mouse embryonic stem cells (ESCs) and fully functional naive ESCs; to manipulate primed cells into a naive state. METHODS We have cultured 3 lines of cells from different mouse strains that have been shown to be naive or primed as determined by generating germline-transmitting chimeras. Cells were put through a battery of tests to measure the different features. RNA from cells was analyzed using microarrays, to determine a priority list of the differentially expressed genes. These were later validated by quantificational real-time polymerase chain reaction. Viral cassettes were created to induce expression of differentially expressed genes in the primed cells through lentiviral transduction. Primed reprogrammed cells were subjected to in-vivo incorporation studies. RESULTS Most results show that both primed and naive cells have similar features (morphology, proliferation rates, stem cell genes expressed). However, there were some genes that were differentially expressed in the naïve cells relative to the primed cells. Key upregulated genes in naïve cells include ESRRB, ERAS, ATRX, RNF17, KLF-5, and MYC. After over-expressing some of these genes the primed cells were able to incorporate into embryos in-vivo, re-acquiring a feature previously absent in these cells. CONCLUSION Although there are no notable phenotypic differences, there are key differences in gene expression between these naïve and primed stem cells. These differences can be overcome through overexpression. PMID:27822342

  5. Lung epithelial cells induce both phenotype alteration and senescence in breast cancer cells.

    PubMed

    Furukawa, Masashi; Wheeler, Sarah; Clark, Amanda M; Wells, Alan

    2015-01-01

    The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells. Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype. Co-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture. Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.

  6. Type II alveolar epithelial cell in vitro culture in aerobiosis.

    PubMed

    Aerts, C; Voisin, C; Wallaert, B

    1988-08-01

    A method of Type II alveolar epithelial cell culture in aerobiosis has been developed. Isolation of Type II cells was performed by digesting guinea-pig lung tissue with crude trypsin and elastase and using discontinuous Percoll density gradients. The Type II cells, as identified by light and electron microscopy, were cultured in aerobiosis for up to six days, in direct contact with the atmosphere in conditions mimicking those present in the lower respiratory tract. Significant activities of cellular superoxide dismutase (SOD), manganese dependent superoxide dismutase (Mn-SOD), catalase and glutathione peroxidase (GSH-Px) were found at the time of isolation. In contrast, cell glutathione content varied widely from one experiment to another. Changes of antioxidant enzymes were evaluated during cell culture in aerobiosis. SOD, Mn-SOD and catalase were significantly decreased after three days but were not significantly different between a three day and six day culture. Antioxidant changes did not influence the cell culture. In marked contrast, decrease in cell glutathione was associated with rapid cell death, whereas good cell survival was obtained at high levels of cell glutathione. Cell culture in aerobiosis will permit a precise evaluation of the effects of gases, particularly oxidant gases, on a primary culture of Type II alveolar epithelial cells.

  7. Host epithelial geometry regulates breast cancer cell invasiveness.

    PubMed

    Boghaert, Eline; Gleghorn, Jason P; Lee, KangAe; Gjorevski, Nikolce; Radisky, Derek C; Nelson, Celeste M

    2012-11-27

    Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment.

  8. Langerin negative dendritic cells promote potent CD8+ T-cell priming by skin delivery of live adenovirus vaccine microneedle arrays

    PubMed Central

    Bachy, Veronique; Hervouet, Catherine; Becker, Pablo D.; Chorro, Laurent; Carlin, Leo M.; Herath, Shanthi; Papagatsias, Timos; Barbaroux, Jean-Baptiste; Oh, Sea-Jin; Benlahrech, Adel; Athanasopoulos, Takis; Dickson, George; Patterson, Steven; Kwon, Sung-Yun; Geissmann, Frederic; Klavinskis, Linda S.

    2013-01-01

    Stabilization of virus protein structure and nucleic acid integrity is challenging yet essential to preserve the transcriptional competence of live recombinant viral vaccine vectors in the absence of a cold chain. When coupled with needle-free skin delivery, such a platform would address an unmet need in global vaccine coverage against HIV and other global pathogens. Herein, we show that a simple dissolvable microneedle array (MA) delivery system preserves the immunogenicity of vaccines encoded by live recombinant human adenovirus type 5 (rAdHu5). Specifically, dried rAdHu5 MA immunization induced CD8+ T-cell expansion and multifunctional cytokine responses equipotent with conventional injectable routes of immunization. Intravital imaging demonstrated MA cargo distributed both in the epidermis and dermis, with acquisition by CD11c+ dendritic cells (DCs) in the dermis. The MA immunizing properties were attributable to CD11c+ MHCIIhi CD8αneg epithelial cell adhesion molecule (EpCAMneg) CD11b+ langerin (Lang; CD207)neg DCs, but neither Langerhans cells nor Lang+ DCs were required for CD8+ T-cell priming. This study demonstrates an important technical advance for viral vaccine vectors progressing to the clinic and provides insights into the mechanism of CD8+ T-cell priming by live rAdHu5 MAs. PMID:23386724

  9. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

    PubMed Central

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M.; Collins, Jane E.; Davies, Donna E.; Morgan, Hywel; Swindle, Emily J.

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  10. Molecular mechanisms of membrane polarity in renal epithelial cells.

    PubMed

    Campo, C; Mason, A; Maouyo, D; Olsen, O; Yoo, D; Welling, P A

    2005-01-01

    Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.

  11. Epithelial Cell Proliferation Contributes to Airway Remodeling in Severe Asthma

    PubMed Central

    Cohen, Lance; E, Xueping; Tarsi, Jaime; Ramkumar, Thiruvamoor; Horiuchi, Todd K.; Cochran, Rebecca; DeMartino, Steve; Schechtman, Kenneth B.; Hussain, Iftikhar; Holtzman, Michael J.; Castro, Mario

    2007-01-01

    Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. Measurements and Main Results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p = 0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p = 0.002) and Ki67 was increased (p < 0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p < 0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p = 0.002), suggesting increased cell death. Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe

  12. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  13. Spraying Respiratory Epithelial Cells to Coat Tissue-Engineered Constructs

    PubMed Central

    Thiebes, Anja Lena; Albers, Stefanie; Klopsch, Christian; Jockenhoevel, Stefan; Cornelissen, Christian G.

    2015-01-01

    Abstract Applying cells in a spray can overcome current hurdles in coating tissue engineered constructs with a thin layer of endo- or epithelial cells. We report here a structured study on the influences of spray application with a medical spray device on vascular smooth muscle cells (vSMCs) and respiratory epithelial cells (RECs) with and without fibrin gel. Next to viability and cytotoxicity assays, the in vitro differentiation capacity after spray processing was analyzed. For vSMC, no influence of air pressures till 0.8 bar could be shown, whereas the viability decreased for higher pressures. The viability of RECs was reduced to 88.5% with 0.4 bar air pressure. Lactate dehydrogenase-levels in the culture medium increased the first day after spraying but normalized afterward. In the short term, no differences by means of morphology and expression-specific markers for vSMCs and RECs were seen between the control and study group. In addition, in a long-term study for 28 days with the air–liquid interface, RECs differentiated and built up an organized epithelial layer with ciliary development that was comparable to the control for cells sprayed without fibrin gel. When spraying within fibrin gel, ciliary development was lower at 28 days. Thus, spraying of vSMCs and RECs was proved to be a suitable method for tissue engineering. Especially for RECs, this application is of special significance when coating luminal structures or other unfavorable topographies. PMID:26309803

  14. Effects of weaning on intestinal crypt epithelial cells in piglets

    PubMed Central

    Yang, Huansheng; Xiong, Xia; Wang, Xiaocheng; Li, Tiejun; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells in the crypt proliferate in piglets in response to weaning. However, the underlying mechanism has been unclear. We examined 40 piglets from eight litters (five piglets per litter) that were weaned at the age of 14 d, and one piglet from each litter was randomly selected for closer investigation. Based on the distended intestinal sac method, we isolated crypt epithelial cells from the mid-jejunum on Days 0, 1, 3, 5, and 7 post-weaning. Protein expression was analyzed using either isobaric tags for relative and absolute quantification or western blotting. Proteins related to the cell cycle, organization of the cellular macromolecular complex subunit, localization of cellular macromolecules, Golgi vesicle transport, fatty acid metabolism, oxidative phosphorylation, and translational initiation were mainly down-regulated, while those involved in glycolysis, cell cycle arrest, protein catabolism, and cellular amino acid metabolism were up-regulated. The amount of proteins active in the mTOR signaling pathway was generally decreased over time. These results indicate that weaning influences energy metabolism, cellular macromolecule organization and localization, and protein metabolism, thereby affecting the proliferation of intestinal epithelial cells in weaned piglets. Moreover, those cellular processes are possibly controlled by that signaling pathway. PMID:27830738

  15. Expression of inducible nitric oxide in human lung epithelial cells.

    PubMed

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  16. Asbestos exposure increases human bronchial epithelial cell fibrinolytic activity.

    PubMed

    Gross, T J; Cobb, S M; Gruenert, D C; Peterson, M W

    1993-03-01

    Chronic exposure to asbestos fibers results in fibrotic lung disease. The distal pulmonary epithelium is an early target of asbestos-mediated injury. Local plasmin activity may be important in modulating endoluminal inflammatory responses in the lung. We studied the effects of asbestos exposure on cell-mediated plasma clot lysis as a marker of pericellular plasminogen activation. Exposing human bronchial epithelial (HBE) cells to 100 micrograms/ml of asbestos fibers for 24 h resulted in increased plasma clot lysis. Fibrinolytic activity was augmented in a dose-dependent fashion, was not due to secreted protease, and occurred only when there was direct contact between the plasma clot and the epithelial monolayer. Further analysis showed that asbestos exposure increased HBE cell-associated urokinase-type plasminogen activator (uPA) activity in a time-dependent manner. The increased cell-associated PA activity could be removed by acid washing. The increase in PA activity following asbestos exposure required new protein synthesis because it was abrogated by treatment with either cycloheximide or actinomycin D. Therefore, asbestos exposure increases epithelial-mediated fibrinolysis by augmenting expression of uPA activity at the cell surface by mechanisms that require new RNA and protein synthesis. These observations suggest a novel mechanism whereby exposure of the distal epithelium to inhaled particulates may result in a chronic inflammatory response that culminates in the development of fibrotic lung disease.

  17. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  18. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  19. Generation of Spheres from Dental Epithelial Stem Cells

    PubMed Central

    Natsiou, Despoina; Granchi, Zoraide; Mitsiadis, Thimios A.; Jimenez-Rojo, Lucia

    2017-01-01

    The in vitro three-dimensional sphere model has already been established as an important tool in fundamental sciences. This model facilitates the study of a variety of biological processes including stem cell/niche functions and tissue responses to injury and drugs. Here we describe the complete protocol for the in vitro formation of spheres originated from the epithelium of rodent incisors. In addition, we show that in these spheres cell proliferation is maintained, as well as the expression of several key molecules characterizing stem cells such as Sox2 and p63. These epithelial dentospheres could be used as an in vitro model system for stem cell research purposes. PMID:28154538

  20. Hepatic stellate cells promote upregulation of epithelial cell adhesion molecule and epithelial-mesenchymal transition in hepatic cancer cells.

    PubMed

    Nagahara, Teruya; Shiraha, Hidenori; Sawahara, Hiroaki; Uchida, Daisuke; Takeuchi, Yasuto; Iwamuro, Masaya; Kataoka, Junro; Horiguchi, Shigeru; Kuwaki, Takeshi; Onishi, Hideki; Nakamura, Shinichiro; Takaki, Akinobu; Nouso, Kazuhiro; Yamamoto, Kazuhide

    2015-09-01

    Microenvironment plays an important role in epithelial-mesenchymal transition (EMT) and stemness of cells in hepatocellular carcinoma (HCC). Epithelial cell adhesion molecule (EpCAM) is known as a tumor stemness marker of HCC. To investigate the relationship between microenvironment and stemness, we performed an in vitro co-culture assay. Four HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) were co-cultured with the TWNT-1 immortalized hepatic stellate cells (HSCs), which create a microenvironment with HCC. Cell proliferation ability was analyzed by flow cytometry (FCM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while migration ability was assessed by a wound healing assay. Expression of EpCAM was analyzed by immunoblotting and FCM. HCC cell lines were co-cultured with TWNT-1 treated with small interfering RNA (siRNA) for TGF-β and HB-EGF; we then analyzed proliferation, migration ability and protein expression using the methods described above. Proliferation ability was unchanged in HCC cell lines co-cultured with TWNT-1. Migration ability was increased in HCC cell lines (HepG2, Hep3B, HuH-7 and PLC/PRF/5) directly (216.2±67.0, 61.0±22.0, 124.0±66.2 and 51.5±40.3%) and indirectly (102.5±22.0, 84.6±30.9, 86.1±25.7 and 73.9±29.7%) co-cultured with TWNT-1 compared with the HCC uni-culture. Immunoblot analysis revealed increased EpCAM expression in the HCC cell lines co-cultured with TWNT-1. Flow cytometry revealed that the population of E-cadherin-/N-cadherin+ and EpCAM-positive cells increased and accordingly, EMT and stemness in the HCC cell line were activated. These results were similar in the directly and indirectly co-cultured samples, indicating that humoral factors were at play. Conversely, HCC cell lines co-cultured with siRNA‑treated TWNT-1 showed decreased migration ability, a decreased population of EpCAM-positive and E-cadherin-/N-cadherin+ cells. Taken together, humoral factors secreted from TWNT-1

  1. Rhinovirus-Induced IL-1β Release from Bronchial Epithelial Cells Is Independent of Functional P2X7

    PubMed Central

    Shi, Lei; Manthei, David M.; Guadarrama, Arturo G.; Lenertz, Lisa Y.

    2012-01-01

    Airway epithelial cell defenses to viral infections are often compromised in disease or injury. Danger molecules, including ATP, are released during infection and contribute to nucleotide receptor–dependent inflammatory responses, largely through P2X7. Although respiratory epithelium has been shown to express a variety of nucleotide receptors, the functional contribution of P2X7 to the epithelial cell inflammatory response is unclear. We used human donor bronchial epithelial cells (BECs) and primary brushed epithelium to explore responses upon nucleotide and Toll-like receptor stimulation. P2X7 messenger RNA and protein were observed in unprimed BECs, whereas inflammatory cytokine stimulation increased both messenger RNA and protein. Functional pore activity characteristic of P2X7 was observed in BECs, and IL-1β was rapidly released by BECs after Toll-like receptor 3 agonist, polyinosine-polycytidylic acid, priming followed by ATP administration, although no change was observed in IL-18 release. BECs produced more IL-1β after stimulation with polyinosine-polycytidylic acid than LPS, showing a different preferential response than monocytes. In addition, blockade of nucleotide receptors with oxidized ATP significantly increased human rhinovirus (HRV) recovered 24 hours after infection in BECs, whereas 2′-3′-O-(4-benzoylbenzoyl) ATP treatment of brushed epithelial cells and respiratory cell lines nonsignificantly decreased HRV recovery. IL-1β release was detected after HRV infection in both BECs and brushed cells, but BzATP did not significantly increase IL-1β release further. BEC processing of pro–IL-1β to the mature, cleaved, 17-kD form was confirmed by Western blotting. These results support the expression of functional P2X7 in human lung epithelium, although its role in epithelial pathogen defense is likely independent of IL-1 family cytokine processing. PMID:22493010

  2. Prime Knowledge about Primes

    ERIC Educational Resources Information Center

    Eisenberg, Theodore

    2007-01-01

    Several proofs demonstrating that there are infinitely many primes, different types of primes, tests of primality, pseudo primes, prime number generators and open questions about primes are discussed in Section 1. Some of these notions are elaborated upon in Section 2, with discussions of the Riemann zeta function and how algorithmic complexity…

  3. Prime Knowledge about Primes

    ERIC Educational Resources Information Center

    Eisenberg, Theodore

    2007-01-01

    Several proofs demonstrating that there are infinitely many primes, different types of primes, tests of primality, pseudo primes, prime number generators and open questions about primes are discussed in Section 1. Some of these notions are elaborated upon in Section 2, with discussions of the Riemann zeta function and how algorithmic complexity…

  4. Epigenetics in Intestinal Epithelial Cell Renewal

    PubMed Central

    Roostaee, Alireza; Benoit, Yannick D.; Boudjadi, Salah

    2016-01-01

    A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. J. Cell. Physiol. 231: 2361–2367, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27061836

  5. Progenitor Cells in Proximal Airway Epithelial Development and Regeneration

    PubMed Central

    Lynch, Thomas J.; Engelhardt, John F.

    2015-01-01

    Multiple distinct epithelial domains are found throughout the airway that are distinguishable by location, structure, function, and cell-type composition. Several progenitor cell populations in the proximal airway have been identified to reside in confined microenvironmental niches including the submucosal glands (SMGs), which are embedded in the tracheal connective tissue between the surface epithelium and cartilage, and basal cells that reside within the surface airway epithelium (SAE). Current research suggests that regulatory pathways that coordinate development of the proximal airway and establishment of progenitor cell niches may overlap with pathways that control progenitor cell responses during airway regeneration following injury. SMGs have been shown to harbor epithelial progenitor cells, and this niche is dysregulated in diseases such as cystic fibrosis. However, mechanisms that regulate progenitor cell proliferation and maintenance within this glandular niche are not completely understood. Here we discuss glandular progenitor cells during development and regeneration of the proximal airway and compare properties of glandular progenitors to those of basal cell progenitors in the SAE. Further investigation into glandular progenitor cell control will provide a direction for interrogating therapeutic interventions to correct aberrant conditions affecting the SMGs in diseases such as cystic fibrosis, chronic bronchitis, and asthma. PMID:24818588

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

    PubMed Central

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

    2016-01-01

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

  7. Azithromycin kills invasive Aggregatibacter actinomycetemcomitans in gingival epithelial cells.

    PubMed

    Lai, Pin-Chuang; Walters, John D

    2013-03-01

    Aggregatibacter actinomycetemcomitans invades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelial A. actinomycetemcomitans. [(3)H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers with A. actinomycetemcomitans (Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited by l-carnitine and several other organic cations. Cell incubation in medium containing 5 μg/ml azithromycin yielded steady-state intracellular concentrations of 144 μg/ml in SCC-25 cells and 118 μg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of both A. actinomycetemcomitans strains. Treatment of infected Smulow-Glickman cells with 0.125 μg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 μg/ml azithromycin killed ≥82% of the CFU of both strains (P < 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (P < 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelial A. actinomycetemcomitans and is shared with organic cations.

  8. Differentiation of cultured epithelial cells: response to toxic agents.

    PubMed Central

    Rice, R H; LaMontagne, A D; Petito, C T; Rong, X H

    1989-01-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Great differences were evident even among those cells derived from stratified squamous epithelia (epidermal, esophageal, vaginal, forestomach) despite their expression of aryl hydrocarbon hydroxylase activities to similar degrees. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAMP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Although expressing keratinocyte character (transglutaminase activity and envelope forming ability), the cells thus retain some hormonal character that may be modulated by cAMP-dependent kinase activity. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents. Images FIGURE 1. FIGURE 4. PMID:2466642

  9. Epstein-Barr Virus Transcytosis through Polarized Oral Epithelial Cells

    PubMed Central

    Herrera, Rossana; Palefsky, Joel M.

    2013-01-01

    Although Epstein-Barr virus (EBV) is an orally transmitted virus, viral transmission through the oropharyngeal mucosal epithelium is not well understood. In this study, we investigated how EBV traverses polarized human oral epithelial cells without causing productive infection. We found that EBV may be transcytosed through oral epithelial cells bidirectionally, from both the apical to the basolateral membranes and the basolateral to the apical membranes. Apical to basolateral EBV transcytosis was substantially reduced by amiloride, an inhibitor of macropinocytosis. Electron microscopy showed that virions were surrounded by apical surface protrusions and that virus was present in subapical vesicles. Inactivation of signaling molecules critical for macropinocytosis, including phosphatidylinositol 3-kinases, myosin light-chain kinase, Ras-related C3 botulinum toxin substrate 1, p21-activated kinase 1, ADP-ribosylation factor 6, and cell division control protein 42 homolog, led to significant reduction in EBV apical to basolateral transcytosis. In contrast, basolateral to apical EBV transcytosis was substantially reduced by nystatin, an inhibitor of caveolin-mediated virus entry. Caveolae were detected in the basolateral membranes of polarized human oral epithelial cells, and virions were detected in caveosome-like endosomes. Methyl β-cyclodextrin, an inhibitor of caveola formation, reduced EBV basolateral entry. EBV virions transcytosed in either direction were able to infect B lymphocytes. Together, these data show that EBV transmigrates across oral epithelial cells by (i) apical to basolateral transcytosis, potentially contributing to initial EBV penetration that leads to systemic infection, and (ii) basolateral to apical transcytosis, which may enable EBV secretion into saliva in EBV-infected individuals. PMID:23698302

  10. Elevated tropomyosin expression is associated with epithelial-mesenchymal transition of lens epithelial cells.

    PubMed

    Kubo, Eri; Hasanova, Nailia; Fatma, Nigar; Sasaki, Hiroshi; Singh, Dhirendra P

    2013-01-01

    Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α-smooth muscle actin (α-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.

  11. Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

    PubMed

    Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

    2015-04-01

    The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

  12. Iodide handling by the thyroid epithelial cell.

    PubMed

    Nilsson, M

    2001-01-01

    Iodination of thyroglobulin, the key event in the synthesis of thyroid hormone, is an extracellular process that takes place inside the thyroid follicles at the apical membrane surface that faces the follicular lumen. The supply of iodide involves two steps of TSH-regulated transport, basolateral uptake and apical efflux, that imprint the polarized phenotype of the thyroid cell. Iodide uptake is generated by the sodium/iodide symporter present in the basolateral plasma membrane. A candidate for the apical iodide-permeating mechanism is pendrin, a chloride/iodide transporting protein recently identified in the apical membrane. In physiological conditions, transepithelial iodide transport occurs without intracellular iodination, despite the presence of large amounts of thyroglobulin and thyroperoxidase inside the cells. The reason is that hydrogen peroxide, serving as electron acceptor in iodide-protein binding and normally produced at the apical cell surface, is rapidly degraded by cytosolic glutathione peroxidase once it enters the cells. Iodinated thyroglobulin in the lumen stores not only thyroid hormone but iodine incorporated in iodotyrosine residues as well. After endocytic uptake and degradation of thyroglobulin, intracellular deiodination provides a mechanism for recycling of iodide to participate in the synthesis of new thyroid hormone at the apical cell surface.

  13. Effects of ozone on the nasal mucosa (epithelial cells).

    PubMed

    Dokic, D; Howarth, H P

    2006-12-01

    Ozone is a significant public health concern worldwide. Despite increasing evidence for the role of the bronchial epithelial cells in the generation of proinflammatory cytokines there is little information on the biological relevance of ozone induced release of cytokines in nasal airway inflammation. We have investigated the effect of ozone on the nasal mucosa using immunohistochemical staining of nasal biopsies taken 6h after exposure to either 400 ppb ozone or filtered. We found that ozone significantly increases the number of neutrophils in the epithelium (p=0.03), and expression of NF-kB (p<0.03), TNF-a (p<0.05), IL-1b (p<0.03), IL-8 (p<0.007), IL-6 (p<0.02), GM-CSF (p<0.02) and ICAM-1 (p <0.01) in the epithelial cells 6h after exposure. Furthermore, we found a significant correlations between IL-8 expression and number of neutrophils (r=0.85, p< 0.002) and NF-kB and TNF-a expression (r=0.77, p<0.009) in the epithelium. These results suggest that ozone-induced inflammation of the nasal mucosa may be a consequence of increased synthesis and release of epithelial cell-derived cytokines and adhesion molecules which influence the activity of inflammatory cells.

  14. Serratia marcescens is injurious to intestinal epithelial cells

    PubMed Central

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens. PMID:25426769

  15. Brucella pinnipedialis in hooded seal (Cystophora cristata) primary epithelial cells.

    PubMed

    Larsen, Anett Kristin; Godfroid, Jacques; Nymo, Ingebjørg Helena

    2016-01-25

    Marine Brucella spp. have been isolated from numerous pinniped and cetacean species, but pathological findings in association with infection with Brucella pinnipedialis in pinnipeds have been sparse. The capacity of brucellae to survive and replicate within host macrophages underlies their important ability to produce chronic infections, but previous work has shown that B. pinnipedialis spp. are rapidly eliminated from hooded seal (Cystophora cristata) alveolar macrophages. To investigate if multiplication could take place in other hooded seal cell types, primary epithelial cells were isolated, verified to express the epithelial marker cytokeratin and challenged with three different strains of B. pinnipedialis; B. pinnipedialis sp. nov., B. pinnipedialis hooded seal strain B17, and B. pinnipedialis hooded seal strain 22F1. All strains were steadily eliminated and the amounts of intracellular bacteria were reduced to less than one-third by 48 h post infection. Intracellular presence was verified using immunocytochemistry. So far, intracellular multiplication in seal cells has not been documented for B. pinnipedialis. The lack of intracellular survival in macrophages, as well as in epithelial cells, together with the fact that pathological changes due to B. pinnipedialis infection is not yet identified in seals, suggests that the bacteria may only cause a mild, acute and transient infection. These findings also contribute to substantiate the hypothesis that seals may not be the primary host of B. pinnipedialis and that the transmission to seals are caused by other species in the marine environment.

  16. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    PubMed Central

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  17. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells.

    PubMed

    Duss, Stephan; Brinkhaus, Heike; Britschgi, Adrian; Cabuy, Erik; Frey, Daniel M; Schaefer, Dirk J; Bentires-Alj, Mohamed

    2014-06-10

    Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer.

  18. Murine Langerin+ dermal dendritic cells prime CD8+ T cells while Langerhans cells induce cross-tolerance

    PubMed Central

    Flacher, Vincent; Tripp, Christoph H; Mairhofer, David G; Steinman, Ralph M; Stoitzner, Patrizia; Idoyaga, Juliana; Romani, Nikolaus

    2014-01-01

    Skin dendritic cells (DCs) control the immunogenicity of cutaneously administered vaccines. Antigens targeted to DCs via the C-type lectin Langerin/CD207 are cross-presented to CD8+ T cells in vivo. We investigated the relative roles of Langerhans cells (LCs) and Langerin+ dermal DCs (dDCs) in different vaccination settings. Poly(I:C) and anti-CD40 agonist antibody promoted cytotoxic responses upon intradermal immunization with ovalbumin (OVA)-coupled anti-Langerin antibodies (Langerin/OVA). This correlated with CD70 upregulation in Langerin+ dDCs, but not LCs. In chimeric mice where Langerin targeting was restricted to dDCs, CD8+ T-cell memory was enhanced. Conversely, providing Langerin/OVA exclusively to LCs failed to prime cytotoxicity, despite initial antigen cross-presentation to CD8+ T cells. Langerin/OVA combined with imiquimod could not prime CD8+ T cells and resulted in poor cytotoxicity in subsequent responses. This tolerance induction required targeting and maturation of LCs. Altogether, Langerin+ dDCs prime long-lasting cytotoxic responses, while cross-presentation by LCs negatively influences CD8+ T-cell priming. Moreover, this highlights that DCs exposed to TLR agonists can still induce tolerance and supports the existence of qualitatively different DC maturation programs. PMID:25085878

  19. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    USDA-ARS?s Scientific Manuscript database

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  20. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  1. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  2. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

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

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left–right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left–right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left–right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  3. Multipotent Capacity of Immortalized Human Bronchial Epithelial Cells

    PubMed Central

    Delgado, Oliver; Kaisani, Aadil A.; Spinola, Monica; Xie, Xian-Jin; Batten, Kimberly G.; Minna, John D.; Wright, Woodring E.; Shay, Jerry W.

    2011-01-01

    While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer. PMID:21760947

  4. Epithelial cell polarity determinant CRB3 in cancer development.

    PubMed

    Li, Pingping; Mao, Xiaona; Ren, Yu; Liu, Peijun

    2015-01-01

    Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development.

  5. Oral Epithelial Cell Responses to Multispecies Microbial Biofilms

    PubMed Central

    Peyyala, R.; Kirakodu, S.S.; Novak, K.F.; Ebersole, J.L

    2013-01-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms. PMID:23300185

  6. [Advances in Classification and Research Methods of Lung Epithelial Stem 
and Progenitor Cells].

    PubMed

    Deng, Minhua; Li, Jinhua; Gan, Ye; Chen, Ping

    2017-02-20

    Isolation and characterization of lung epithelial stem and progenitor cells and understanding of their specific role in lung physiopathology are critical for preventing and controlling lung diseases including lung cancer. In this review, we summarized recent advances in classification and research methods of lung epithelial stem and progenitor cells. Lung epithelial stem and progenitor cells were region-specific, which primarily included basal cells and duct cells in proximal airway, Clara cells, variant Clara cells, bronchioalveolar stem cells and induced krt5+ cells in bronchioles, type II alveolar cells and type II alveolar progenitor cells in alveoli. The research methods of lung epithelial stem and progenitor cells were mainly focused on lung injury models, lineage-tracing experiments, three dimensional culture, transplantation, chronic labeled cells and single-cell transcriptome analysis. Lastly, the potential relationship between lung epithelial stem and progenitor cells and lung cancer as well as lung cancer stem cell-targeted drug development were briefly reviewed.

  7. Interaction between submicron COD crystals and renal epithelial cells

    PubMed Central

    Peng, Hua; Ouyang, Jian-Ming; Yao, Xiu-Qiong; Yang, Ru-E

    2012-01-01

    Objectives This study aims to investigate the adhesion characteristics between submicron calcium oxalate dihydrate (COD) with a size of 150 ± 50 nm and African green monkey kidney epithelial cells (Vero cells) before and after damage, and to discuss the mechanism of kidney stone formation. Methods Vero cells were oxidatively injured by hydrogen peroxide to establish a model of injured cells. Scanning electron microscopy was used to observe Vero–COD adhesion. Inductively coupled plasma emission spectrometry was used to quantitatively measure the amount of adhered COD microcrystals. Nanoparticle size analyzer and laser scanning confocal microscopy were performed to measure the change in the zeta potential on the Vero cell surface and the change in osteopontin expression during the adhesion process, respectively. The level of cell injury was evaluated by measuring the changes in malonaldehyde content, and cell viability during the adhesion process. Results The adhesion capacity of Vero cells in the injury group to COD microcrystals was obviously stronger than that of Vero cells in the control group. After adhesion to COD, cell viability dropped, both malonaldehyde content and cell surface zeta potential increased, and the fluorescence intensity of osteopontin decreased because the osteopontin molecules were successfully covered by COD. Submicron COD further damaged the cells during the adhesion process, especially for Vero cells in the control group, leading to an elevated amount of attached microcrystals. Conclusion Submicron COD can further damage injured Vero cells during the adhesion process. The amount of attached microcrystals is proportional to the degree of cell damage. The increased amount of microcrystals that adhered to the injured epithelial cells plays an important role in the formation of early-stage kidney stones. PMID:22973095

  8. Maintenance of Epithelial Stem Cells by Cbl Proteins

    DTIC Science & Technology

    2013-09-01

    our research findings during the entire grant period (Sept. 2010 – Aug. 2013). 1. Analysis of Cbl functions in progenitor-type mammary epithelial...catenin pathway, but further investigation is required to establish this. 2. Analysis of Cbl functions in vivo using gene mutant mouse models We...Nandwani N, Gu H, Band V, Band H. Rapidly fatal myeloproliferative disorders in mice with deletion of Casitas B-cell lymphoma (Cbl) and Cbl-b in

  9. Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells

    PubMed Central

    Pettengill, Matthew A.; Lam, Verissa W.; Ollawa, Ikechukwu; Marques-da-Silva, Camila; Ojcius, David M.

    2012-01-01

    Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia. PMID:23119027

  10. Characterization of foamy epithelial surface cells in the canine endometrium.

    PubMed

    Bartel, C; Tichy, A; Walter, I

    2014-06-01

    In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, β-catenin and E-cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E-cadherin and β-catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid-accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.

  11. Norepinephrine potentiates proinflammatory responses of human vaginal epithelial cells.

    PubMed

    Brosnahan, Amanda J; Vulchanova, Lucy; Witta, Samantha R; Dai, Yuying; Jones, Bryan J; Brown, David R

    2013-06-15

    The vaginal epithelium provides a barrier to pathogens and recruits immune defenses through the secretion of cytokines and chemokines. Several studies have shown that mucosal sites are innervated by norepinephrine-containing nerve fibers. Here we report that norepinephrine potentiates the proinflammatory response of human vaginal epithelial cells to products produced by Staphylococcus aureus, a pathogen that causes menstrual toxic shock syndrome. The cells exhibit immunoreactivity for catecholamine synthesis enzymes and the norepinephrine transporter. Moreover, the cells secrete norepinephrine and dopamine at low concentrations. These results indicate that norepinephrine may serve as an autocrine modulator of proinflammatory responses in the vaginal epithelium.

  12. Epithelial stem cells are formed by small-particles released from particle-producing cells

    PubMed Central

    Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

    2017-01-01

    Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

  13. Role of medullary progenitor cells in epithelial cell migration and proliferation

    PubMed Central

    Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

    2014-01-01

    This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

  14. Erythropoietin Induces an Epithelial to Mesenchymal Transition-like Process in Mammary Epithelial Cells MCF10A.

    PubMed

    Ordoñez-Moreno, Alejandra; Rodriguez-Monterrosas, Cecilia; Cortes-Reynosa, Pedro; Perez-Carreon, Julio Isael; Perez Salazar, Eduardo

    2017-03-01

    Anemia is associated with chemotherapy treatment in cancer patients. Erythropoietin (EPO) has been used to treat anemia of cancer patients, because it stimulates erythropoiesis. However, treatment of breast cancer patients with EPO has been associated with poor prognosis and decrease of survival. Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells are transdifferentiated to a mesenchymal state. It has been implicated in tumor progression, because epithelial cells acquire the capacity to execute the multiple steps of invasion/metastasis process. However, the role of EPO on EMT process in human mammary epithelial cells has not been studied. In the present study, we demonstrate that EPO promotes a decrease of E-cadherin expression, an increase of N-cadherin, vimentin and Snail2 expression, activation of FAK and Src kinases and an increase of MMP-2 and MMP-9 secretions. Moreover, EPO induces an increase of NFκB DNA binding activity, an increase of binding of p50 and p65 NFκB subunits to Snail1 promoter, migration and invasion in mammary non-tumorigenic epithelial cells MCF10A. In summary, these findings demonstrate, for the first time, that EPO induces an EMT-like process in mammary non-tumorigenic epithelial cells. This article is protected by copyright. All rights reserved.

  15. Notch as a Driver of Gastric Epithelial Cell Proliferation.

    PubMed

    Demitrack, Elise S; Samuelson, Linda C

    2017-05-01

    The gastric epithelium is sustained by a population of stem cells that replenish the various mature epithelial lineages throughout adulthood. Regulation of stem and progenitor cell proliferation occurs via basic developmental signaling pathways, including the Notch pathway, which recently was described to promote gastric stem cell proliferation in both mice and human beings. Current cancer theory proposes that adult stem cells that maintain gastrointestinal tissues accumulate mutations that promote cancerous growth, and that basic signaling pathways, such as Notch, which stimulate stem cell proliferation, can promote tumorigenesis. Accordingly, constitutive Notch activation leads to unchecked cellular proliferation and gastric tumors in genetic mouse models. Furthermore, there is emerging evidence suggesting that the Notch pathway may be activated in some human gastric cancers, supporting a potential role for Notch in gastric tumorigenesis. In this review, we first summarize the current understanding of gastric stem cells defined by genetic mouse studies, followed by discussion of the literature regarding Notch pathway regulation of gastric stem cell function in the mouse and human beings. Notch action to maintain gastric epithelial cell homeostasis and the cellular consequences of dysregulated signaling to promote tumorigenesis are discussed, including studies associating Notch activation with human gastric cancer. Finally, we compare and contrast Notch function in the stomach with other gastrointestinal tissues, including the intestine, to highlight the sensitivity of the stomach to Notch-induced tumors.

  16. A dynamic podosome-like structure of epithelial cells.

    PubMed

    Spinardi, Laura; Rietdorf, Jens; Nitsch, Lucio; Bono, Maria; Tacchetti, Carlo; Way, Michael; Marchisio, Pier Carlo

    2004-05-01

    Focal contacts and hemidesmosomes are cell-matrix adhesion structures of cultured epithelial cells. While focal contacts link the extracellular matrix to microfilaments, hemidesmosomes make connections with intermediate filaments. We have analyzed hemidesmosome assembly in 804G carcinoma cells. Our data show that hemidesmosomes are organized around a core of actin filaments that appears early during cell adhesion. These actin structures look similar to podosomes described in cells of mesenchymal origin. These podosome-like structures are distinct from focal contacts and specifically contain Arp3 (Arp2/3 complex), cortactin, dynamin, gelsolin, N-WASP, VASP, Grb2 and src-like kinase(s). The integrin alpha3beta1 is localized circularly around F-actin cores and co-distributes with paxillin, vinculin, and zyxin. We also show that the maintenance of the actin core and hemidesmosomes is dependent on actin polymerization, src-family kinases, and Grb2, but not on microtubules. Video microscopy analysis reveals that assembly of hemidesmosomes is preceded by recruitment of beta4 integrin subunit to the actin core before its positioning at hemidesmosomes. When 804G cells are induced to migrate, actin cores as well as hemidesmosomes disappear and beta4 integrin subunit becomes co-localized with dynamic actin at leading edges. We show that podosome-like structures are not unique to cells of mesenchymal origin, but also appear in epithelial cells, where they seem to be related to basement membrane adhesion.

  17. High glucose modifies heparansulphate synthesis by mouse glomerular epithelial cells.

    PubMed

    Morano, S; Guidobaldi, L; Cipriani, R; Gabriele, A; Pantellini, F; Medici, F; D'Erme, M; Di Mario, U

    1999-01-01

    Alterations in proteoglycan metabolism are involved in the pathogenesis of diabetic nephropathy. The aim of this study is to evaluate the effects of high glucose on proteoglycan production and to find a reliable in vitro model for the study of diabetic nephropathy. A clone of mouse glomerular epithelial cells was cultured in media containing elevated (30 mmol) and physiological (5 mmol) glucose, or iso-osmolar (30 mmol) mannitol concentrations. We evaluated the synthesis of 35SO4-labeled molecules and the amount of proteoglycans by Sepharose CL6B and DEAE-Sephacel chromatographies. A clear decrease (56%) in total cell-layer proteoglycan synthesis was induced by 30 mmol glucose, in comparison with normal glucose. A reduction of 25% in medium associated proteoglycan synthesis was observed in high glucose cultured cells. After Sepharose CL6B, in cells cultured in high glucose, cell layer heparansulphate proteoglycan-I (Kav 6B 0. 04) synthesis was reduced by about 81%, heparansulphate proteoglycan-II (Kav 6B 0.21) by about 87% and heparansulphate glycosaminoglycan (Kav 0.4-0.8) by about 91%, respectively. In mannitol-incubated cells the reductions observed were less evident and not significantly different from those in normal glucose. These results indicate that (1) glomerular epithelial cells play a central role in proteoglycan synthesis, (2) high glucose modifies the amount and influences the different species production of these macromolecules, while osmotic forces seem to be only partially involved in these effects, and (3) this cellular clone of glomerular epithelial cells can represent a reliable in vitro model for the study of the mechanisms involved in diabetic nephropathy. Copyright 1999 John Wiley & Sons, Ltd.

  18. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    PubMed Central

    Yaghi, Asma; Dolovich, Myrna B.

    2016-01-01

    Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations. PMID:27845721

  19. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

    PubMed Central

    Corbeil, L B; Hodgson, J L; Jones, D W; Corbeil, R R; Widders, P R; Stephens, L R

    1989-01-01

    Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective. Images PMID:2471692

  20. Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

    PubMed Central

    Xing, Yan; Hogquist, Kristin A.

    2014-01-01

    The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution. PMID:25145384

  1. Characterization of Human Mammary Epithelial Stem Cells

    DTIC Science & Technology

    2008-10-01

    9 Appendix……………………………………………………………………………… 10 Eirew,P., Stingl,J., Raouf,A., Turashvili,G., Aparicio ,S., Emerman,J.T., and Eaves,C.J. A...Peter Eirew, John Stingl, Afshin Raouf, Gulisa Turashvili, Samuel Aparicio , Joanne Emerman and Connie Eaves. A method for quantifying normal human...Eirew, Afshin Raouf, John Stingl, Gulisa Turashvili, Allen Delaney, Joanne Emerman, Marco Marra and Samuel Aparicio . “Stem Cells in the Mammary Gland

  2. Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

    PubMed

    Ghosh, Moumita; Ahmad, Shama; White, Carl W; Reynolds, Susan D

    2017-01-01

    Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

  3. Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

    PubMed Central

    Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

    2013-01-01

    Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

  4. Synthesis of sulfated oligosaccharides by cystic fibrosis trachea epithelial cells.

    PubMed

    Mendicino, J; Sangadala, S

    1999-11-01

    The mucin glycoproteins in tracheal mucus of patients with cystic fibrosis is more highly sulfated than the corresponding secretions from healthy individuals [16]. In order to further characterize these differences in sulfation and possibly also glycosylation patterns, we compared the structures of sulfated mucin oligosaccharides synthesized by continuously cultured human tracheal cells transformed by simian virus 40. The synthesis of highly sulfated oligosaccharide chains in mucins secreted by normal human epithelial and submucosal cell lines were compared with mucins formed by cystic fibrosis tracheal epithelial and submucosal cell lines. The epithelial cell lines from cystic fibrosis trachea showed a higher rate of sulfate uptake and a significantly higher rate of synthesis and sulfation of high molecular weight chains. Mucins synthesized by each cell line in the presence of 35SO4 were isolated and oligosaccharide chains were released by beta-elimination and separated by ion exchange chromatography and gel filtration. The sulfated high molecular weight chains synthesized by the cystic fibrosis cell lines were characterized by methylation analysis and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GlcNAc in a ratio of 1:2:2.2 and only one galactosaminitol residue for about every 150-200 sugar residues present. The average molecular size of oligosaccharide chains in these fractions was between 30,000-40,000 daltons. These studies show that increased sulfation of oligosaccharides in mucins synthesized by cells from cystic fibrosis trachea is accompanied by a significant increase in the extension of a basic branched structure present in many of the lower molecular weight oligosaccharides.

  5. Prion infection of epithelial Rov cells is a polarized event.

    PubMed

    Paquet, Sophie; Sabuncu, Elifsu; Delaunay, Jean-Louis; Laude, Hubert; Vilette, Didier

    2004-07-01

    During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells.

  6. Prion Infection of Epithelial Rov Cells Is a Polarized Event

    PubMed Central

    Paquet, Sophie; Sabuncu, Elifsu; Delaunay, Jean-Louis; Laude, Hubert; Vilette, Didier

    2004-01-01

    During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells. PMID:15194791

  7. Neurotransmitter Influence on Human Meibomian Gland Epithelial Cells

    PubMed Central

    Kam, Wendy R.

    2011-01-01

    Purpose. A striking characteristic of the human meibomian gland is its rich sensory, sympathetic, and parasympathetic innervation, yet the functional relevance of these nerve fibers remains unknown. Acting on the hypothesis that neurotransmitters are released in the vicinity of the gland, act on glandular receptors, and influence the production, secretion, and/or delivery of meibomian gland secretions to the ocular surface, the goal in this study was to begin to determine whether neurotransmitters influence the meibomian gland. Methods. Immortalized human meibomian gland epithelial (SLHMG) cells were examined for the presence of vasoactive intestinal peptide (VIP) and muscarinic acetylcholine (mACh) receptor transcripts and proteins. Cells were also exposed to VIP, carbachol, forskolin, and/or 3-isobutyl-1-methylxanthine (IBMX) to determine whether these agents, alone or in combination, modulate the adenylyl cyclase pathway, the accumulation of intracellular free calcium ([Ca2+]i), or cell proliferation. Results. Results demonstrate that SLHMG cells transcribe and translate VIP and mACh receptors; VIP, with either IBMX or forskolin, activates the adenylyl cyclase pathway, and the effect of VIP and forskolin together is synergistic; both VIP and carbachol increase intracellular [Ca2+] in SLHMG cells; and VIP with forskolin stimulates SLHMG cell proliferation. Conclusions. This study shows that parasympathetic neurotransmitters and their agonists influence the function of human meibomian gland epithelial cells. It remains to be determined whether this action alters the production, secretion, and/or delivery of meibum to the ocular surface. PMID:21969302

  8. The intestinal epithelial cell cycle: uncovering its 'cryptic' nature.

    PubMed

    McKernan, Declan P; Egan, Laurence J

    2015-03-01

    To discuss the recent landmark findings that have increased our understanding not only of the role of the epithelial cell cycle in the homeostasis of the small intestine, but also its relevance to inflammation and cancer. Recent data have unveiled novel information on protein interactions directly involved in the cell cycle as well as in the pathways that transduce external environmental signals to the cell cycle. A growing body of the recent evidence confirms the importance of food as well as hormonal regulation in the gut on cell cycle. Information on the contribution of the epithelial microenvironment, including the microbiota, has grown substantially in the recent years as well as on the gene-environment interactions and the multiple epigenetic mechanisms involved in regulating cell-cycle proteins and signalling. Finally, further studies investigating the dysregulation of the cell cycle during inflammation and proliferation have increased our understanding of the pathophysiology of chronic inflammatory diseases and cancer. This review highlights some of the most recent advances that further emphasize the importance of the cell cycle in the small intestine during homeostasis as well as in inflammation and cancer.

  9. Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells

    PubMed Central

    He, Yinghong; Esser, Philipp; Heinemann, Anja; Bruckner-Tuderman, Leena; Has, Cristina

    2011-01-01

    Kindlins are a novel family of intracellular adaptor proteins in integrin-containing focal adhesions. Kindlin-1 and -2 are expressed in the skin, but whether and how they cooperate in adult epithelial cells have remained elusive. We uncovered the overlapping roles of kindlin-1 and -2 in maintaining epithelial integrity and show that the phenotype of kindlin-1-deficient cells can be modulated by regulating kindlin-2 gene expression and vice versa. The experimental evidence is provided by use of human keratinocyte cell lines that express both kindlins, just kindlin-1 or kindlin-2, or none of them. Double deficiency of kindlin-1 and -2 had significant negative effects on focal adhesion formation and actin cytoskeleton organization, cell adhesion, survival, directional migration, and activation of β1 integrin, whereas deficiency of one kindlin only showed variable perturbation of these functions. Cell motility and formation of cell-cell contacts were particularly affected by lack of kindlin-2. These results predict that kindlin-1 and -2 can functionally compensate for each other, at least in part. The high physiologic and pathologic significance of the compensation was emphasized by the discovery of environmental regulation of kindlin-2 expression. UV-B irradiation induced loss of kindlin-2 in keratinocytes. This first example of environmental regulation of kindlin expression has implications for phenotype modulation in Kindler syndrome, a skin disorder caused by kindlin-1 deficiency. PMID:21356350

  10. Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

    PubMed Central

    Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

    2016-01-01

    Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

  11. Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state.

    PubMed

    Soady, Kelly; Smalley, Matthew J

    2012-09-10

    Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells.

  12. M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells.

    PubMed

    Cui, Jiajun; Xu, Wenhua; Chen, Jian; Li, Hui; Dai, Lu; Frank, Jacqueline A; Peng, Shaojun; Wang, Siying; Chen, Gang

    2017-03-28

    The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages.

  13. M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells

    PubMed Central

    Li, Hui; Dai, Lu; Frank, Jacqueline A.; Peng, Shaojun; Wang, Siying; Chen, Gang

    2017-01-01

    The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages. PMID:28423485

  14. Transdifferentiation of Peripheral Blood Mononuclear Cells into Epithelial-Like Cells

    PubMed Central

    Medina, Abelardo; Kilani, Ruhangiz T.; Carr, Nicholas; Brown, Erin; Ghahary, Aziz

    2007-01-01

    Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3ς for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3ς and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P ≤ 0.01). The depletion of 14-3-3ς from these conditioned media by immunoprecipitation reduced the effect by 39.5% (P value, 0.05). Therefore, the releasable 14-3-3ς from PBMC-derived epithelial-like cells is involved in this process. Our findings provide new insights into the PBMC transdifferentiation to generate epithelial-like cells and subsequently release of 14-3-3ς that will disclose new therapeutic alternatives for different dermal clinical settings. PMID:17717137

  15. Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

    PubMed Central

    Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

    2016-01-01

    The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

  16. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles.

    PubMed

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica; Camussi, Giovanni

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs.

  17. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

    PubMed Central

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs. PMID:27409796

  18. Influences of nanomaterials on the barrier function of epithelial cells.

    PubMed

    Ali, Shariq; Rytting, Erik

    2014-01-01

    Recent advances in nanotechnology have led to exciting opportunities in medicine, energy, manufacturing, and other fields. Nevertheless, it is important to adequately assess the potential impacts of nanomaterial exposure. This chapter focuses on the interactions of nanomaterials with epithelial barriers in the lungs, intestine, kidneys, skin, and placenta. Methods for determining transepithelial electrical resistance and paracellular permeability are described. Effects on cell viability and barrier integrity depend on the chemical nature of the nanomaterial, nanoparticle size, surface coatings, and concentration. Disruption of tight junctions can affect permeability and interfere with normal regulatory processes of the epithelial barrier. Future research is needed to better understand the possibilities and the limits of novel approaches in nanotechnology.

  19. Efficient cultivation conditions for human limbal epithelial cells.

    PubMed

    Kim, Mee Kum; Lee, Jae Lim; Oh, Joo Youn; Shin, Mi Sun; Shin, Kyeong Seon; Wee, Won Ryang; Lee, Jin Hak; Park, Ki Sook; Son, Young Sook

    2008-10-01

    To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

  20. Epithelial cell extrusion during fluid transport in canine small intestine.

    PubMed

    Lee, J S

    1977-04-01

    Epithelial cell extrusion during fluid transport was studied under both in vitro and in vivo conditions. The rate of cell extrusion from the villus tips in vitro increased by about onefold in the villi with obstruction of lymph flow associated with the increase of lymph and tissue fluid pressure. When lymph pressure in the jejunal and ileal villi was increased to 6.4 +/- .2 and 12.3 +/- .5 mmHg, respectively, by injection of Ringer solution into the central lacteals, fluid leaked out of the villi and a shedding of epithelium occurred. Vigorous villus spasmodic contraction induced by cocaine or atropine also caused a shedding of epithelium. Cells always appeared in the lumen of intestine in vivo either during fluid absorption or secretion. A copious secretion of fluid, increase of cell loss, and congestion of blood in the villi occurred by the action of cholera toxin, MgSO4, and choline chloride. The rate of cell loss was highest during fluid secretion induced by an elevation of tissue fluid pressure such as at high venous pressure or during intra-arterial histamine infusion. It is thus concluded that elevated tissue fluid pressure is involved in epithelial cell extrusion during fluid transport.

  1. TCDD exposure disrupts mammary epithelial cell differentiation and function

    PubMed Central

    Collins, Loretta L.; Lew, Betina J.; Lawrence, B. Paige

    2011-01-01

    Mammary gland growth and differentiation during pregnancy is a developmental process that is sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a widespread environmental contaminant and a potent ligand for the aryl hydrocarbon receptor (AhR). We demonstrate reduced β-casein protein induction in mouse mammary glands and in cultured SCp2 mammary epithelial cells following exposure to TCDD. SCp2 cells exposed to TCDD also show reduced cell clustering and less alveolar-like structure formation. SCp2 cells express transcriptionally active AhR, and exposure to TCDD induces expression of the AhR target gene CYP1B1. Exposure to TCDD during pregnancy reduced expression of the cell adhesion molecule E-cadherin in the mammary gland and decreased phosphorylation of STAT5, a known regulator of β-casein gene expression. These data provide morphological and molecular evidence that TCDD-mediated AhR activation disrupts structural and functional differentiation of the mammary gland, and present an in vitro model for studying the effects of TCDD on mammary epithelial cell function. PMID:19490989

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

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

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

  3. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

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

    2010-03-15

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

  4. Epithelial mesenchymal transition in lung cancer cells: A quantitative analysis.

    PubMed

    Sarkar, Atasi; Barui, A; Sengupta, S; Chatterjee, J; Ghorai, S; Mukherjee, Anirban

    2015-01-01

    Cellular auto-fluorescence along with morphological and cytoskeletal features were assessed in lung cancer cells undergoing induced epithelial mesenchymal transition (EMT). During EMT progression, significant increase was observed in cellular aspect ratio (AR), filamentous (F)-actin and green auto-fluorescence intensities while blue intensity decreased. These features were provided to a kernel classification framework. The classification accuracy were impressive, thus these features along with the classification technique can be considered as suitable tools for automated grading of lung cancer cells undergoing EMT progression.

  5. Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.

    PubMed

    Luissint, Anny-Claude; Parkos, Charles A; Nusrat, Asma

    2016-10-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte-epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  6. Inflammation and the Intestinal Barrier: Leukocyte–Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair

    PubMed Central

    Luissint, Anny-Claude; Parkos, Charles A.; Nusrat, Asma

    2017-01-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte–epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  7. Erythropoietin priming improves the vasculogenic potential of G-CSF mobilized human peripheral blood mononuclear cells.

    PubMed

    Kang, Jeehoon; Yun, Ji-Yeon; Hur, Jin; Kang, Jin-A; Choi, Jae-Il; Ko, Seung Bum; Lee, Jaewon; Kim, Ju-Young; Hwang, In-Chang; Park, Young-Bae; Kim, Hyo-Soo

    2014-10-01

    From our previous clinical trials, intracoronary infusion of granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells ((mob)PBMCs) proved to be effective in improving myocardial contractility and reducing infarct volume in acute myocardial infarction. We tested the effect of priming (mob)PBMCs with erythropoietin (EPO) to augment its therapeutic efficacy. (mob)PBMCs were obtained from healthy volunteers after a 3-day subcutaneous injection of G-CSF (10 μg/kg). About 40% of (mob)PBMCs were EPO receptor (EPOR) (+) and responded to 6 h EPO-priming (10 IU/mL) by increasing the expression of vasculogenic factors (i.e. IL8, IL10, bFGF, PDGF, MMP9) and adhesion molecules (i.e. integrin αV, β1, β2, β8) through the JAK2 and Akt pathway. These responses were also observed in PBMCs from elderly patients with coronary disease. The conditioned media from EPO-primed (mob)PBMCs contained various cytokines such as IL8, IL10, TNFα, and PDGF, which enhanced the migration and tube formation capability of endothelial cells. EPO-primed (mob)PBMCs also showed increased adhesion on endothelial cells or fibronectin. Augmented vasculogenic potential of EPO-primed (mob)PBMCs was confirmed in a Matrigel plug assay, ischaemic hindlimb, and myocardial infarction models of athymic nude mice. There were two action mechanisms: (i) cellular effects confirmed by direct incorporation of human (mob)PBSCs into mouse vasculature and (ii) indirect humoral effects confirmed by the therapeutic effect of the supernatant of EPO-primed (mob)PBMCs. Brief ex vivo EPO-priming is a novel method to augment the vasculogenic potential of human (mob)PBMCs, which would help to achieve better results after intracoronary infusion in myocardial infarction patients. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  8. Distinct terminal and cell body mechanisms in the nociceptor mediate hyperalgesic priming.

    PubMed

    Ferrari, Luiz F; Araldi, Dioneia; Levine, Jon D

    2015-04-15

    Hyperalgesic priming, a form of neuroplasticity in nociceptors, is a model of the transition from acute to chronic pain in the rat, which involves signaling from the site of an acute tissue insult in the vicinity of the peripheral terminal of a nociceptor to its cell body that, in turn, induces a signal that travels back to the terminal to mediate a marked prolongation of prostaglandin E2-induced hyperalgesia. In the present experiments, we studied the underlying mechanisms in the cell body and compared them to the mechanisms in the nerve terminal. Injection of a cell-permeant cAMP analog, 8-bromo cAMP, into the dorsal root ganglion induced mechanical hyperalgesia and priming with an onset more rapid than when induced at the peripheral terminal. Priming induced by intraganglion 8-bromo cAMP was prevented by an oligodeoxynucleotide antisense to mRNA for a transcription factor, cAMP response element-binding protein (CREB), and by an inhibitor of importin, which is required for activated CREB to get into the nucleus. While peripheral administration of 8-bromo cAMP also produced hyperalgesia, it did not produce priming. Conversely, interventions administered in the vicinity of the peripheral terminal of the nociceptor that induces priming-PKCε activator, NGF, and TNF-α-when injected into the ganglion produce hyperalgesia but not priming. The protein translation inhibitor cordycepin, injected at the peripheral terminal but not into the ganglion, reverses priming induced at either the ganglion or peripheral terminal of the nociceptor. These data implicate different mechanisms in the soma and terminal in the transition to chronic pain.

  9. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

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

    EPA Science Inventory

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

  11. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

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

    EPA Science Inventory

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

  13. A culture filtrate of Phytophthora infestans primes defense reaction in potato cell suspensions.

    PubMed

    Val, F; Desender, S; Bernard, K; Potin, P; Hamelin, G; Andrivon, D

    2008-06-01

    Priming of defense reactions by an elicitor results in an enhanced ability of the plant to respond to subsequent pathogen challenges. We previously showed that application of lipopolysaccharides (LPS) to potato cell suspensions causes apoplastic acidification, but does not stimulate lipoxygenase (LOX) activity. Here, we tested the ability of various elicitors to prime and elicit defense reactions in potato cell suspensions. Adding 20 microg ml(1) LPS, laminarin, harpin N, or a concentrated culture filtrate (CCF) of Phytophthora infestans to cell cultures 18 h before a second elicitation with LPS did not alter the intensity of apoplastic acidification compared with a single LPS application. Conversely, high concentrations (200 or 400 microg ml(1)) of LPS, laminarin, and harpin N activated LOX in cells pretreated with 1 microg ml(1) CCF, but not in cells pretreated with LPS, laminarin, or harpin N. LOX response was maximal in pretreated cells of potato cv. Bintje when the second elicitation occurred 18 to 24 h after CCF application. These results showed that LOX activation is primed in potato cells by CCF, but not by LPS, harpin N, or laminarin. Finally, bioassays showed a slightly greater reduction of rot weight in half tubers treated with CCF followed by LPS before inoculation with Pectobacterium atrosepticum than in half tubers treated with either preparation alone, indicating a priming effect of CCF on both LOX induction and disease suppression.

  14. Pepsin promotes proliferation of laryngeal and pharyngeal epithelial cells

    PubMed Central

    Johnston, Nikki; Yan, Justin C.; Hoekzema, Craig R.; Samuels, Tina L.; Stoner, Gary D.; Blumin, Joel H.; Bock, Jonathan M.

    2013-01-01

    Objective/Hypothesis Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis. Study Design Translational research study Methods Normal human laryngeal primary epithelial cell cultures and hypopharyngeal FaDu SCC cells were exposed to human pepsin and analyzed by Human Cancer PathwayFinder and miRNA Superarrays, flow cytometry and Western blot to determine the effect of pepsin on carcinogenesis. Laryngeal biopsy specimens, taken from cancer patients and normal control subjects, were analyzed for the presence of pepsin by Western blot. Results Microarray analysis demonstrated that pepsin significantly altered the expression of 27 genes implicated in carcinogenesis and also affected the expression of 22 microRNAs known to be altered in human head and neck cancers. Pepsin increased proliferation in both FaDu SCC cells and cultured normal laryngeal epithelial primary cells by increasing S phase distribution on flow cytometry analysis in a time and dose dependent manner. Furthermore, pepsin was detected in 60% (3/5) human laryngeal cancer biopsies, absent in all (0/5) normal control specimens. Conclusion These data support a role for refluxed pepsin in the promotion of epithelial proliferation and carcinogenesis of the larynx and pharynx. PMID:22570308

  15. Interleukin-23 Increases Intestinal Epithelial Cell Permeability In Vitro.

    PubMed

    Heinzerling, Nathan P; Donohoe, Deborah; Fredrich, Katherine; Gourlay, David M; Liedel, Jennifer L

    2016-06-01

    Background Breast milk has a heterogeneous composition that differs between mothers and changes throughout the first weeks after birth. The proinflammatory cytokine IL-23 has a highly variable expression in human breast milk. We hypothesize that IL-23 found in human breast milk is biologically active and promotes epithelial barrier dysfunction. Methods The immature rat small intestinal epithelial cell line, IEC-18, was grown on cell inserts or standard cell culture plates. Confluent cultures were exposed to human breast milk with high or low levels of IL-23 and barrier function was measured using a flux of fluorescein isothiocyanate-dextran (FD-70). In addition, protein and mRNA expression of occludin and ZO-1 were measured and immunofluorescence used to stain occludin and ZO-1. Results Exposure to breast milk with high levels of IL-23 caused an increase flux of FD-70 compared with both controls and breast milk with low levels of IL-23. The protein expression of ZO-1 but not occludin was decreased by exposure to high levels of IL-23. These results correlate with immunofluorescent staining of ZO-1 and occludin which show decreased staining of occludin in both the groups exposed to breast milk with high and low IL-23. Conversely, cells exposed to high IL-23 breast milk had little peripheral staining of ZO-1 compared with controls and low IL-23 breast milk. Conclusion IL-23 in human breast milk is biologically active and negatively affects the barrier function of intestinal epithelial cells through the degradation of tight junction proteins. Georg Thieme Verlag KG Stuttgart · New York.

  16. Expression of cell adhesion complexes in epithelial cells seeded on biomaterial surfaces.

    PubMed

    Räisänen, L; Könönen, M; Juhanoja, J; Varpavaara, P; Hautaniemi, J; Kivilahti, J; Hormia, M

    2000-01-01

    Clinical studies indicate that soft tissue responses around dental implants vary, depending on the material used. It is therefore also possible that there are differences in how epithelial cells attach to various biomaterial surfaces. We studied the adhesion of cultured epithelial cells to five different dental material surfaces and to glass. The efficacy of adhesion was evaluated by using scanning electron microscopy (SEM) and immunofluorescence microscopy (IF) with antibodies to vinculin and alpha(6)beta(4) integrin, two cell surface molecules that are functional in epithelial cell adhesion. Our results indicate that epithelial cells adhere and spread more avidly on metallic surfaces (titanium, Ti(6)Al(4)V titanium alloy, dental gold alloy) than on ceramic surfaces (dental porcelain, aluminum oxide). As revealed by SEM, cells on metallic surfaces had a flattened morphology and formed multicellular islands. On porcelain and aluminum oxide most cells were round and adhesion occurred as single cells. Surface coverage was over twofold on metallic surfaces as compared to ceramic surfaces. IF of cells grown on metallic surfaces revealed vinculin in well-organized focal contacts and alpha(6)beta(4) integrin in punctate patterns typical of prehemidesmosomes. On porcelain and aluminum oxide surfaces the cells were mostly round and showed less well-organized adhesion complexes. Our results indicate that smooth metallic biomaterial surfaces are optimal for epithelial cell adhesion and spreading. These findings may have clinical implications in the design of transgingival implant structures. Copyright 2000 John Wiley & Sons, Inc.

  17. Microvesicles released from tumor cells disrupt epithelial cell morphology and contractility.

    PubMed

    Bordeleau, Francois; Chan, Bryan; Antonyak, Marc A; Lampi, Marsha C; Cerione, Richard A; Reinhart-King, Cynthia A

    2016-05-24

    During tumor progression, cancer cells interact and communicate with non-malignant cells within their local microenvironment. Microvesicles (MV) derived from human cancer cells play an important role in mediating this communication. Another critical aspect of cancer progression involves widespread ECM remodeling, which occur both at the primary and metastatic sites. ECM remodeling and reorganization within the tumor microenvironment is generally attributed to fibroblasts. Here, using MCF10a cells, a well-characterized breast epithelial cell line that exhibits a non-malignant epithelial phenotype, and MVs shed by aggressive MDA-MB-231 carcinoma cells, we show that non-malignant epithelial cells can participate in ECM reorganization of 3D collagen matrices following their treatment with cancer cell-derived MVs. In addition, MVs trigger several changes in epithelial cells under 3D culture conditions. Furthermore, we show that this ECM reorganization is associated with an increase in cellular traction force following MV treatment, higher acto-myosin contractility, and higher FAK activity. Overall, our findings suggest that MVs derived from tumor cells can contribute to ECM reorganization occurring within the tumor microenvironment by enhancing the contractility of non-malignant epithelial cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Human epithelial cell cultures from superficial limbal explants.

    PubMed

    Ghoubay-Benallaoua, D; Basli, E; Goldschmidt, P; Pecha, F; Chaumeil, C; Laroche, L; Borderie, V

    2011-02-01

    To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer. Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR). The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm(2) and 429 µm(2), and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human corneal stromal

  19. Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

    PubMed

    Hyun, Dong Won; Kim, Yun Hee; Koh, Ah Young; Lee, Hyun Ju; Wee, Won Ryang; Jeon, Saewha; Kim, Mee Kum

    2017-03-01

    The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  20. Urban particulate matter suppresses priming of T helper type 1 cells by granulocyte/macrophage colony-stimulating factor-activated human dendritic cells.

    PubMed

    Matthews, Nick C; Faith, Alex; Pfeffer, Paul; Lu, Haw; Kelly, Frank J; Hawrylowicz, Catherine M; Lee, Tak H

    2014-02-01

    Urban particulate matter (UPM) exacerbates asthmatic lung inflammation and depresses lung immunity. Lung dendritic cells (DCs) react to airway particulates, and have a critical role in linking innate and adaptive immunity, but the direct effects of UPM on DCs, that have been activated by granulocyte/macrophage colony-stimulating factor (GM-CSF), a product of stimulated normal human bronchial epithelial cells, has not been investigated. Human blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of GM-CSF with and without LPS, and DC maturation was assessed by flow cytometry. DC stimulatory capacity and priming of 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester-labeled naive CD4 T cells was investigated using the allogeneic mixed lymphocyte reaction. T cell proliferation and effector function were assessed using flow cytometry and secreted cytokines were measured by combined bead array. UPM enhanced DC maturation in an LPS-independent manner. DCs activated by UPM plus GM-CSF (UPM + GM-CSF DCs) induced higher naive CD4 T cell proliferation in the allogeneic mixed lymphocyte reaction than DCs pretreated by GM-CSF alone (GM-CSF DCs), and elicited both substantially lower levels of IFN-γ, IL-13, and IL-5 secretion, and lower frequencies of alloantigen-specific T helper (Th) type 1 effector cells than naive CD4 T cells primed by GM-CSF DCs. UPM-stimulated DCs produced IL-6 and TNF-α. Neutralization of IL-6 decreased naive CD4 T cell proliferation stimulated by UPM + GM-CSF DCs, and significantly increased the frequency of alloantigen-specific Th1 effector cells, but did not reverse UPM-induced inhibition of IFN-γ secretion. We conclude that UPM enhances GM-CSF-induced DC maturation and stimulatory capacity, but inhibits the generation of Th1 cells. Thus, UPM exposure may impair Th1 responses to pulmonary pathogens.

  1. Protocadherin-1 Localization and Cell-Adhesion Function in Airway Epithelial Cells in Asthma

    PubMed Central

    Faura Tellez, Grissel; Willemse, Brigitte W. M.; Brouwer, Uilke; Nijboer-Brinksma, Susan; Vandepoele, Karl; Noordhoek, Jacobien A.; Heijink, Irene; de Vries, Maaike; Smithers, Natalie P.; Postma, Dirkje S.; Timens, Wim; Wiffen, Laura; van Roy, Frans; Holloway, John W.; Lackie, Peter M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2016-01-01

    Background The asthma gene PCDH1 encodes Protocadherin-1, a putative adhesion molecule of unknown function expressed in the airway epithelium. Here, we characterize the localization, differential expression, homotypic adhesion specificity and function of PCDH1 in airway epithelial cells in asthma. Methods We performed confocal fluorescence microscopy to determine subcellular localization of PCDH1 in 16HBE cells and primary bronchial epithelial cells (PBECs) grown at air-liquid interface. Next, to compare PCDH1 expression and localization in asthma and controls we performed qRT-PCR and fluorescence microscopy in PBECs and immunohistochemistry on airway wall biopsies. We examined homotypic adhesion specificity of HEK293T clones overexpressing fluorescently tagged-PCDH1 isoforms. Finally, to evaluate the role for PCDH1 in epithelial barrier formation and repair, we performed siRNA knockdown-studies and measured epithelial resistance. Results PCDH1 localized to the cell membrane at cell-cell contact sites, baso-lateral to adherens junctions, with increasing expression during epithelial differentiation. No differences in gene expression or localization of PCDH1 isoforms expressing the extracellular domain were observed in either PBECs or airway wall biopsies between asthma patients and controls. Overexpression of PCDH1 mediated homotypic interaction, whereas downregulation of PCDH1 reduced epithelial barrier formation, and impaired repair after wounding. Conclusions In conclusion, PCDH1 is localized to the cell membrane of bronchial epithelial cells baso-lateral to the adherens junction. Expression of PCDH1 is not reduced nor delocalized in asthma even though PCDH1 contributes to homotypic adhesion, epithelial barrier formation and repair. PMID:27701444

  2. Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

    PubMed

    Martínez-Martínez, Eduardo; Uribe-Querol, Eileen; Galván-Hernández, Claudio I; Gutiérrez-Ospina, Gabriel

    2016-01-01

    We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology.

  3. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  4. Bombesin-like peptide receptors in human bronchial epithelial cells.

    PubMed

    Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

    1996-01-01

    Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

  5. Effects of amniotic epithelial cell transplantation in endothelial injury

    PubMed Central

    Vácz, Gabriella; Cselenyák, Attila; Cserép, Zsuzsanna; Benkő, Rita; Kovács, Endre; Pankotai, Eszter; Lindenmair, Andrea; Wolbank, Susanne; Schwarz, Charlotte M.; Horváthy, Dénes B.; Kiss, Levente; Hornyák, István; Lacza, Zsombor

    2016-01-01

    Purpose Human amniotic epithelial cells (hAECs) are promising tools for endothelial repair in vascular regenerative medicine. We hypothesized that these epithelial cells are capable of repairing the damaged endothelial layer following balloon injury of the carotid artery in adult male rats. Results Two days after injury, the transplanted hAECs were observed at the luminal side of the arterial wall. Then, 4 weeks after the injury, significant intimal thickening was observed in both untreated and cell implanted vessels. Constriction was decreased in both implanted and control animals. Immunohistochemical analysis showed a few surviving cells in the intact arterial wall, but no cells were observed at the site of injury. Interestingly, acetylcholine-induced dilation was preserved in the intact side and the sham-transplanted injured arteries, but it was a trend toward decreased vasodilation in the hAECs’ transplanted vessels. Conclusion We conclude that hAECs were able to incorporate into the arterial wall without immunosuppression, but failed to improve vascular function, highlighting that morphological implantation does not necessarily result in functional benefits and underscoring the need to understand other mechanisms of endothelial regeneration. PMID:28180006

  6. Urban particulate matter increases human airway epithelial cell IL-1β secretion following scratch wounding and H1N1 influenza A exposure in vitro.

    PubMed

    Hirota, Jeremy A; Marchant, David J; Singhera, Gurpreet K; Moheimani, Fatemeh; Dorscheid, Delbert R; Carlsten, Christopher; Sin, Don; Knight, Darryl

    2015-01-01

    The airway epithelium represents the first line of defense against inhaled environmental insults including air pollution, allergens, and viruses. Epidemiological and experimental evidence has suggested a link between air pollution exposure and the symptoms associated with respiratory viral infections. We hypothesized that multiple insults integrated by the airway epithelium NLRP3 inflammasome would result in augmented IL-1β release and downstream cytokine production following respiratory virus exposure. We performed in vitro experiments with a human airway epithelial cell line (HBEC-6KT) that involved isolated or combination exposure to mechanical wounding, PM10, house dust mite, influenza A virus, and respiratory syncytial virus. We performed confocal microscopy to image the localization of PM10 within HBEC-6KT and ELISAs to measure soluble mediator production. Airway epithelial cells secrete IL-1β in a time-dependent fashion that is associated with internalization of PM10 particles. PM10 exposure primes human airway epithelial cells to subsequent models of cell damage and influenza A virus exposure. Prior PM10 exposure had no effect on IL-1β responses to RSV exposure. Finally we demonstrate that PM10-priming of human airway epithelial cell IL-1β and GM-CSF responses to influenza A exposure are sensitive to NLRP3 inflammasome inhibition. Our results suggest the NLRP3 inflammasome may contribute to exaggerated immune responses to influenza A virus following periods of poor air quality. Intervention strategies targeting the NLRP3 inflammasome in at risk individuals may restrict poor air quality priming of mucosal immune responses that result from subsequent viral exposures.

  7. Direct activation of antigen-presenting cells is required for CD8+ T-cell priming and tumor vaccination

    PubMed Central

    Kratky, Wolfgang; Reis e Sousa, Caetano; Oxenius, Annette; Spörri, Roman

    2011-01-01

    Successful priming of adaptive immune responses is crucially dependent on innate activation signals that convert resting antigen-presenting cells (APCs) into immunogenic ones. APCs expressing the relevant innate pattern recognition receptors can be directly activated by pathogen-associated molecular patterns (PAMPs) to become competent to prime T-cell responses. Alternatively, it has been suggested that APCs could be activated indirectly by proinflammatory mediators synthesized by PAMP-exposed cells. However, data obtained with CD4+ T cells suggest that inflammatory signals often cannot substitute for direct pattern recognition in APC activation for the priming of T helper responses. To test whether the same is true for CD8+ T cells, we studied cytotoxic T lymphocyte development in vitro and in mixed chimeric mice in which coexisting APCs can either present a preprocessed model antigen or directly recognize a given PAMP, but not both. We show that indirectly activated APCs promote antigen-specific proliferation of naïve CD8+ T cells but fail to support their survival and cytotoxic T lymphocyte differentiation. Furthermore, CD8+ T cells primed by indirectly activated APCs are unable to reject tumors. Thus, inflammation cannot substitute for direct recognition of single PAMPs in CD8+ T-cell priming. These findings have important practical implications for vaccine design, indicating that adjuvants must be judiciously chosen to trigger the relevant pattern recognition receptors in APCs. PMID:21987815

  8. L-Myc expression by dendritic cells is required for optimal T-cell priming

    PubMed Central

    Wumesh, KC; Satpathy, Ansuman T.; Rapaport, Aaron S.; Briseño, Carlos G.; Wu, Xiaodi; Albring, Jörn C.; Russler-Germain, Emilie V.; Kretzer, Nicole M.; Durai, Vivek; Persaud, Stephen P.; Edelson, Brian T.; Loschko, Jakob; Cella, Marina; Allen, Paul M.; Nussenzweig, Michel C.; Colonna, Marco; Sleckman, Barry P.; Murphy, Theresa L.; Murphy, Kenneth M.

    2014-01-01

    The transcription factors c-Myc and N-Myc encoded by Myc and Mycn, respectively, regulate cellular growth1 and are required for embryonic development2,3. A third paralog, Mycl1, is dispensable for normal embryonic development but its normal biologic function has remained unclear4. To examine the in vivo function of Mycl1, we generated an inactivating Mycl1gfp allele that also reports Mycl1 expression. We found that Mycl1 was selectively expressed in dendritic cells (DCs) of the immune system and controlled by IRF8, and that during DC development, Mycl1 expression was initiated in the common DC progenitor5 (CDP) concurrent with reduction in c-Myc expression. Mature DCs lacked expression of c-Myc and N-Myc, but maintained L-Myc expression even in the presence of inflammatory signals, such as GM-CSF. All DC subsets developed in Mycl1-deficient mice, but several DC subsets, such as migratory CD103+ cDCs in the lung and liver, were significantly reduced at steady state. Importantly, loss of L-Myc by DCs caused a significant decrease in the in vivo T-cell priming during infection by Listeria monocytogenes and vesicular stomatitis virus. The replacement of c-Myc by L-Myc in immature DCs may provide for Myc transcriptional activity in the setting of inflammation that is required for optimal T-cell priming6. PMID:24509714

  9. Urokinase induces activation of STAT3 in lung epithelial cells.

    PubMed

    Shetty, Sreerama; Rao, Gadiparthi N; Cines, Douglas B; Bdeir, Khalil

    2006-10-01

    Urokinase-type plasminogen activator (uPA) is a serine protease that plays a major role in diverse physiological and pathological processes. Studies from our laboratory have shown that exposure of human lung epithelial cells to uPA induces proliferation. To understand uPA mitogenic signaling events, we sought to elucidate its effects on tyrosine phosphorylation in a human bronchial epithelial cell line (Beas2B). uPA induced tyrosine phosphorylation of several proteins in a time-dependent manner. One of these proteins was identified as the 91-kDa signal transduction activator transcription (Stat)3 moiety. Tyrosine phosphorylation of Stat3 by uPA was time dependent. uPA induced Stat3-DNA binding activity in a time-dependent manner. uPA-induced Stat3 activation does not require uPA catalytic activity, as the uPA amino-terminal fragment alone was as potent as active two-chain uPA (tcuPA) in causing this effect. Single-chain uPA likewise induced tyrosine phosphorylation of Stat3 to a similar extent as intact tcuPA. Plasmin did not alter uPA-induced Stat3 activation. Furthermore, transfection of Beas2B cells with dominant-negative Stat3 blocked uPA-induced DNA synthesis. These results reveal for the first time that the uPA-uPAR interaction leads to activation of Stat3, independent of its catalytic activity but dependent on its interaction with its receptor, uPAR, leading to DNA synthesis in lung epithelial cells.

  10. In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

    PubMed Central

    Isaacson, R E; Fusco, P C; Brinton, C C; Moon, H W

    1978-01-01

    Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili. PMID:357285

  11. In vitro adhesion of Escherichia coli to porcine small intestinal epithelial cells: pili as adhesive factors.

    PubMed

    Isaacson, R E; Fusco, P C; Brinton, C C; Moon, H W

    1978-08-01

    Escherichia coli strains with pili (K99 or 987P) known to facilitate intestinal colonization adhered in vitro to porcine intestinal epithelial cells. These strains adhered equally to both ileal and jejunal epithelial cells. A laboratory E. coli strain that has type 1 pili also adhered to porcine intestinal epithelial cells. When nonpiliated cells derived from 987P+, K99+, or type 1 pilus+ strains were used for in vitro adhesion assays, they failed to adhere. The attachment of piliated bacteria to epithelial cells was a saturable process that plateaued at 30 to 40 bacterial cells attached per epithelial cell. Competitive inhibition of bacterial cell attachment to epithelial cells with purified pili showed that only purified 987P competed against the 987P+ strain and only purified type 1 pili competed against the type 1 pilus+ strain. Competition between a K99+ strain and K99 was not consistently achieved. K99+, 987P+, and type 1 pilus+ bacteria could be prevented from adhering to epithelial cells by Fab fragments specific for K99, 987P, or type 1 pili, respectively. Fab fragments specific for non-K99 bacterial surface antigens did not inhibit adhesion of the K99+ strain. It is concluded that adhesion of E. coli to porcine intestinal epithelial cells in vitro is mediated by pili and that the epithelial cells used apparently had different receptors for different pili.

  12. Bimodal Analysis of Mammary Epithelial Cell Migration in Two Dimensions

    PubMed Central

    Potdar, Alka A.; Lu, Jenny; Jeon, Junhwan; Weaver, Alissa M.; Cummings, Peter T.

    2013-01-01

    Cell migration paths of mammary epithelial cells (expressing different versions of the promigratory tyrosine kinase receptor Her2/Neu) were analyzed within a bimodal framework that is a generalization of the run-and-tumble description applicable to bacterial migration. The mammalian cell trajectories were segregated into two types of alternating modes, namely, the “directional-mode” (mode I, the more persistent mode, analogous to the bacterial run phase) and the “re-orientation-mode” (mode II, the less persistent mode, analogous to the bacterial tumble phase). Higher resolution (more pixel information, relative to cell size) and smaller sampling intervals (time between images) were found to give a better estimate of the deduced single cell dynamics (such as directional-mode time and turn angle distribution) of the various cell types from the bimodal analysis. The bimodal analysis tool permits the deduction of short-time dynamics of cell motion such as the turn angle distributions and turn frequencies during the course of cell migration compared to standard methods of cell migration analysis. We find that the two-hour mammalian cell tracking data do not fall into the diffusive regime implying that the often-used random motility expressions for mammalian cell motion (based on assuming diffusive motion) are invalid over the time steps (fraction of minute) typically used in modeling mammalian cell migration. PMID:18982450

  13. Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells.

    PubMed

    Tumminia, S J; Rao, P V; Zigler, J S; Russell, P

    1993-12-08

    Xenobiotic regulatory elements have been identified for enzymes which ameliorate oxidative damage in cells. Zeta (zeta)-crystallin, a taxon-specific enzyme/crystallin shown to be a novel NADPH-dependent quinone reductase, is found in a number of tissues and cell types. This study shows that zeta-crystallin is present in mouse lens epithelium, as well as in the alpha TN4 mouse lens epithelial cell line. To determine whether zeta-crystallin is an inducible quinone reductase, cell cultures were exposed to the xenobiotics, 1,2-naphthoquinone and beta-naphthoflavone. Assays of cellular homogenates showed that quinone reductase activity was stimulated greater than 70% and 90%, respectively, over the control cells. This observed activity was sensitive to dicumarol, a potent inhibitor of quinone reductase activity. 1,2-Naphthoquinone- and beta-naphthoflavone-exposed cells were found to exhibit 1.47- and 1.68-fold increases, respectively, in zeta-crystallin protein concentration. A comparable increase in zeta-crystallin mRNA was indicative of an induction in zeta-crystallin expression in response to naphthalene challenge. Lens epithelial cells were also checked for DT-diaphorase, a well-known cellular protective enzyme which can catalyze the two-electron reduction of quinones. Slot blot analyses indicated that alpha TN4 cells exposed to 1,2-naphthoquinone and beta-naphthoflavone exhibited 2.71- and 6.81-fold increases in DT-diaphorase concentration when compared to the control cells. The data suggest that while DT-diaphorase is most likely responsible for the majority of the observed increase in quinone reductase activity, the zeta-crystallin gene also undergoes activation which is apparently mediated by a xenobiotic-responsive element.

  14. Novel strategies to enforce an epithelial phenotype in mesenchymal cells

    PubMed Central

    Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

    2014-01-01

    E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

  15. Organization of the cytokeratin network in an epithelial cell.

    PubMed

    Portet, Stéphanie; Arino, Ovide; Vassy, Jany; Schoëvaërt, Damien

    2003-08-07

    The cytoskeleton is a dynamic three-dimensional structure mainly located in the cytoplasm. It is involved in many cell functions such as mechanical signal transduction and maintenance of cell integrity. Among the three cytoskeletal components, intermediate filaments (the cytokeratin in epithelial cells) are the best candidates for this mechanical role. A model of the establishment of the cytokeratin network of an epithelial cell is proposed to study the dependence of its structural organization on extracellular mechanical environment. To implicitly describe the latter and its effects on the intracellular domain, we use mechanically regulated protein synthesis. Our model is a hybrid of a partial differential equation of parabolic type, governing the evolution of the concentration of cytokeratin, and a set of stochastic differential equations describing the dynamics of filaments. Each filament is described by a stochastic differential equation that reflects both the local interactions with the environment and the non-local interactions via the past history of the filament. A three-dimensional simulation model is derived from this mathematical model. This simulation model is then used to obtain examples of cytokeratin network architectures under given mechanical conditions, and to study the influence of several parameters.

  16. Cholinergic regulation of epithelial sodium channels in rat alveolar type 2 epithelial cells.

    PubMed

    Takemura, Yoshizumi; Helms, My N; Eaton, Amity F; Self, Julie; Ramosevac, Semra; Jain, Lucky; Bao, Hui-Fang; Eaton, Douglas C

    2013-03-15

    We and others have shown that epithelial Na(+) channels (ENaC) in alveolar type 2 (AT2) cells are activated by β2 agonists, steroid hormones, elevated oxygen tension, and by dopamine. Although acetylcholine receptors (AChRs) have been previously described in the lung, there are few reports of whether cholinergic agonists alter sodium transport in the alveolar epithelium. Therefore, we investigated how cholinergic receptors regulate ENaC activity in primary cultures of rat AT2 cells using cell-attached patch-clamp recordings to assess ENaC activity. We found that the muscarinic agonists, carbachol (CCh) and oxotremorine, activated ENaC in a dose-dependent manner but that nicotine did not. CCh-induced activation of ENaC was blocked by atropine. Western blotting and immunohistochemistry suggested that muscarinic M2 and M3 receptors (mAChRs) but not nicotinic receptors were present in AT2 cells. Endogenous RhoA and GTP-RhoA increased in response to CCh and the increase was reduced by pretreatment with atropine. We showed that Y-27632, an inhibitor of Rho-associated protein kinase (ROCK), abolished endogenous ENaC activity and inhibited the activation of ENaC by CCh. We also showed that ROCK signaling was necessary for ENaC stability in 2F3 cells, a model for AT2 cells. Our results showed that muscarinic agonists activated ENaC in rat AT2 cells through M2 and/or M3 mAChRs probably via a RhoA/ROCK signaling pathway.

  17. Two distinct secretory vesicle-priming steps in adrenal chromaffin cells.

    PubMed

    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R; Rettig, Jens

    2010-09-20

    Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

  18. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

    PubMed

    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  19. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  20. Galvanotactic control of collective cell migration in epithelial monolayers

    NASA Astrophysics Data System (ADS)

    Cohen, Daniel J.; James Nelson, W.; Maharbiz, Michel M.

    2014-04-01

    Many normal and pathological biological processes involve the migration of epithelial cell sheets. This arises from complex emergent behaviour resulting from the interplay between cellular signalling networks and the forces that physically couple the cells. Here, we demonstrate that collective migration of an epithelium can be interactively guided by applying electric fields that bias the underlying signalling networks. We show that complex, spatiotemporal cues are locally interpreted by the epithelium, resulting in rapid, coordinated responses such as a collective U-turn, divergent migration, and unchecked migration against an obstacle. We observed that the degree of external control depends on the size and shape of the cell population, and on the existence of physical coupling between cells. Together, our results offer design and engineering principles for the rational manipulation of the collective behaviour and material properties of a tissue.

  1. Quantification of regenerative potential in primary human mammary epithelial cells.

    PubMed

    Linnemann, Jelena R; Miura, Haruko; Meixner, Lisa K; Irmler, Martin; Kloos, Uwe J; Hirschi, Benjamin; Bartsch, Harald S; Sass, Steffen; Beckers, Johannes; Theis, Fabian J; Gabka, Christian; Sotlar, Karl; Scheel, Christina H

    2015-09-15

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49f(hi)/EpCAM(-) population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis.

  2. Quantification of regenerative potential in primary human mammary epithelial cells

    PubMed Central

    Linnemann, Jelena R.; Miura, Haruko; Meixner, Lisa K.; Irmler, Martin; Kloos, Uwe J.; Hirschi, Benjamin; Bartsch, Harald S.; Sass, Steffen; Beckers, Johannes; Theis, Fabian J.; Gabka, Christian; Sotlar, Karl; Scheel, Christina H.

    2015-01-01

    We present an organoid regeneration assay in which freshly isolated human mammary epithelial cells are cultured in adherent or floating collagen gels, corresponding to a rigid or compliant matrix environment. In both conditions, luminal progenitors form spheres, whereas basal cells generate branched ductal structures. In compliant but not rigid collagen gels, branching ducts form alveoli at their tips, express basal and luminal markers at correct positions, and display contractility, which is required for alveologenesis. Thereby, branched structures generated in compliant collagen gels resemble terminal ductal-lobular units (TDLUs), the functional units of the mammary gland. Using the membrane metallo-endopeptidase CD10 as a surface marker enriches for TDLU formation and reveals the presence of stromal cells within the CD49fhi/EpCAM− population. In summary, we describe a defined in vitro assay system to quantify cells with regenerative potential and systematically investigate their interaction with the physical environment at distinct steps of morphogenesis. PMID:26071498

  3. Limbal stem cells: Central concepts of corneal epithelial homeostasis.

    PubMed

    Yoon, Jinny J; Ismail, Salim; Sherwin, Trevor

    2014-09-26

    A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface.

  4. Gradient isolation of glial cells: evidence that flat epithelial cells are astroglial cell precursors.

    PubMed

    Meller, K

    1987-07-01

    Discontinuous gradients of metrizamide were used to separate the cell components of monolayers of primary cultures of embryonic rat brains. These primary cell cultures were of two types: long-term cultures (more than a year) of embryonic rat brain, which contained several glial cell types, and monolayers of cell cultures (several weeks old), which contained a complex population of cells, including neuronal elements. The gradient separation produces fractions of pure flat epithelial cells that are able to survive and proliferate. After a few days, all flat epithelial cells become confluent and show a positive reaction to glial fibrillary acidic protein (GFAP); this indicates that these cells astroglial precursor cells. Following their maintenance in vitro for several months, all cultures give rise to a pure population of astrocytes identified not only by their characteristic morphology, but also by their content of GFAP. It is proposed that the differentiation controls are dependent on cell interactions that are influenced by the composition of the cell population and/or the molecular growth and differentiation factors released by these cells into the medium.

  5. Density dependent polarized secretion of a prostatic epithelial cell line.

    PubMed

    Djakiew, D; Pflug, B; Delsite, R; Lynch, J H; Onoda, M

    1992-01-01

    The polarized secretions (apical/basal) of newly synthesized total protein and proteases from prostatic epithelial sheets of PA-III cells grown in dual compartment chambers were investigated at various cell densities and culture conditions. PA-III cells grown in a serum free defined medium (SFDM) form morphologically polarized monolayers of epithelial cells. These cells secreted their 35S-methionine labeled total protein in a predominantly apical direction (apical/basal ratio, 4-8 fold), with a lesser proportion of protein secreted apically at lower cell densities of the PA-III cell monolayer. PA-III cells grown in 5% fetal calf serum (FCS) are morphologically squamous, comparable to the anaplastic phenotype, and exhibited an inversion of polarized total protein secretion (apical/basal ratio, 0.4-0.9 fold), with an increased proportion of total protein secreted in a basal direction at lower cell densities. Since the culture of PA-III cells in FCS may approximate the anaplastic phenotype we investigated the polarized secretion of proteases from these cells at various cell densities, and compared them with the secretory pattern of protease secretion from polarized PA-III cells cultured in SFDM. At lower cell densities of the PA-III cells grown in FCS the polarity of protease secretion was inverted such that metalloproteinases, tissue type plasminogen activator, and a 72 kD gelatinase were secreted in a predominantly basal direction, as well as urokinase and a gelatinase of 26 kD that were secreted more or less equally into the apical and basal compartments of the chambers. On the other hand, for cultures of PA-III cells grown in SFDM the aforementioned proteases exhibited predominantly an apically directed polarity of secretion. These results suggest that the anaplastic phenotype characterized by a loss of polarized structure may also be characterized by a functional loss or inversion of polarized secretion. The consequences of such a loss or inversion of polarized

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

    SciTech Connect

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

    2008-04-01

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

  7. Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

    PubMed

    Ruane, Peter T; Berneau, Stéphane C; Koeck, Rebekka; Watts, Jessica; Kimber, Susan J; Brison, Daniel R; Westwood, Melissa; Aplin, John D

    2017-09-01

    How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model? Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium. In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models. This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells. Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001. Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5

  8. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    DTIC Science & Technology

    2012-04-01

    algorithm for CpG-island detection. BMC Bioinformatics 7: 446. 17. Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol...it does not have a CpG island according to the original criteria (Gardiner-Garden and Frommer 1987). H3K4me3 and H3Ac are present in miR-205...culture of normal human mammary epithelial cells. Cancer Res 69: 7557–7568. Gardiner-GardenM, Frommer M. 1987. CpG islands in vertebrate genomes. J Mol

  9. A Common Stem Cell for Murine Cortical and Medullary Thymic Epithelial Cells?

    PubMed Central

    Van Soest, Peter; Platenburg, Peter Paul; Van Ewijk, Willem

    1995-01-01

    We have addressed the question whether the epithelial stroma in the thymus is derived from a common stem cell or whether cortical and medullary epithelial cells are derived from different embryonic stem cells emerging, for example, from endoderm and ectoderm. By the use of rapidly expanding cultures of thymic epithelial cells (TEC) from 14 to 16 day-old murine fetuses and by specific antibodies against cortical and medullary epithelium, respectively, we were able to demonstrate a small subpopulation of double-labeled TEC in the cultures. These cells were not present in TEC cultures initiated from thymuses of neonatal mice. Double-labeled TEC were also found in tissue sections from fetal thymuses. These findings may indicate that TEC populations of the cortex and the medulla are derived from a common stem cell, with potential for differentiation toward both cortical and medullary TEC. PMID:9700364

  10. Cellular and Nuclear Alignment Analysis for Determining Epithelial Cell Chirality.

    PubMed

    Raymond, Michael J; Ray, Poulomi; Kaur, Gurleen; Singh, Ajay V; Wan, Leo Q

    2016-05-01

    Left-right (LR) asymmetry is a biologically conserved property in living organisms that can be observed in the asymmetrical arrangement of organs and tissues and in tissue morphogenesis, such as the directional looping of the gastrointestinal tract and heart. The expression of LR asymmetry in embryonic tissues can be appreciated in biased cell alignment. Previously an in vitro chirality assay was reported by patterning multiple cells on microscale defined geometries and quantified the cell phenotype-dependent LR asymmetry, or cell chirality. However, morphology and chirality of individual cells on micropatterned surfaces has not been well characterized. Here, a Python-based algorithm was developed to identify and quantify immunofluorescence stained individual epithelial cells on multicellular patterns. This approach not only produces results similar to the image intensity gradient-based method reported previously, but also can capture properties of single cells such as area and aspect ratio. We also found that cell nuclei exhibited biased alignment. Around 35% cells were misaligned and were typically smaller and less elongated. This new imaging analysis approach is an effective tool for measuring single cell chirality inside multicellular structures and can potentially help unveil biophysical mechanisms underlying cellular chiral bias both in vitro and in vivo.

  11. Versican regulates metastasis of epithelial ovarian carcinoma cells and spheroids

    PubMed Central

    2014-01-01

    Background Epithelial ovarian carcinoma is a deadly disease characterized by overt peritoneal metastasis. Individual cells and multicellular aggregates, or spheroids, seed these metastases, both commonly found in ascites. Mechanisms that foster spheroid attachment to the peritoneal tissues preceding formation of secondary lesions are largely unknown. Methods Cell culture models of SKOV-3, OVCAR3, OVCAR4, Caov-3, IGROV-1, and A2780 were used. In this report the role of versican was examined in adhesion of EOC spheroids and cells to peritoneal mesothelial cell monolayers in vitro as well as in formation of peritoneal tumors using an in vivo xenograft mouse model. Results The data demonstrate that versican is instrumental in facilitating cell and spheroid adhesion to the mesothelial cell monolayers, as its reduction with specific shRNAs led to decreased adhesion. Furthermore, spheroids with reduced expression of versican failed to disaggregate to complete monolayers when seeded atop monolayers of peritoneal mesothelial cells. Failure of spheroids lacking versican to disaggregate as efficiently as controls could be attributed to a reduced cell migration that was observed in the absence of versican expression. Importantly, both spheroids and cells with reduced expression of versican demonstrated significantly impaired ability to generate peritoneal tumors when injected intraperitoneally into athymic nude mice. Conclusions Taken together these data suggest that versican regulates the development of peritoneal metastasis originating from cells and spheroids. PMID:24999371

  12. Barrier Epithelial Cells and the Control of Type 2 Immunity.

    PubMed

    Hammad, Hamida; Lambrecht, Bart N

    2015-07-21

    Type-2-cell-mediated immunity, rich in eosinophils, basophils, mast cells, CD4(+) T helper 2 (Th2) cells, and type 2 innate lymphoid cells (ILC2s), protects the host from helminth infection but also drives chronic allergic diseases like asthma and atopic dermatitis. Barrier epithelial cells (ECs) represent the very first line of defense and express pattern recognition receptors to recognize type-2-cell-mediated immune insults like proteolytic allergens or helminths. These ECs mount a prototypical response made up of chemokines, innate cytokines such as interleukin-1 (IL-1), IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), as well as the alarmins uric acid, ATP, HMGB1, and S100 proteins. These signals program dendritic cells (DCs) to mount Th2-cell-mediated immunity and in so doing boost ILC2, basophil, and mast cell function. Here we review the general mechanisms of how different stimuli trigger type-2-cell-mediated immunity at mucosal barriers and how this leads to protection or disease.

  13. Cellular and Nuclear Alignment Analysis for Determining Epithelial Cell Chirality

    PubMed Central

    Raymond, Michael J.; Ray, Poulomi; Kaur, Gurleen; Singh, Ajay V.; Wan, Leo Q.

    2015-01-01

    Left-right (LR) asymmetry is a biologically conserved property in living organisms that can be observed in the asymmetrical arrangement of organs and tissues and in tissue morphogenesis, such as the directional looping of the gastrointestinal tract and heart. The expression of LR asymmetry in embryonic tissues can be appreciated in biased cell alignment. Previously an in vitro chirality assay was reported by patterning multiple cells on microscale defined geometries and quantified the cell phenotype–dependent LR asymmetry, or cell chirality. However, morphology and chirality of individual cells on micropatterned surfaces has not been well characterized. Here, a Python-based algorithm was developed to identify and quantify immunofluorescence stained individual epithelial cells on multicellular patterns. This approach not only produces results similar to the image intensity gradient-based method reported previously, but also can capture properties of single cells such as area and aspect ratio. We also found that cell nuclei exhibited biased alignment. Around 35% cells were misaligned and were typically smaller and less elongated. This new imaging analysis approach is an effective tool for measuring single cell chirality inside multicellular structures and can potentially help unveil biophysical mechanisms underlying cellular chiral bias both in vitro and in vivo. PMID:26294010

  14. Visualizing lens epithelial cell proliferation in whole lenses

    PubMed Central

    Shui, Ying-Bo; Beebe, David C.

    2010-01-01

    Purpose To develop a means to image cells in S-phase of the cell cycle while preserving the anatomic relationships within the lens. Methods Mice were injected with the thymidine analog, EdU. Whole lenses were removed, fixed and permeabilized. Cells that had incorporated EdU into their DNA were chemically labeled using fluorescent azides and “click” chemistry. Double labeling was performed with antibodies to other antigens, like phospho-histoneH3, a marker of mitotic cells. The position of labeled cells and lens anatomy was viewed using a simple device to position and flatten the lens. Results The nuclei of cells in S-phase of the cell cycle were intensely stained without the use of antibodies. Stained cells were readily localized with reference anatomic landmarks, like the transition zone. Whole lenses could be assayed by rotating the lens on the microscope stage. Double-labeling permitted the co-localization of markers in cycling cells. Conclusions EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens. PMID:20664699

  15. ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming.

    PubMed

    Segura, Elodie; Nicco, Carole; Lombard, Bérangère; Véron, Philippe; Raposo, Graça; Batteux, Frédéric; Amigorena, Sebastian; Théry, Clotilde

    2005-07-01

    Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes, which transfer functional major histocompatibility complex (MHC)-peptide complexes to other DCs. Since immature and mature DCs induce different functional T-cell responses (ie, tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that exosomes secreted by lipopolysaccharide (LPS)-treated mature DCs are 50- to 100-fold more potent to induce antigen-specific T-cell activation in vitro than exosomes from immature DCs. In vitro, exosomes from mature DCs transfer to B lymphocytes the ability to prime naive T cells. In vivo, only mature exosomes trigger effector T-cell responses, leading to fast skin graft rejection. Proteomic and biochemical analyses revealed that mature exosomes are enriched in MHC class II, B7.2, intercellular adhesion molecule 1 (ICAM-1), and bear little milk-fat globule-epidermal growth factor-factor VIII (MFG-E8) as compared with immature exosomes. Functional analysis using DC-derived exosomes from knock-out mice showed that MHC class II and ICAM-1 are required for mature exosomes to prime naive T cells, whereas B7.2 and MFG-E8 are dispensable. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.

  16. Liver Sinusoidal Endothelial Cell-Mediated CD8 T Cell Priming Depends on Co-Inhibitory Signal Integration over Time

    PubMed Central

    Kaczmarek, Julita; Homsi, Yahya; van Üüm, Jan; Metzger, Christina; Knolle, Percy A.; Kolanus, Waldemar; Lang, Thorsten; Diehl, Linda

    2014-01-01

    The initiation of adaptive immunity requires cell-to-cell contact between T cells and antigen-presenting cells. Together with immediate TCR signal transduction, the formation of an immune synapse (IS) is one of the earliest events detected during T cell activation. Here, we show that interaction of liver sinusoidal endothelial cells (LSEC) with naive CD8 T cells, which induces CD8 T cells without immediate effector function, is characterized by a multi-focal type IS. The co-inhibitory molecule B7H1, which is pivotal for the development of non-responsive LSEC-primed T cells, did not alter IS structure or TCRβ/CD11a cluster size or density, indicating that IS form does not determine the outcome of LSEC-mediated T cell activation. Instead, PD-1 signaling during CD8 T cell priming by LSEC repressed IL-2 production as well as sustained CD25 expression. When acting during the first 24 h of LSEC/CD8 T cell interaction, CD28 co-stimulation inhibited the induction of non-responsive LSEC-primed T cells. However, after more than 36 h of PD-1 signaling, CD28 co-stimulation failed to rescue effector function in LSEC-primed T cells. Together, these data show that during LSEC-mediated T cell priming, integration of co-inhibitory PD-1 signaling over time turns on a program for CD8 T cell development, that cannot be overturned by co-stimulatory signals. PMID:24924593

  17. Alveolar epithelial cells orchestrate DC function in murine viral pneumonia

    PubMed Central

    Unkel, Barbara; Hoegner, Katrin; Clausen, Björn E.; Lewe-Schlosser, Peter; Bodner, Johannes; Gattenloehner, Stefan; Janßen, Hermann; Seeger, Werner; Lohmeyer, Juergen; Herold, Susanne

    2012-01-01

    Influenza viruses (IVs) cause pneumonia in humans with progression to lung failure. Pulmonary DCs are key players in the antiviral immune response, which is crucial to restore alveolar barrier function. The mechanisms of expansion and activation of pulmonary DC populations in lung infection remain widely elusive. Using mouse BM chimeric and cell-specific depletion approaches, we demonstrated that alveolar epithelial cell (AEC) GM-CSF mediates recovery from IV-induced injury by affecting lung DC function. Epithelial GM-CSF induced the recruitment of CD11b+ and monocyte-derived DCs. GM-CSF was also required for the presence of CD103+ DCs in the lung parenchyma at baseline and for their sufficient activation and migration to the draining mediastinal lymph nodes (MLNs) during IV infection. These activated CD103+ DCs were indispensable for sufficient clearance of IVs by CD8+ T cells and for recovery from IV-induced lung injury. Moreover, GM-CSF applied intratracheally activated CD103+ DCs, inducing increased migration to MLNs, enhanced viral clearance, and attenuated lung injury. Together, our data reveal t