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Sample records for epithelial cell culture

  1. Applications of mouse airway epithelial cell culture for asthma research.

    PubMed

    Horani, Amjad; Dickinson, John D; Brody, Steven L

    2013-01-01

    Primary airway epithelial cell culture provides a valuable tool for studying cell differentiation, cell-cell interactions, and the role of immune system factors in asthma pathogenesis. In this chapter, we discuss the application of mouse tracheal epithelial cell cultures for the study of asthma biology. A major advantage of this system is the ability to use airway epithelial cells from mice with defined genetic backgrounds. The in vitro proliferation and differentiation of mouse airway epithelial cells uses the air-liquid interface condition to generate well-differentiated epithelia with characteristics of native airways. Protocols are provided for manipulation of differentiation, induction of mucous cell metaplasia, genetic modification, and cell and pathogen coculture. Assays for the assessment of gene expression, responses of cells, and analysis of specific cell subpopulations within the airway epithelium are included. PMID:23943446

  2. 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

  3. Morphological appearances of human lens epithelial cells in culture.

    PubMed

    Power, W; Neylan, D; Collum, L

    1993-01-01

    A system for culturing human lens epithelial cells in the laboratory was developed. The morphological appearances of the cells was studied using phase contrast, scanning and transmission electron microscopy. Cell marker studies using monoclonal antibodies to cytokeratin, vimentin and epithelial membrane antigen were also performed. There was a marked increase in cell size as a function of time in culture. After 3 to 4 weeks cells showed early signs of ageing. By 6 to 8 weeks the majority of the cells had become very irregular in shape and demonstrated irregularities of the plasma membrane and intra-cytoplasmic vacuole formation. The cells stained strongly for vimentin and epithelial membrane antigen. Staining with cytokeratin was somewhat weaker. This culture technique provides us with a suitable model for studying the growth behavior of these cells. PMID:7512459

  4. 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

  5. Microfluidic approaches for epithelial cell layer culture and characterisation.

    PubMed

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

    2014-07-01

    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 using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  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. Culture and characterization of human junctional epithelial cells.

    PubMed

    Matsuyama, T; Izumi, Y; Sueda, T

    1997-03-01

    This study was undertaken to establish a culture of junctional epithelial cells derived from gingival tissue attached to the tooth surface and to characterize these cells immunocytochemically and ultrastructurally. Primary cultures of cells were obtained from the junctional tissue explanted on type I collagen-coated dishes and immersed in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Cells were subcultured with conditioned serum-free keratinocyte medium (keratinocyte-SFM + 5% FBS) on dishes coated with solubilized extract of the basement membrane. After 24 hours, the medium was changed to keratinocyte-SFM (0.09 mM Ca2+). The cell-doubling time was 40.5 hours. As a control, cells from gingival tissue were cultured by the same method. Cells from junctional tissue and gingival tissue were compared immunocytochemically using monoclonal antibodies to keratin, vimentin, and desmoplakins I and II and using Dolichos biflorus agglutinin (DBA). The keratin AE1 and AE3 was expressed by all of culture cells. The vimentin (specific for the intermediate filament of mesenchymal cells) was also expressed by all cells. The expression pattern of keratin 19 was observed not only by cells from junctional tissue but also by cells from gingival tissue. All keratin peptides were expressed in both cells. However, DBA reacted only with cells from the junctional tissue. Anti-desmoplakin I and II reacted with both cells, however, the staining patterns differed. DBA-positive cultured epithelial cells from the junctional tissue showed poor tonofilament bundles and were rich in cytoplasmic organelles. These findings suggest that junctional epithelial cells can be isolated from junctional tissue and cultured under improved conditions. PMID:9100198

  8. Culture and immortalization of pancreatic ductal epithelial cells.

    PubMed

    Lawson, Terence; Ouellette, Michel; Kolar, Carol; Hollingsworth, Michael

    2005-01-01

    Some populations of the epithelial cells from the duct and ductular network of the mammalian pancreas have been isolated and maintained in vitro for up to 3 mo. These cells express many of the surface factors that are unique to them in vivo. They also retain significant drug- and carcinogen-metabolizing capacity in vitro. In this chapter we review the progression of the methods for the isolation, culture and maintenance in vitro for these cells from the earliest when only duct/ductular fragments were obtainable to the current ones which provide epithelial cells. The critical steps in the isolation process are identified and strategies are provided to facilitate these steps. These include the selection of tissue digestive enzymes, the importance of extensive mincing before culture and the importance of roles of some co-factors used in the culture medium. PMID:15542901

  9. 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. PMID:23475606

  10. Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype.

    PubMed

    Khan, Ayyad Z; Utheim, Tor P; Jackson, Catherine J; Reppe, Sjur; Lyberg, Torstein; Eidet, Jon R

    2016-06-01

    Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman's correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia. PMID:27329312

  11. 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.

  12. Three-Dimensional Cultures of Mouse Mammary Epithelial Cells

    PubMed Central

    Mroue, Rana; Bissell, Mina J.

    2013-01-01

    The mammary gland is an ideal “model organism” for studying tissue specificity and gene expression in mammals: it is one of the few organs that develop after birth and it undergoes multiple cycles of growth, differentiation and regression during the animal’s lifetime in preparation for the important function of lactation. The basic “functional differentiation” unit in the gland is the mammary acinus made up of a layer of polarized epithelial cells specialized for milk production surrounded by myoepithelial contractile cells, and the two-layered structure is surrounded by basement membrane. Much knowledge about the regulation of mammary gland development has been acquired from studying the physiology of the gland and of lactation in rodents. Culture studies, however, were hampered by the inability to maintain functional differentiation on conventional tissue culture plastic. We now know that the microenvironment, including the extracellular matrix and tissue architecture, plays a crucial role in directing functional differentiation of organs. Thus, in order for culture systems to be effective experimental models, they need to recapitulate the basic unit of differentiated function in the tissue or organ and to maintain its three-dimensional (3D) structure. Mouse mammary culture models evolved from basic monolayers of cells to an array of complex 3D systems that observe the importance of the microenvironment in dictating proper tissue function and structure. In this chapter, we focus on how 3D mouse mammary epithelial cultures have enabled investigators to gain a better understanding of the organization, development and function of the acinus, and to identify key molecular, structural, and mechanical cues important for maintaining mammary function and architecture. The accompanying chapter of Vidi et al. describes 3D models developed for human cells. Here, we describe how mouse primary epithelial cells and cell lines—essentially those we use in our

  13. Human alveolar epithelial type II cells in primary culture.

    PubMed

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-02-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  14. Human alveolar epithelial type II cells in primary culture

    PubMed Central

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-01-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  15. Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

    PubMed

    Mathias, Neil R; Wu, Sharon K; Kim, Kwang-Jin; Lee, Vincent H L

    2005-01-01

    The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or

  16. Lymphohaemopoietic antigens of cultured human glomerular epithelial cells.

    PubMed Central

    van der Woude, F. J.; Michael, A. F.; Muller, E.; van der Hem, G. K.; Vernier, R. L.; Kim, Y.

    1989-01-01

    Glomerular visceral epithelial cells (GVEC) from normal human glomeruli were grown in tissue culture. Cell surface markers were studied by immunofluorescence microscopy using antibodies against lymphohaemopoietic differentiation antigens which are known to be present early (BA-1, OKB2, BA-2) and late (J5, anti CR1) in renal ontogenesis. Like foetal human glomerular epithelium, the cultured cells reacted with BA-1 and OKB2 (identifying an antigen expressed on B cells and polymorphonuclear leucocytes), and BA-2 (leukaemia-associated antigen), but were consistently negative for CR1 (C3b receptor); J5 which identifies the common acute lymphoblastic leukaemia antigen (CALLA) stained variably. Reactivity with antimyosin or anti factor VIII were absent. The cells produced an extracellular matrix containing laminin, type IV collagen, and fibronectin. This study supports the notion that GVEC undergo dedifferentiation as shown by the acquisition of lymphohaemopoietic differentiation antigens present early in renal ontogeny. In addition, the production of extracellular matrix constituents in vitro may be useful for the investigation of human glomerular basement membranes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:2647119

  17. In Vitro Culture and Characterization of a Mammary Epithelial Cell Line from Chinese Holstein Dairy Cow

    PubMed Central

    Hu, Han; Wang, Jiaqi; Bu, Dengpan; Wei, Hongyang; Zhou, Linyun; Li, Fadi; Loor, Juan J.

    2009-01-01

    Background The objective of this study was to establish a culture system and elucidate the unique characteristics of a bovine mammary epithelial cell line in vitro. Methodology Mammary tissue from a three year old lactating dairy cow (ca. 100 d relative to parturition) was used as a source of the epithelial cell line, which was cultured in collagen-coated tissue culture dishes. Fibroblasts and epithelial cells successively grew and extended from the culturing mammary tissue at the third day. Pure epithelial cells were obtained by passages culture. Principal Findings The strong positive immunostaining to cytokeratin 18 suggested that the resulting cell line exhibited the specific character of epithelial cells. Epithelial cells cultured in the presence of 10% FBS, supraphysiologic concentrations of insulin, and hydrocortisone maintained a normal diploid chromosome modal number of 2n = 60. Furthermore, they were capable of synthesizing β-casein (CSN2), acetyl-CoA carboxylase-α (ACACA) and butyrophilin (BTN1A1). An important finding was that frozen preservation in a mixture of 90% FBS and 10% DMSO did not influence the growth characteristics, chromosome number, or protein secretion of the isolated epithelial cell line. Conclusions The obtained mammary epithelial cell line had normal morphology, growth characteristics, cytogenetic and secretory characteristics, thus, it might represent an useful tool for studying the function of Chinese Holstein dairy cows mammary epithelial cell (CMECs). PMID:19888476

  18. Interactions between airway epithelial cells and dendritic cells during viral infections using an in vitro co-culture model

    EPA Science Inventory

    Rationale: Historically, single cell culture models have been limited in pathological and physiological relevance. A co-culture model of dendritic cells (DCs) and differentiated human airway epithelial cells was developed to examine potential interactions between these two cell t...

  19. Cytokeratin changes in cell culture systems of epithelial cells isolated from oral mucosa: a short review.

    PubMed

    Gasparoni, Alberto; Squier, Christopher Alan; Fonzi, Luciano

    2005-01-01

    In the past three decades, many studies have analyzed ultrastructural and molecular markers of differentiation in squamous stratified epithelial tissues. In these tissues, epithelial cells migrating from the basal layer to the upper layers undergo drastic changes, which involve membrane-associated proteins, DNA synthesis, phenotypic aspects, lipid composition, and cytoskeletal components. Cytoskeletal components include a large and heterogeneous group, including intermediate filaments, components of the cornified envelope, and of the stratum corneum. When grown in mono- and multilayer cell cultures, epithelial cells isolated from the oral mucosa may reproduce many of the biochemical and morphological aspects of epithelial tissue in vivo. In the present paper, we examine phenotypic changes, development of suprabasal layer, and Involucrin expression occurring in differentiating oral epithelial cells, based on literature review and original data. PMID:16277157

  20. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    SciTech Connect

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  1. Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency

    PubMed Central

    Paaske Utheim, Tor; Aass Utheim, Øygunn; Khan, Qalb-E-Saleem; Sehic, Amer

    2016-01-01

    The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells. PMID:26938569

  2. Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

    PubMed

    Harvey, H A; Ketterer, M R; Preston, A; Lubaroff, D; Williams, R; Apicella, M A

    1997-06-01

    In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected. PMID:9169783

  3. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  4. Recognition of Major Histocompatibility Complex Antigens on Cultured Human Biliary Epithelial Cells by Alloreactive Lymphocytes

    PubMed Central

    Saidman, Susan L.; Duquesnoy, Rene J.; Zeevi, Adriana; Fung, John J.; Starzl, Thomas E.; Demetris, A. Jake

    2010-01-01

    We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-γ-interferon monoclonal antibody. In addition, recombinant human γ-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell–induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody–blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID:1704868

  5. Epithelial cell cultures from normal and cancerous human tissues.

    PubMed

    Owens, R B; Smith, H S; Nelson-Rees, W A; Springer, E L

    1976-04-01

    Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal. PMID:176412

  6. 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

  7. Differentiation of adult rat bone marrow stem cells into epithelial progenitor cells in culture.

    PubMed

    Shu, Chang; Li, Ting Yu; Tsang, Lai Ling; Fok, Kin Lam; Lo, Pui Shan; Zhu, Jin Xia; Ho, Lo Sze; Chung, Yiu Wa; Chan, Hsiao Chang

    2006-10-01

    We have previously obtained monoclonal bone marrow stem cells from adult rats (rMSCs) and induced them into phenotypic neurons. In the present study, we aimed to induce rMSCs into epithelial cells by culturing them onto compartmentalized permeable supports, which have been used for growing a variety of polarized epithelia in culture. Hematoxylin staining showed that after 4 days grown on permeable supports, rMSCs formed an epithelial-like monolayer. Immunofluorescence of the permeably-supported monolayers, but not the rMSCs grown in culture flasks, showed positive signals for epithelial markers, cytokeratin 5 & 8. RT-PCR results also showed the mRNA expression of epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) as well as tight junction protein ZO-1 in the rMSC-derived monolayers grown on permeable supports but absent from those grown in culture flasks. However, western blot only detected protein expression of ZO-1 but not ENaC nor CFTR. The short-circuit current measurements showed that the rMSC-derived monolayers grown on permeable supports exhibited a trans-monolayer resistance of 30-50 Omega cm(2); however, the monolayers did not respond to activators or blockers of CFTR or ENaC. The results suggest that compartmentalized or polarized culture conditions provide a suitable environment for rMSCs to differentiate into epithelial progenitor cells with tight junction formation; however, this condition is not sufficient for functional expression of epithelial ion channels associated with well-differentiated epithelia. PMID:16877014

  8. Epithelial monolayer culture system for real‐time single‐cell analyses

    PubMed Central

    Seo, Jong Bae; Moody, Mark; Koh, Duk‐Su

    2014-01-01

    Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single‐cell and subcellular levels, and can be extended to other cell types with minor modifications. PMID:24771696

  9. Human corneal stromal stem cells support limbal epithelial cells cultured on RAFT tissue equivalents

    PubMed Central

    Kureshi, Alvena K; Dziasko, Marc; Funderburgh, James L; Daniels, Julie T

    2015-01-01

    Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63α, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease. PMID:26531048

  10. Characterization of Long-Term Cultured Murine Submandibular Gland Epithelial Cells

    PubMed Central

    Tsunoda, Kazuyuki; Nakagawa, Taneaki; Tsubota, Kazuo

    2016-01-01

    Purpose Human and rat salivary gland cell lines derived from tumors or genetic modification are currently available for research. Here, we attempted to culture and characterize long-term cultured cells spontaneously derived from wild type murine submandibular glands (SGs). Methods SGs were removed from 3-week-old C57B/6J female mice and dissociated by collagenase type 1 and hyaluronidase digestion. Isolated SG epithelial cells were cultured in low calcium, serum-free growth media in the presence of cholera toxin (CT) during early passages. Single-cell colonies were isolated by limiting dilution culture after 25 passages. Early- and late-stage cell cultures were characterized for keratin 14, keratin 18, α-smooth muscle actin, and p63 by immunostaining and quantitative real-time PCR analysis. Results SG epithelial cells cultured in optimized media maintained their proliferative ability and morphology for over 80 passages. Long-term cultured cells expressed keratin 14, keratin 18, and p63, indicative of an epithelial phenotype. Conclusions Epithelial cells originating from wild type murine SGs could be cultured for longer periods of time and remain phenotypically similar to ductal basal epithelium. PMID:26800086

  11. Immunohistochemical characterisation of epithelial cells of rodent harderian glands in primary culture

    PubMed Central

    DJERIDANE, YASMINA; SIMONNEAUX, VALERIE; KLOSEN, PAUL; VIVIEN-ROELS, BERTHE; PEVET, PAUL

    1999-01-01

    The aims of the current investigation were (1) to establish an efficient procedure for the isolation of rodent harderian gland cells and to define conditions for maintenance of viable differentiated cells; (2) to compare the in vitro growth pattern of cultured epithelial cells; and (3) to characterise the cultured epithelial cells from 3 rodent species: Wistar rats, Syrian hamsters and Djungarian hamsters. We have established primary culture conditions that permit the maintenance of viable and differentiated secretory cells from adult rodent harderian gland. This study demonstrates that the cell growth pattern is faster in hamsters than in rats and despite morphological changes, epithelial cells reestablish their distinctive (biochemical/metabolic) phenotype as indicated by lipid-containing vacuoles, porphyrin pigment and serotonin and tryptophan hydroxylase labelling. PMID:10634691

  12. Association of ICAM-1 with the cytoskeleton in rat alveolar epithelial cells in primary culture.

    PubMed

    Barton, W W; Wilcoxen, S E; Christensen, P J; Paine, R

    1996-11-01

    Intercellular adhesion molecule-1 ICAM-1) is a transmembrane adhesion protein that is expressed constitutively on the apical surface of type I cells in vivo and on type II cells in vitro as they spread in culture, assuming type I cell-like characteristics. To investigate the possible interaction of ICAM-1 with the alveolar epithelial cell cytoskeleton, rat type II cells in primary culture were extracted with nonionic detergent, and residual ICAM-1 associated with the cytoskeletal remnants was determined using immunofluorescence microscopy, immunoprecipitation, and cell-based enzyme-linked immunosorbent assay. A large fraction of alveolar epithelial cell ICAM-1 remained associated with the cytoskeleton after detergent extraction, whereas two other transmembrane molecules, transferrin receptor and class II major histocompatibility complex, were completely removed. ICAM-1 was redistributed on the cell surface after the disruption of actin filaments with cytochalasin B, suggesting interaction with the actin cytoskeleton. In contrast, ICAM-1 was completely detergent soluble in rat pulmonary artery endothelial cells, human umbilical vein endothelial cells, and rat alveolar macrophages. The association of ICAM-1 with the alveolar epithelial cell cytoskeleton was not altered after stimulation with inflammatory cytokines. However, detergent resistant ICAM-1 was significantly increased after crosslinking of ICAM-1 on the cell surface, suggesting that this cytoskeletal association may be modulated by interactions of alveolar epithelial cells with inflammatory cells. The association of ICAM-1 with the cytoskeleton in alveolar epithelial cells may provide a fixed intermediary between mobile inflammatory cells and the alveolar surface. PMID:8944713

  13. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells.

    PubMed

    Drummond, Coyne G; Nickerson, Cheryl A; Coyne, Carolyn B

    2016-01-01

    Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and encounters the

  14. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    PubMed Central

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  15. Changes in the gene expression pattern of cytokeratins in human respiratory epithelial cells during culture.

    PubMed

    Endres, Michaela; Leinhase, Iris; Kaps, Christian; Wentges, Marek; Unger, Manja; Olze, Heidi; Ringe, Jochen; Sittinger, Michael; Rotter, Nicole

    2005-05-01

    The replacement of extensive tracheal defects resulting from intensive care medicine, trauma or large resections is still challenged by the re-epithelialization of an autologous or alloplastic trachea replacement. Therefore, this study was performed to investigate the potential of culture-expanded human respiratory epithelial cells (hREC) to regenerate a functional epithelium for tracheal tissue engineering. hREC from seven male nasal turbinates were freshly isolated, expanded on a collagenous matrix and subsequently cultured in high-density multi-layers to allow epithelial differentiation. The composition of epithelial cells in native respiratory epithelial tissue and culture-expanded hREC was analyzed by histological staining with Alcian blue and by immunohistochemical staining of cytokeratin pairs CK1/10 and CK5/14 with the antibodies 34betaE12 and CD44v6. Differentiation of culture-expanded hREC was further characterized by gene expression analysis of cytokeratins CK5, CK13, CK14 and CK18 using semi-quantitative real-time RT-PCR technique. Histological and immunohistochemical staining of culture-expanded hREC demonstrated basal cells covering the collagenous matrix. These cells formed a cellular multi-layer, which was composed of a basal layer of undifferentiated basal cells and an upper layer of cells differentiating along the squamous metaplasia and ciliated cell lineage. Lineage development of culture-expanded hREC was further documented by the induction of cytokeratins CK13 and CK18. Our results suggest that culture-expanded hREC have the potential to colonize collagen-coated biomaterials and to regenerate epithelial cell types for tracheal tissue engineering. PMID:15549340

  16. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  17. Isolation and Culture of Alveolar Epithelial Type I and Type II Cells from Rat Lungs

    PubMed Central

    Gonzalez, Robert F.; Dobbs, Leland G.

    2014-01-01

    The pulmonary alveolar epithelium, comprised of alveolar Type I (TI) and Type II (TII) cells, covers more than 99% of the internal surface area of the lungs. The study of isolated and cultured alveolar epithelial TI and TII cells has provided a large amount of information about the functions of both cell types. This chapter provides information about methods for isolating and culturing both of these cell types from rat lungs. PMID:23097106

  18. Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing.

    PubMed

    Peramo, Antonio; Feinberg, Stephen E; Marcelo, Cynthia L

    2013-09-01

    Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking. PMID:23149549

  19. Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

    PubMed Central

    Rother, Jan; Büchsenschütz-Göbeler, Matthias; Nöding, Helen; Steltenkamp, Siegfried; Samwer, Konrad; Janshoff, Andreas

    2015-01-01

    The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m−1, and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells. PMID:25566882

  20. Cytotoxicity of Shiga toxin for primary cultures of human colonic and ileal epithelial cells.

    PubMed Central

    Moyer, M P; Dixon, P S; Rothman, S W; Brown, J E

    1987-01-01

    Shiga toxin purified from Shigella dysenteriae 1 was cytotoxic to cultured epithelial cells from human colon and ileum. The cytotoxicity, which affected only about 50% of treated cells, was neutralized by rabbit antiserum monospecific for Shiga toxin and mediated by protein synthesis inhibition. PMID:3570477

  1. Long-term culture and partial characterization of dog gallbladder epithelial cells.

    PubMed

    Oda, D; Lee, S P; Hayashi, A

    1991-05-01

    We describe the successful isolation and maintenance of primary cultures of dog gallbladder epithelial cells. The surgically removed gallbladder was treated with trypsin/EDTA for 45 minutes and epithelial cells were collected and resuspended in Eagle's minimum essential medium with 10% fetal calf serum, and plated on Vitrogen-coated culture dishes. Each gallbladder yielded approximately 12 to 15 x 10(6) columnar epithelial cells, greater than 95% of which were viable by trypan blue exclusion. In culture, cells maintained their polarity. They were arranged and grew in small and tight clusters that coalesced at confluency. When examined using transmission electron microscopy, prominent and numerous microville were identified on the apical portion of the plasma membrane. Cells were connected by well-formed desmosomes. Scanning electron microscopy revealed clusters of polyhedral cells with numerous papillary projections. Immunohistochemical studies demonstrated uniform staining of cells to keratin 35BH11 and AE1. Histochemical studies were positive for gamma-glutamyl transpeptidase and negative for glucose-6-phosphatase and albumin. Cells incorporated [3H]uridine into intracellular proteins and [14C]glucosamine into tissue and secreted mucous glycoproteins linearly over 2 to 24 hours. Flow cytometry studies demonstrated a consistent and reproducible number of cells (10 to 12%) at S-phase. However, the number of cells at S-phase was dramatically reduced to almost negligible as cells reached confluency. This method of culturing primary dog gallbladder epithelial cells is highly reproducible and reliable. These cells preserve their state of differentiation, polarity, histochemical and immunohistochemical profile, morphologic, and metabolic integrity with repeated passaging or after being frozen. [3H]Thymidine uptake is well maintained, although doubling time shows a trend of decreased cell duplication with time. This technique offers the opportunity to study the

  2. Cell and Molecular Biology of Ataxia Telangiectasia Heterozygous Human Mammary Epithelial Cells Irradiated in Culture

    NASA Technical Reports Server (NTRS)

    Richmond, Robert C.

    2001-01-01

    Autologous isolates of cell types from obligate heterozygotes with the autosomal disorder ataxia-telangiectasia (A-T)were used to begin a tissue culture model for assessing pathways of radiation-induced cancer formation in this target tissue. This was done by establishing cultures of stromal fibroblasts and long-term growth human mammary epithelial cells (HMEC) in standard 2-dimensional tissue culture in order to establish expression of markers detailing early steps of carcinogenesis. The presumptive breast cancer susceptibility of A-T heterozygotes as a sequel to damage caused by ionizing radiation provided reason to study expression of markers in irradiated HMEC. Findings from our study with HMEC have included determination of differences in specific protein expression amongst growth phase (e.g., log vs stationary) and growth progression (e.g., pass 7 vs pass 9), as well as differences in morphologic markers within populations of irradiated HMEC (e.g., development of multinucleated cells).

  3. Estrogen inhibits cell cycle progression and retinoblastoma phosphorylation in rhesus ovarian surface epithelial cell culture

    SciTech Connect

    Wright, Jay W.; Stouffer, Richard L.; Rodland, Karin D.

    2003-10-31

    Estrogen promotes the growth of some ovarian cancer cells at nanomolar concentrations, but has been shown to inhibit growth of normal ovarian surface epithelial (OSE) cells at micromolar concentrations (1μg/ml). OSE cells express the estrogen receptor (ER)-α, and are the source of 90% of various cancers. The potential sensitivity of OSE cells to estrogen stresses the importance of understanding the estrogen-dependent mechanisms at play in OSE proliferation and transformation, as well as in anticancer treatment. We investigated the effects of estradiol on cell proliferation in vitro, and demonstrate an intracellular locus of action of estradiol in cultured rhesus ovarian surface epithelial (RhOSE) cells. We show that ovarian and breast cells are growth-inhibited by micromolar concentration of estradiol and that this inhibition correlates with estrogen receptor expression. We further show that normal rhesus OSE cells do not activate ERK or Akt in response to estradiol nor does estradiol block the ability of serum to stimulate ERK or induce cyclin D expression. Contrarily, estradiol inhibits serum-dependent retinoblastoma protein (Rb) phosphorylation and blocks DNA synthesis. This inhibition does not formally arrest cells and is reversible within hours of estrogen withdrawal. Our data are consistent with growth inhibition by activation of Rb and indicate that sensitivity to hormone therapy in anticancer treatment can be modulated by cell cycle regulators downstream of the estrogen receptor.

  4. Isolation and Culture of Human Endometrial Epithelial Cells and Stromal Fibroblasts

    PubMed Central

    Chen, Joseph C.; Roan, Nadia R.

    2016-01-01

    Purification and culture of endometrial epithelial cells (eEC) and stromal fibroblasts (eSF) from endometrial biopsies allows for downstream cell-specific in vitro studies. The utility of this protocol is the ease with which cells are purified without contamination from unwanted cell types, and the ability to use patient-paired eEC and eSF in experiments. These methods have been previously published, but here the protocol has been updated for maximum efficiency. PMID:27347495

  5. IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

    PubMed Central

    Vilela, Ricardo Chaves; Benchimol, Marlene

    2013-01-01

    Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells. PMID:23440124

  6. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

    PubMed

    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression. PMID:23667900

  7. EFFECT OF PHORBOL ESTERS ON CLONAL CULTURES OF HUMAN, HAMSTER, AND RAT RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth of epithelial cells from rat, hamster, and human respiratory tract has been measured by monitoring colony formation in culture. TPA and its active derivatives stimulated colony formation of ...

  8. Establishment of three-dimensional cultures of human pancreatic duct epithelial cells

    SciTech Connect

    Gutierrez-Barrera, Angelica M.; Menter, David G.; Abbruzzese, James L.; Reddy, Shrikanth A.G. . E-mail: sa08366@wotan.mdacc.tmc.edu

    2007-07-06

    Three-dimensional (3D) cultures of epithelial cells offer singular advantages for studies of morphogenesis or the role of cancer genes in oncogenesis. In this study, as part of establishing a 3D culture system of pancreatic duct epithelial cells, we compared human pancreatic duct epithelial cells (HPDE-E6E7) with pancreatic cancer cell lines. Our results show, that in contrast to cancer cells, HPDE-E6E7 organized into spheroids with what appeared to be apical and basal membranes and a luminal space. Immunostaining experiments indicated that protein kinase Akt was phosphorylated (Ser473) and CTMP, a negative Akt regulator, was expressed in both HPDE-E6E7 and cancer cells. However, a nuclear pool of CTMP was detectable in HPDE-E6E7 cells that showed a dynamic concentrated expression pattern, a feature that further distinguished HPDE-E637 cells from cancer cells. Collectively, these data suggest that 3D cultures of HPDE-E6E7 cells are useful for investigating signaling and morphological abnormalities in pancreatic cancer cells.

  9. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures.

    PubMed

    Doyle, Timothy E; Goodrich, Jeffrey B; Ambrose, Brady J; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H

    2010-11-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  10. Culture of hormone-dependent functional epithelial cells from rat thyroids.

    PubMed Central

    Ambesi-Impiombato, F S; Parks, L A; Coon, H G

    1980-01-01

    Primary cultures of rat thyroid cells were made in medium supplemented with 0.1--0.5% calf serum and containing six hormones or growth factors: insulin, thyrotropin, transferrin, hydrocortisone, somatostatin, and glycyl-L-histidyl-L-lysine acetate. The FRTL strain was purified by successive colonial isolations and was found to maintain highly differentiated features (secretion into the culture medium of physiological amounts of thyroglobulin and concentration of iodide by 100-fold). The FRTL strain has been observed for more than 3 years in continuous culture. It has maintained the same biochemical and morphological characteristics that typified the primary cultures of thyroid follicular cells immediately after their enzymatic release from the rat thyroid. Thyroid epithelial cells that were grown under more conventional cell culture conditions failed to retain these specialized characteristics. We show that maintenance in vitro of these specialized functions of rat thyroid follicular cells is dependent on low serum concentrations and supplementation with hormones in the primary cultures. Our observations indicate that this culture strategem may be aplicable to the general problem of maintenance of differentiated characteristics in cultures of other epithelial cells. Images PMID:6106191

  11. Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis.

    PubMed

    Wiszniewski, Ludovic; Jornot, Lan; Dudez, Tecla; Pagano, Alessandra; Rochat, Thierry; Lacroix, Jean Silvain; Suter, Susanne; Chanson, Marc

    2006-01-01

    The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies. PMID:16179582

  12. Enhanced growth medium and method for culturing human mammary epithelial cells

    DOEpatents

    Stampfer, Martha R.; Smith, Helene S.; Hackett, Adeline J.

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  13. Mitochondria are targets for the antituberculosis drug rifampicin in cultured epithelial cells.

    PubMed

    Erokhina, M V; Kurynina, A V; Onishchenko, G E

    2013-10-01

    Rifampicin is a widely used drug for antituberculosis therapy. Its target is the bacterial RNA polymerase. After entry into the human or mammalian organism, rifampicin is accumulated in cells of epithelial origin (kidneys, liver, lungs) where it induces apoptosis, necrosis, and fibrosis. The purpose of this study was to determine the intracellular mechanisms leading to rifampicin-induced pathological changes and cell death. We analyzed the survival and state of the chondriome of cultured epithelial cells of the SPEV line under the influence of rifampicin. Our data show that the drug induces pronounced pathological changes in the network and ultrastructure of mitochondria, and their dysfunction results in excessive production of reactive oxygen species and release of cytochrome c. These data suggest the initiation of the mitochondrial pathway of apoptosis. Simultaneously, we observed inhibition of cell proliferation and changes in morphology of the epithelial cells toward fibroblast-like appearance, which could indicate induction of epithelial-mesenchymal transition. Thus, mitochondria are the main potential target for rifampicin in cells of epithelial origin. We suggest that similar mechanisms of pathological changes can be induced in vivo in organs and tissues accumulating rifampicin during chemotherapy of bacterial infectious diseases. PMID:24237150

  14. Human Airway Primary Epithelial Cells Show Distinct Architectures on Membrane Supports Under Different Culture Conditions.

    PubMed

    Min, Kyoung Ah; Rosania, Gus R; Shin, Meong Cheol

    2016-06-01

    To facilitate drug development for lung delivery, it is highly demanding to establish appropriate airway epithelial cell models as transport barriers to evaluate pharmacokinetic profiles of drug molecules. Besides the cancer-derived cell lines, as the primary cell model, normal human bronchial epithelial (NHBE) cells have been used for drug screenings because of physiological relevance to in vivo. Therefore, to accurately interpret drug transport data in NHBE measured by different laboratories, it is important to know biophysical characteristics of NHBE grown on membranes in different culture conditions. In this study, NHBE was grown on the polyester membrane in a different medium and its transport barrier properties as well as cell architectures were fully characterized by functional assays and confocal imaging throughout the days of cultures. Moreover, NHBE cells on inserts in a different medium were subject to either of air-interfaced culture (AIC) or liquid-covered culture (LCC) condition. Cells in the AIC condition were cultivated on the membrane with medium in the basolateral side only, whereas cells with medium in apical and basolateral sides under the LCC condition. Quantitative microscopic imaging with biophysical examination revealed distinct multilayered architectures of differentiated NHBE cells, suggesting NHBE as functional cell barriers for the lung-targeting drug transport. PMID:26818810

  15. Differential crosstalk between epithelial cells, dendritic cells and bacteria in a co-culture model.

    PubMed

    Zoumpopoulou, Georgia; Tsakalidou, Effie; Dewulf, Joelle; Pot, Bruno; Grangette, Corinne

    2009-04-30

    Intestinal epithelial cells (IECs) provide a primary physical barrier against commensal and pathogenic bacteria, but the influence of IECs in the regulation of the associated mucosal immune system remains largely unknown. The network of dendritic cells (DCs) in the vicinity of IECs is known to play a crucial role in the regulation of gut homeostasis. We investigated the cross-talk between murine IECs (m-IC(cl2) cell line), bone marrow derived DCs and different bacteria using an in vitro Transwell co-culture model. IECs responded poorly to different gram-positive lactic acid bacteria (LAB) and to a Staphylococcus aureus strain. In contrast two Escherichia coli strains, including the probiotic strain Nissle 1917, strongly activated IECs, as evidenced by the induction of different chemokines. While a differential maturation of DCs is observed upon direct stimulation with the various bacteria, DC maturation across the epithelial barrier was only observed upon challenge of the apical surface of the IECs with both E. coli strains and LPS. These results suggested that the capacity of bacteria to induce pro-inflammatory signals through the epithelial barrier is not linked to their pathogenic or commensal status, but seem to be dependent on the presence of specific surface factors. As already reported, we confirmed that m-IC(cl2) cells are highly susceptible to LPS. It is highly possible, at least in this model, that free LPS is the "specific factor" key to activate IEC or BMDC. Moreover, IECs are broadly unresponsive to gram-positive bacterial components, notably TLR-2 ligands, in contrast to gram-negative bacterial components. These results suggest that the gut epithelium will sense the commensal bacteria in a different way, and seems to be unresponsive to gram positive bacteria in particular to LAB. PMID:19264370

  16. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

    PubMed Central

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

  17. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.

    PubMed

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

  18. Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

    PubMed Central

    Amemiya, Takeshi; Nakamura, Takahiro; Yamamoto, Toshiro; Kinoshita, Shigeru; Kanamura, Narisato

    2015-01-01

    The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects. PMID:25915046

  19. Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

    PubMed Central

    Utheim, Oeygunn; Islam, Rakibul; Lyberg, Torstein; Roald, Borghild; Eidet, Jon Roger; de la Paz, Maria Fideliz; Dartt, Darlene A.; Raeder, Sten; Utheim, Tor Paaske

    2015-01-01

    Aim/Purpose of the Study To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Materials and Methods Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). Results The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). Conclusion Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days

  20. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche

    PubMed Central

    Ootani, Akifumi; Li, Xingnan; Sangiorgi, Eugenio; Ho, Quoc T; Ueno, Hiroo; Toda, Shuji; Sugihara, Hajime; Fujimoto, Kazuma; Weissman, Irving L; Capecchi, Mario R; Kuo, Calvin J

    2010-01-01

    The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the γ-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein–coupled receptor-5–positive (Lgr5+) and B lymphoma moloney murine leukemia virus insertion region homolog-1–positive (Bmi1+) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5+ cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche. PMID:19398967

  1. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.

    PubMed

    Ootani, Akifumi; Li, Xingnan; Sangiorgi, Eugenio; Ho, Quoc T; Ueno, Hiroo; Toda, Shuji; Sugihara, Hajime; Fujimoto, Kazuma; Weissman, Irving L; Capecchi, Mario R; Kuo, Calvin J

    2009-06-01

    The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche. PMID:19398967

  2. Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures.

    PubMed

    Costabile, Valeria; Duraturo, Francesca; Delrio, Paolo; Rega, Daniela; Pace, Ugo; Liccardo, Raffaella; Rossi, Giovanni Battista; Genesio, Rita; Nitsch, Lucio; Izzo, Paola; De Rosa, Marina

    2015-05-01

    Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E‑cadherin decreases, resulting in loss of cell‑cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real‑time PCR and immunofluorescence were performed to investigate the expression of E‑cadherin, vimentin, β‑catenin, cytokeratin‑20 and ‑18, Twist1, Snail, CD44, cyclooxygenase‑2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl‑containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E‑cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin‑18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3β (GSK3β) inhibitor LiCl, the cells began to express E‑cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET‑reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal‑to‑epithelial transition (MET). Our observation indicates that LiCl, a GSK3β inhibitor, induces MET in vitro, suggesting that LiCl and GSK3β could represent

  3. Epithelial morphogenesis of MDCK cells in three-dimensional collagen culture is modulated by interleukin-8.

    PubMed

    Wells, Erika K; Yarborough, OrLando; Lifton, Richard P; Cantley, Lloyd G; Caplan, Michael J

    2013-05-15

    Epithelial morphogenesis is dependent upon a variety of factors, many of which involve complex interactions between cells and their surrounding environments. We analyzed the patterns of differential gene expression associated with Madin-Darby canine kidney (MDCK) renal epithelial cells grown within a collagen gel in three-dimensional (3D) culture compared with those grown atop a collagen gel in two-dimensional (2D) culture. Under these conditions, MDCK cells spontaneously formed either hollow spherical cysts or flat monolayer sheets, respectively. Microarray analysis of gene expression revealed a twofold or greater expression difference in 732 gene sets from MDCK cysts compared with monolayers (false discovery rate or FDR-adjusted P values <0.05). Interleukin-8 (IL-8) was reproducibly found to be among the genes whose expression was most dramatically upregulated, and this behavior was verified through real-time PCR analysis. The level of IL-8 protein expression was significantly increased in 3D MDCK cultures compared with that detected in cells in 2D culture. Hepatocyte growth factor (HGF) induces MDCK cells in 3D culture to form linear tubule-like structures. We found that HGF stimulation caused MDCK cells in 3D culture to decrease the expression of IL-8 at both the mRNA and protein levels. Furthermore, the addition of recombinant IL-8 to HGF-stimulated 3D MDCK cultures was sufficient to partially reverse the tubulogenic effects of HGF, resulting in the formation of cystic structures. These data suggest that IL-8 participates in the formation of cystic structures by MDCK cells in 3D culture and that HGF may stimulate tubulogenesis through the suppression of IL-8. PMID:23485708

  4. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells

    PubMed Central

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W.

    2016-01-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, +H, 56Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004; +H P = 0.049; 56Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation—implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure. PMID:27539227

  5. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells.

    PubMed

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W

    2016-01-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, (+)H, (56)Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004; (+)H P = 0.049; (56)Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation-implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure. PMID:27539227

  6. Three-dimensional Culture Conditions Lead to Decreased Radiation Induced Crytoxicity in Human Mammary Epithelial Cells

    SciTech Connect

    Sowa, Marianne B.; Chrisler, William B.; Zens, Kyra D.; Ashjian, Emily J.; Opresko, Lee K.

    2010-05-01

    For both targeted and non-targeted exposures, the cellular responses to ionizing radiation have predominantly been measured in two dimensional monolayer cultures. Although convenient for biochemical analysis, the true interactions in vivo depend upon complex interactions between cells themselves and the surrounding extra cellular matrix. This study directly compares the influence of culture conditions on radiation induced cytotoxicity following exposure to low-LET ionizing radiation. Using a three dimensional (3D) human mammary epithelial tissue model, we have found a protective effect of 3D cell culture on cell survival after irradiation. The initial state of the cells (i.e., 2D vs. 3D culture) at the time of irradiation does not alter survival, nor does the presence of extracellular matrix during and after exposure to dose, but long term culture in 3D which offers significant reduction in cytotoxicity at a given dose (e.g. ~4 fold increased survival at 5 Gy). The cell cycle delay induced following exposure to 2 and 5 Gy was almost identical between 2D and 3D culture conditions and cannot account for the observed differences in radiation responses. However the amount of apoptosis following radiation exposure is significantly decreased in 3D culture relative to the 2D monolayer after the same dose. A likely mechanism of the cytoprotective effect afforded by 3D culture conditions is the down regulation of radiation induced apoptosis in 3D structures

  7. Three-Dimensional Culture of Functional Adult Rabbit Lacrimal Gland Epithelial Cells on Decellularized Scaffold.

    PubMed

    Lin, Hui; Sun, Guoying; He, Hong; Botsford, Benjamin; Li, Mackenzie; Elisseeff, Jennifer H; Yiu, Samuel C

    2016-01-01

    Aqueous tear-deficient dry eye disease is a multifactorial chronic disorder, in which the lacrimal gland fails to produce enough tears to maintain a healthy ocular surface. Some severe cases may develop corneal damage and significant vision loss. Treatment primarily involves palliation using ocular surface lubricants, but can only provide temporary relief. Construction of a bioengineered lacrimal gland having functional secretory epithelial cells is a potentially promising option for providing long-term relief to severe dry eye patients. Using sphere-forming culture techniques, we cultured adult rabbit lacrimal gland progenitor cells and prepared a lacrimal gland scaffold by decellularization. When progenitor cells were seeded onto the decellularized scaffold, they formed duct- and acinar-like structures in the three-dimensional culture system. Lacrimal gland epithelial cells showed good cell viability, cell differentiation, and secretory function in decellularized lacrimal gland matrix, as indicated by morphology, immunostaining, and β-hexosaminidase secretion assay. This study demonstrated the potential suitability of utilizing tissue-specific progenitor cells and a tissue-derived bioscaffold for lacrimal gland restoration. PMID:26414959

  8. Mechanophysical Stimulations of Mucin Secretion in Cultures of Nasal Epithelial Cells

    PubMed Central

    Even-Tzur Davidovich, Nurit; Kloog, Yoel; Wolf, Michael; Elad, David

    2011-01-01

    Nasal epithelial cells secret mucins and are exposed in vivo to airflow-induced mechanophysical stresses, including wall shear stress (WSS), temperature, and humidity. In this work, human nasal epithelial cells cultured under air-liquid interface conditions were subjected to fields of airflow-induced oscillatory WSS at different temperature and humidity conditions. Changes in mucin secretion due to WSS were measured and the role of the cytoskeleton in mucin secretion was explored. Mucin secretion significantly increased in response to WSS in a magnitude-dependent manner with respect to static cultures and independently of the airflow temperature and humidity. In static cultures, mucin secretion decreased at high humidity with or without elevation of the temperature with respect to cultures at a comfortable climate. In cultures exposed to WSS, mucin secretion increased at high temperature with respect to cultures at comfortable climate conditions. The polymerization of actin microfilaments was shown to increase mucin secretion under WSS, whereas the dynamics of microtubule polymerization did not affect secretion. In conclusion, the data in this study show that mucin secretion is sensitive to oscillatory WSS as well as high temperature and humidity conditions. PMID:21689518

  9. Cell Lineage Identification and Stem Cell Culture in a Porcine Model for the Study of Intestinal Epithelial Regeneration

    PubMed Central

    Gonzalez, Liara M.; Williamson, Ian; Piedrahita, Jorge A.; Blikslager, Anthony T.; Magness, Scott T.

    2013-01-01

    Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/‘reserve’ stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease. PMID:23840480

  10. Effects of steroids on the secretion of immunoregulatory factors by thymic epithelial cell cultures.

    PubMed Central

    Stimson, W H; Crilly, P J

    1981-01-01

    Rat thymic epithelial cells were cultured for 39 days in the presence of various concentrations of oestradiol, testosterone, progesterone and corticosterone and the supernatants assessed for effects on the stimulation of cells from the thymus, bone marrow, lymph nodes and spleen, with several agents. All the steroids, except progesterone, were found to significantly regulate the secretion of immunoregulatory factors by the epithelial cells at physiological levels but the effects were dose dependent. Fractionation of active supernatants indicated that the capacity to enhance or depress cellular proliferation was mainly associated with substances having molecular weights greater than 30,000 or less than 1000, respectively. This study supports the idea that certain steroids can influence the immune response indirectly through the thymus. PMID:7298074

  11. Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses.

    PubMed

    Desmarets, Lowiese M B; Theuns, Sebastiaan; Olyslaegers, Dominique A J; Dedeurwaerder, Annelike; Vermeulen, Ben L; Roukaerts, Inge D M; Nauwynck, Hans J

    2013-01-01

    Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79-1683 and WSU 79-1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79-1683 still replicated significantly more efficient compared to FCoV WSU 79-1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

  12. Lens and cataract: clastogenic responses in epithelial cells of the organ-cultured rat lens

    SciTech Connect

    Geard, C.R.; Worgul, B.V.

    1987-01-01

    The epithelial cells of the vertebrate lens have an unique character and a probable involvement in cataract formation, which could be initiated by exogenous stimuli. Individual rat lenses were organ-cultured, and the effects of mitomycin C and gamma rays on sister chromatid exchanges (SCE), chromosomal aberrations, and cellular kinetics assessed in cells from the epithelial monolayer. SCE showed about a 5.5-fold increase over the mitomycin C dose range (0, 17, 83, 170 nM), while chromosomal aberrations increased 38-fold. In cells from untreated lenses, SCE were 1600 times more frequent than aberrations and at a level consistent with in vivo assessments in other cell types. Gamma rays (up to 4 Gy) had a greater inhibiting effect on cellular progression, while 17 nM mitomycin C and 1 Gy induced similar clastogenic responses. This first demonstration of such changes in lens epithelial cells expands on the cell types available for monitoring potential mutagen-carcinogens. Additionally chromosomal changes resulting from lens cellular challenge could be the basis of later cytopathological changes in the lens, of which cataract is the primary concern to humans. Potential cataractogens warrant monitoring, and the study outlined may aid in this endeavor, as well as contributing to an understanding of cataract etiology.

  13. In vitro optimization of the Gallus domesticus oviduct epithelial cells culture.

    PubMed

    Kasperczyk, K; Bajek, A; Joachimiak, R; Walasik, K; Marszalek, A; Drewa, T; Bednarczyk, M

    2012-06-01

    The aim of this experiment was to establish an efficient method for isolation and further culture in vitro of the normal chicken oviduct epithelial cells (COEC) for cell-based research models. Different factors were tested to optimize COEC primary culture for repeatable results: the origin of isolated cells (oviduct Infundibulum or Magnum section); the oviduct tissue dissociation procedure (mechanical scrapping or mincing), tissue digestion times (15, 30 and 45 min), the culture plates coating (colagene I, polystyrene surface or 3T3 feeder layer), the growth media (classic DMEM/Ham's F12 and defined serum-free medium, Lonza Switzerland), incubation temperature (37 °C vs 41°C) and different cell seeding numbers: 0.2M, 0.5M and 1.0M cells/well. The COEC isolated by mincing the Infundibular neck and digestion of tissue for 30 min formed cell aggregates of bright colour and gave proliferating colonies of epithelial-like character which was the best result obtained from all applied procedures in our studies. The fibroblast-like cells considered as contaminants occurred only sporadically up to day 7 of culture. Seeding about 1M cells in 1 mL of serum-free medium onto 12-well dishes gave the optimal growth of colonies resulting in 5 to 7 confluent culture wells from a single oviduct sample. Feeder layer and collagen I did not improve adhesion of the COEC to the culture vessel. Adoption of 37 °C and 41 °C did not reveal apparent differences to the condition of cultured COEC. Cell differentiation and proliferation potential depends on number and replicative capacity of isolated progenitors. The progenitors are responsible for holoclones formation and good culture growth. The percentage of colonies developed from the cells isolated from Infundibulum was greater than that of other samples in our studies. We conclude that the model of COEC primary cultures from different segments of oviduct, in particular infundibulum, should be incorporated to the range of avian cells

  14. Transepithelial electrical resistance (TEER): a functional parameter to monitor the quality of oviduct epithelial cells cultured on filter supports.

    PubMed

    Chen, Shuai; Einspanier, Ralf; Schoen, Jennifer

    2015-11-01

    Cultivation of oviduct epithelial cells on porous filters fosters in vivo-like morphology and functionality. However, due to the optical properties of the filter materials and the cells' columnar shape, cell quality is hard to assess via light microscopy. In this study, we aim to evaluate transepithelial electrical resistance (TEER) measurement as a prognostic quality indicator for the cultivation of porcine oviduct epithelial cells (POEC). POEC were maintained in four different types of media for 3 and 6 w to achieve diverse culture qualities, and TEER was measured before processing samples for histology. Culture quality was scored using morphological criteria (presence of cilia, confluence and cell polarity). We furthermore analyzed the correlation between cellular height (as a measure of apical-basal polarization) and TEER in fully differentiated routine cultures (biological variation) and in cultures with altered cellular height due to hormonal stimulation. Fully differentiated cultures possessed a moderate TEER between 500 and 1100 Ω*cm(2). Only 5% of cultures which exhibited TEER values in this defined range had poor quality. Sub-differentiated cultures showed either very low or excessively high TEER. We unveiled a highly significant (P < 0.0001) negative linear correlation between TEER and epithelial height in well-differentiated cultures (both routine and hormone stimulated group). This may point toward the interaction between tight junction assembly and epithelial apical-basal polarization. In conclusion, TEER is a straightforward quality indicator which could be routinely used to monitor the differentiation status of oviduct epithelial cells in vitro. PMID:26223877

  15. Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in-vitro

    PubMed Central

    Genicio, Nuria; Paramo, Juan Gallo; Shortt, Alex J.

    2015-01-01

    PURPOSE Cultured human limbal epithelial cells (HLEC) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behaviour and long-term fate post transplantation. The aim of this research was to evaluate, in-vitro, quantum dot (QDot) technology as a tool for tracking transplanted HLEC. METHODS In-vitro cultured HLEC were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony forming efficiency assays and expression of CK3, P63alpha and ABCG2. Sheets of cultured HLEC labeled with Qdot nanocrystals were transplanted onto decellularised human corneo-scleral rims in an organ culture model and observed to investigate the behaviour of transplanted cells. RESULTS Qdot labeling had no detrimental effect on HLEC viability or function in-vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks post transplantation in our organ culture model. In addition it was possible to label and observe epithelial cells in intact human corneas using the Rostock corneal module adapted for use with the Heidelberg HRA. CONCLUSIONS This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks post transplantation, however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterisation in in-vivo models are required. PMID:26024089

  16. Differentiation of normal and cultured preneoplastic tracheal epithellal cells in rats: importance of epithelial mesenchymal interactions

    SciTech Connect

    Terzaghi, M.; Klein-Szanto, A.J.P.

    1980-11-01

    Changes in the dependence on mesenchymal tissues for survival and differentiation in inbred F344 female rats were investigated in tracheal epithelial cells exposed to 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). Fresh suspensions of normal tracheal epithelium or cultured preneoplastic cells were inoculated into isolated organ segments (trachea, esophagus, bladder, or small intestine) or into Dacron containers that were then implanted subdermally into isogenic recipients. At various times after cell inoculation and implantation, tissues were removed for histologic evaluation. Normal cells inoculated into frozen-thawed trachea, esophagus, bladder, and intestine yielded a regular mucociliary epithelium. Normal cell inocula did not, however, survive in trachea previously heated (100/sup 0/C), fixed in ethanol, or digested with collagenese; nor did normal cells survive in Dacron containers unless tracheal fibroblasts plus epithelial cells were inoculated together. DMBA- and TPA-exposed cell populations with increased growth capacity in vitro survived and differentiated on all of the above substrates. For survival and differentiation in vivo, preneoplastic cells appeared to have less stringent substrate requirements than did normal cells. Application of the described techniques to the study of changes occurring early in the development of neoplastic disease is discussed.

  17. Azithromycin: assessment of intrinsic cytotoxic effects on corneal epithelial cell cultures

    PubMed Central

    Mencucci, Rita; Pellegrini-Giampietro, Domenico E; Paladini, Iacopo; Favuzza, Eleonora; Menchini, Ugo; Scartabelli, Tania

    2013-01-01

    Purpose To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK). Methods Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours. Results Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test. Conclusion Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process. PMID:23737659

  18. Isolation and Culture of Bovine Oviductal Epithelial Cells for Use in the Anatomy and Physiology Laboratory and Undergraduate Research

    ERIC Educational Resources Information Center

    Way, Amy L.

    2006-01-01

    This article presents methods for the isolation and culture of epithelial cells from the bovine oviduct for use in both research and the teaching laboratory and provides examples of ways that an oviductal cell culture can be incorporated into an undergraduate research program. Cow reproductive tracts are readily available from area butchers, and…

  19. Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

    PubMed Central

    Henry, D N; Del Monte, M; Greene, D A; Killen, P D

    1993-01-01

    Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications. Images PMID:8349800

  20. PRIMARY CULTURE OF CHOROIDAL EPITHELIAL CELLS: CHARACTERIZATION OF AN IN VITRO MODEL OF BLOOD-CSF BARRIER

    PubMed Central

    ZHENG, WEI; ZHAO, QIUQU; GRAZIANO, JOSEPH H.

    2016-01-01

    Summary A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2–5 × 104 cells from pooled plexuses of three to four rats, and a viability of 77–85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2–3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 ± 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood–CSF barrier. PMID:9542634

  1. Biocompatibility of microplates for culturing epithelial renal cells evaluated by a microcalorimetric technique.

    PubMed

    Xie, Y; DePierre, J W; Nässberger, L

    2000-09-01

    In the present study we have developed a microcalorimetric procedure which allows convenient investigation of biocompatibility in a microsystem. We examined the biocompatibility of a porcine renal epithelial tubule cell line LLC-PK1 and a human primary renal epithelial tubule cell (RPTEC) with microplates composed of three different materials, i.e. Thermanox, transparent film and titanium. All three materials showed equal biocompatibility with LLC-PK1 cells, judging from the attainment of steady-state power curves and the same rate of heat production per cell (2.5 microW / microg DNA). The human renal cells were poorly biocompatible with the Thermanox and transparent film. However, on titanium the RPTEC cell did adhere, as demonstrated by a steady-state power curve. The human cells also showed a higher metabolic activity (3.0 microW / microg DNA), than did LLC-PK1 cells cultured on the same type of microplates. In research on biocompatibility there is a need for alternatives to experimental animal investigations. The present technique allows studies of cellular interactions with different biomaterials in a rapid and standardized manner and may therefore prove to be a useful screening procedure. PMID:15348389

  2. Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

    PubMed Central

    Stewart, Ceri E.; Torr, Elizabeth E.; Mohd Jamili, Nur H.; Bosquillon, Cynthia; Sayers, Ian

    2012-01-01

    The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions. PMID:22287976

  3. The mesenchymal substrate influences the epithelial phenotype in a three-dimensional cell culture.

    PubMed

    Merne, Marina; Syrjänen, Stina

    2003-09-01

    Cell-matrix interactions are thought to influence epithelial structure, growth, and differentiation. Three-dimensional cell cultures were used to study the effects of the composition of the dermal equivalent on the morphology of epithelium grown from HaCaT skin keratinocytes. Three commercial preparations, a basement membrane preparation from a tumor (matrix 1), two preparations consisting of collagen type I and III (matrix 3 and 4) and a noncommercial preparation containing collagen type I (matrix 2) were investigated. The effects of fibroblasts of different origin (vaginal mucosa, oral buccal mucosa, and skin) were investigated in connection with matrix 4. The histomorphology and expression of the proteins PCNA, Ki-67, p53, p21, pankeratin, involucrin, cytokeratin 10 (Ck10), Ck17, Ck19 and collagen type IV were evaluated. Three-dimensional cultures of HaCaT cells gave rise to an epithelium with an immature and hyperproliferative character, showing active proliferation with intense PCNA staining. Both matrix 1 and matrix 2 resulted in an epithelium with budding into the matrix and some degree of layering. These epithelia showed only scattered Ck17 and Ck19 expression but a low terminal differentiation potential as indicated by scattered Ck10 and involucrin staining. The epithelia of cocultures with matrices 3 and 4 were positive for Ck17 and Ck19. However, the epithelium on matrix 3 showed strong expression of the terminal differentiation marker Ck10 and involucrin. This methodological study provides evidence of the importance of standardization of the composition of the matrix to avoid confounding effects on epithelial morphology and protein expression in studies using a three-dimensional epithelial culture model. PMID:12898149

  4. Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

    PubMed Central

    Amatangelo, Michael D.; Garipov, Azat; Li, Hua; Conejo-Garcia, Jose R.; Speicher, David W.; Zhang, Rugang

    2013-01-01

    Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy. PMID:23759589

  5. Therapeutic effect of lung mixed culture-derived epithelial cells on lung fibrosis.

    PubMed

    Tanaka, Kensuke; Fujita, Tetsuo; Umezawa, Hiroki; Namiki, Kana; Yoshioka, Kento; Hagihara, Masahiko; Sudo, Tatsuhiko; Kimura, Sadao; Tatsumi, Koichiro; Kasuya, Yoshitoshi

    2014-11-01

    Cell-based therapy is recognized as one of potential therapeutic options for lung fibrosis. However, preparing stem/progenitor cells is complicated and not always efficient. Here, we show easily prepared cell populations having therapeutic capacity for lung inflammatory disease that are named as 'lung mixed culture-derived epithelial cells' (LMDECs). LMDECs expressed surfactant protein (SP)-C and gave rise to type I alveolar epithelial cells (AECs) in vitro and in vivo that partly satisfied type II AEC-like characteristics. An intratracheal delivery of not HEK 293 cells but LMDECs to the lung ameliorated bleomycin (BLM)-induced lung injury. A comprehensive analysis of bronchoalveolar fluid by western blot array revealed that LMDEC engraftment could improve the microenvironment in the BLM-instilled lung in association with stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 signaling axis. SDF-1 enhanced both migration activity and differentiating efficiency of LMDECs. Further classification of LMDECs by flow cytometric study showed that a major population of LMDECs (LMDEC(Maj), 84% of total LMDECs) was simultaneously SP-C(+), CD44(+), CD45(+), and hematopoietic cell lineage(+) and that LMDECs included bronchioalveolar stem cells (BASCs) showing SP-C(+)Clara cell secretory protein(+)stem cell antigen (Sca)1(+) as a small population (1.8% of total LMDECs). CD44(+)-sorted LMDEC(Maj) and Sca1(+)-sorted LMDECs equally ameliorated fibrosis induced by BLM like LMDECs did. However, infiltrated neutrophils were observed in Sca1(+)-sorted LMDEC-treated alveoli that was not typical in LMDEC(Maj)- or LMDEC-treated alveoli. These findings suggest that the protective effect of LMDECs against BLM-induced lung injury depends greatly on that of LMDEC(Maj). Furthermore, the cells expressing both alveolar epithelial and hematopoietic cell lineage markers (SP-C(+)CD45(+)) that have characteristics corresponding to LMDEC(Maj) were observed in the alveoli of lung and

  6. Differentiation of cones in cultured rabbit retina: effects of retinal pigment epithelial cell-conditioned medium.

    PubMed

    Mack, Andreas F; Uhlmann, Daniela; Germer, Angela; Szél, Agoston; Enzmann, Volker; Reichenbach, Andreas

    2003-04-24

    This study was aimed at investigating the postnatal differentiation of cone photoreceptors in the rabbit retina in an organotypic explant culture system. Both short wavelength (S) and middle wavelength (M) cone opsins were expressed in culture but M cones appeared only in retinal explants from the dorsal half of the eye. Stimulating the explants with retinal pigment epithelial cell (RPE) conditioned medium resulted in a suppression of opsin expression despite of an increase of the number of presumptive peanut agglutinin-labeled cones. These results suggest that at birth the immature cones are largely undetermined in terms of their final cone identity although some positional information ('dorsal' vs. 'ventral' retina) is present. Furthermore, factors from RPE may inhibit as well as stimulate different steps of cone cell differentiation. PMID:12676342

  7. A bovine mammary endothelial/epithelial cell culture model of the blood/milk barrier.

    PubMed Central

    Guidry, A J; O'Brien, C N; Douglass, L W

    1998-01-01

    The complex nature of the mammary gland has hampered in-depth studies of the relationship of the circulatory system to cells lining the teat ducts and alveoli of the gland. This study reports an in vitro model of endothelial and epithelial cells separated by a subcellular matrix that simulates the blood milk barrier of the bovine mammary gland. Dual chamber culture dishes with a porous membrane separating the upper and lower chamber were used. Endothelial and epithelial cells were cultured on opposite sides of the porous membrane. A collagen and fibroblast subcellular matrix, separating the 2 cell layers, simulated the in vivo interstitial tissue. Changes in surface binding of anti-bodies to polymorphonuclear neutrophils (PMN) following their migration from the upper to the lower chamber simulated the passage of PMN from blood to milk. Changes in the binding of antibodies to PMN agreed with results observed following the migration of PMN from blood to milk in vivo. This gives credence to the model's potential value for studies where more direct observation of the blood/milk barrier is required. The model will be further tested for its usefulness as an assay for determining: 1) antibiotic diffusion from milk to blood and from blood to milk, 2) cytotoxicity of prophylactic and therapeutic mammary infusion products, 3) factors affecting bacterial adhesion and penetration of mammary epithelial tissue, 4) effectiveness of antibodies present in lacteal secretions in preventing bacterial adhesion, and 5) the feasibility of gene constructs to induce synthesis and secretion of mastitis-preventing compounds and prophylactic and therapeutic compounds for treatment of human disorders. PMID:9553710

  8. Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis.

    PubMed Central

    Cheng, P W; Boat, T F; Cranfill, K; Yankaskas, J R; Boucher, R C

    1989-01-01

    Cystic fibrosis (CF) respiratory epithelia exhibit abnormal anion transport that may be linked to abnormal lung defense. In these studies, we investigated whether primary cultures of CF respiratory epithelial cells regulate abnormally the sulfate content of high molecular weight glycoconjugates (HMG) participating in airways' mucosal defense. HMG, including glycosaminoglycans and mucin-type glycoproteins released spontaneously into medium and HMG released from cell surfaces by trypsin, were metabolically labeled with 35SO4- and [6-3H]-glucosamine (GlcN) or 35SO4- and [3H]serine. All three classes of HMG from CF cells exhibited 35S/3H labeling ratios 1.5-4-fold greater than HMG from normal or disease control cells. Differences for labeling ratios of HMG from CF cells were shown to be the consequence of increased 35SO4- incorporation rather than decreased peptide synthesis and release or HMG glycosylation. The buoyant density of CF mucin-type HMG also was increased, consistent with increased sulfation. These observations suggest that oversulfation of a spectrum of HMG is a genetically determined characteristic of CF epithelial cells and may play an important pathophysiological role by altering the properties of mucous secretions and/or the interactions between selected bacteria and HMG at the airways' surface. Images PMID:2738159

  9. Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos.

    PubMed

    Lin, Han Jen; Wang, Siou Huei; Pan, Yu Hui; Ding, Shih-Torng

    2016-01-01

    We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development. PMID:27022687

  10. Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture.

    PubMed

    Leguen; Carlsson; Perdu-Durand; Prunet; Pärt; Cravedi

    2000-03-01

    The biotransformation of xenobiotics and steroids was investigated in cultured respiratory epithelial cells from rainbow trout (Oncorhynchus mykiss) gills. As a first approach, ethoxyresorufin-O-deethylase (EROD), chosen as a marker of CYP1A activity, was measured in monolayers of adherent cells. The induction of this enzyme was studied in cells exposed to beta-naphthoflavone (BNF) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in concentrations ranging from 10(-6) to 10(-12) M. After 24 h, TCDD showed a maximal induction at a concentration of 10(-9) M while BNF showed a maximal induction at a concentration of 10(-7) M. Concurrently, a variety of substrates involved in cytochrome P450-dependent metabolism as well as phase II reactions, namely ethoxycoumarin, aniline and testosterone were incubated with cultured gill cells for 2 or 8 h and with freshly isolated hepatocytes for comparison. Our results revealed a significant cytochrome P450-dependent activity in gill cells with ethoxycoumarin and aniline, but no hydroxylation was observed with testosterone as substrate. No trace of sulfate conjugate was detected. With 2.5 µM aniline as substrate, 2-hydroxyaniline accounted for 32.1% of the radioactivity after 2 h incubation whereas acetanilide amounted to 6.4%. Significant differences were found between gill cells and isolated hepatocytes in the capacity of these systems to conduct oxidative and conjugating metabolic pathways. Qualitatively, the main difference was observed for testosterone which is hydroxylated in position 6beta and 16beta and conjugated to glucuronic acid in liver cells, whereas reductive biotransformation giving rise to dihydrotestosterone and androstanediol and traces of androstenedione were observed in gill cells. Quantitatively, the biotransformation activity in gill epithelial cells, expressed as pmol/h per mg protein, was between 1.5 and 14% of the activity level observed in isolated hepatocytes, depending on the substrate. PMID:10686323

  11. Development and characterization of a primary culture of chicken embryonic tracheal epithelial cells and their use in avian studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major route of infection of avian influenza is through cells of the airway epithelium. To study the molecular mechanism of infection and early host responses we created a primary chicken tracheal cell culture. Epithelial cells were isolated from the trachea of 18 day old chicken embryos and cult...

  12. Commensal Bacteria Modulate Innate Immune Responses of Vaginal Epithelial Cell Multilayer Cultures

    PubMed Central

    Rose, William A.; McGowin, Chris L.; Spagnuolo, Rae Ann; Eaves-Pyles, Tonyia D.; Popov, Vsevolod L.; Pyles, Richard B.

    2012-01-01

    The human vaginal microbiome plays a critical but poorly defined role in reproductive health. Vaginal microbiome alterations are associated with increased susceptibility to sexually-transmitted infections (STI) possibly due to related changes in innate defense responses from epithelial cells. Study of the impact of commensal bacteria on the vaginal mucosal surface has been hindered by current vaginal epithelial cell (VEC) culture systems that lack an appropriate interface between the apical surface of stratified squamous epithelium and the air-filled vaginal lumen. Therefore we developed a reproducible multilayer VEC culture system with an apical (luminal) air-interface that supported colonization with selected commensal bacteria. Multilayer VEC developed tight-junctions and other hallmarks of the vaginal mucosa including predictable proinflammatory cytokine secretion following TLR stimulation. Colonization of multilayers by common vaginal commensals including Lactobacillus crispatus, L. jensenii, and L. rhamnosus led to intimate associations with the VEC exclusively on the apical surface. Vaginal commensals did not trigger cytokine secretion but Staphylococcus epidermidis, a skin commensal, was inflammatory. Lactobacilli reduced cytokine secretion in an isolate-specific fashion following TLR stimulation. This tempering of inflammation offers a potential explanation for increased susceptibility to STI in the absence of common commensals and has implications for testing of potential STI preventatives. PMID:22412914

  13. Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

    PubMed

    Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

    2009-09-01

    The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads. PMID:19876557

  14. Short communication: Ability of cultured mammary epithelial cells in a bicameral system to secrete milk fat.

    PubMed

    Ernens, I; Clegg, R; Schneider, Y-J; Larondelle, Y

    2007-02-01

    Mammary epithelial cells from lactating cows were cultured onto inserts coated with type I collagen. Every second day, the rates of fatty acid synthesis and secretion were determined by measuring the amount of [14C]-labeled sodium acetate incorporated into lipids over a 4-h period. The [14C]-containing lipids were identified by thin layer chromatography fractionation. In parallel, the integrity of the cell layer was evaluated by measurement of transepithelial electrical resistance. The integrity increased progressively to reach a maximum after 8 d of culture. Cells incorporated acetate into lipids; 1.34% of acetate was incorporated into lipids produced by freshly isolated cells. This percentage decreased to 0.5% after 2 d of culture. Moreover, this capacity decreased with the duration in culture; on d 8, the rate of incorporation dropped to about 3% of that on d 2. In the cell extracts, the [14C]-labeled lipids were mainly triglycerides, although the proportion of diglycerides and phospholipids progressively increased as a part of total newly synthesized lipids. The proportion of triglycerides decreased 0.66 times between d 2 and 8 when the proportion of diglycerides and phospholipids increased 1.33 and 2.18 times, respectively. About 28% of the newly synthesized lipids were secreted within 4 h of incubation. Around 65 to 85% of these labeled lipids were found in the apical compartment, suggesting a partially vectorial secretion. But 58 to 80% of labeled lipids found in the apical and basolateral medium were free fatty acids. Functional tight junctions and incorporation of labeled fatty acids into triglycerides are not compatible with an inferred status of complete dedifferentiation of the cell layer. Moreover, triglyceride secretion seems compromised, probably due to the lack of an appropriate cell environment and cell shape. PMID:17235143

  15. Cationorm shows good tolerability on human HCE-2 corneal epithelial cell cultures.

    PubMed

    Kinnunen, Kati; Kauppinen, Anu; Piippo, Niina; Koistinen, Arto; Toropainen, Elisa; Kaarniranta, Kai

    2014-03-01

    Preservatives have been for a long time known to cause detrimental effects on ocular surface. Cationorm, a preservative-free compound with electrostatic properties is a novel way to solve the problems encountered with traditional benzalkonium chloride (BAK)-containing eye drops. The aim of this study was to evaluate tolerability of the preservative-free cationic emulsion Cationorm in vitro on corneal epithelial cells. The human corneal epithelial cell (HCE-2) culture line was used to study cellular morphology, cytotoxicity and inflammatory responses after Cationorm diluted 1/10 exposure for 5, 15 and 30 min. Exposures to Systane diluted 1/10 with polyquaternium-1/polidronium chloride 0.001% as preservative, BAK 0.001% or C16 (0.0002%) and normal cell culture medium served as positive and negative references. Cell viability was determined by measuring the release of lactate dehydrogenase (LDH) and mitochondrial dehydrogenase activity was evaluated using 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The possible induction of apoptosis was analyzed by measuring the activity of caspase-3, and Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells after the exposure to test compounds. Furthermore, the tendency of the test compounds to produce inflammatory reaction was determined by analyzing the production of proinflammatory cytokines IL-6 and IL-8, and DNA binding of the p65 subunit of transcription factor NF-κB was measured from cell lysates. HCE-2 cells showed no morphological changes after the exposure to Cationorm, but in cells exposed to BAK, clear cytoplasm vacuolization and loose cell-cell contacts were observed in transmission (TEM) or scanning (SEM) electron microscopic analyses. Cell viability, as measured with the release of LDH, indicated a time dependent increase in LDH expression after exposure to all test compounds but especially with BAK. Moreover, Cationorm and BAK time-dependently decreased the

  16. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations.

    PubMed

    Jansen, J; De Napoli, I E; Fedecostante, M; Schophuizen, C M S; Chevtchik, N V; Wilmer, M J; van Asbeck, A H; Croes, H J; Pertijs, J C; Wetzels, J F M; Hilbrands, L B; van den Heuvel, L P; Hoenderop, J G; Stamatialis, D; Masereeuw, R

    2015-01-01

    The bioartificial kidney (BAK) aims at improving dialysis by developing 'living membranes' for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) as a fluorescent substrate. Initial ASP(+) uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a 'living membrane' of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

  17. Human proximal tubule epithelial cells cultured on hollow fibers: living membranes that actively transport organic cations

    PubMed Central

    Jansen, J.; De Napoli, I. E; Fedecostante, M.; Schophuizen, C. M. S.; Chevtchik, N. V.; Wilmer, M. J.; van Asbeck, A. H.; Croes, H. J.; Pertijs, J. C.; Wetzels, J. F. M.; Hilbrands, L. B.; van den Heuvel, L. P.; Hoenderop, J. G.; Stamatialis, D.; Masereeuw, R.

    2015-01-01

    The bioartificial kidney (BAK) aims at improving dialysis by developing ‘living membranes’ for cells-aided removal of uremic metabolites. Here, unique human conditionally immortalized proximal tubule epithelial cell (ciPTEC) monolayers were cultured on biofunctionalized MicroPES (polyethersulfone) hollow fiber membranes (HFM) and functionally tested using microfluidics. Tight monolayer formation was demonstrated by abundant zonula occludens-1 (ZO-1) protein expression along the tight junctions of matured ciPTEC on HFM. A clear barrier function of the monolayer was confirmed by limited diffusion of FITC-inulin. The activity of the organic cation transporter 2 (OCT2) in ciPTEC was evaluated in real-time using a perfusion system by confocal microscopy using 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP+) as a fluorescent substrate. Initial ASP+ uptake was inhibited by a cationic uremic metabolites mixture and by the histamine H2-receptor antagonist, cimetidine. In conclusion, a ‘living membrane’ of renal epithelial cells on MicroPES HFM with demonstrated active organic cation transport was successfully established as a first step in BAK engineering. PMID:26567716

  18. Induction of rat jejunal epithelial cell expression of sucrase-isomaltase by glucocorticoids in primary cell culture and in vivo.

    PubMed

    Yeh, K Y; Yeh, M; Holt, P R

    1989-01-01

    The cell cycle phase that mediates the induction of intestinal sucrase-isomaltase (SI) expression by glucocorticoids was investigated by measuring migration rates of 3H-DNA-labeled and of SI-containing epithelial cells by autoradiography and indirect immunofluorescent staining after simultaneous administration of [3H]thymidine and cortisone to 12-d-old rat pups. By 24 and 48 h, lead 3H-DNA-labeled cells had migrated 7.8 and 12.4 cell positions higher on the villus than lead cells expressing SI. Cell migration rates from 12 to 24 h and 24 to 48 h were 0.68 and 0.97 cell position/h. Thus, commitment to SI expression occurred in cells 11.5-12.8 h after the S phase, which is calculated to be in the G1 phase. To determine whether committed cells need to replicate to express SI, cell differentiation was examined in primary cultures of crypt cells originating from corticosterone-treated rats. About two-thirds of cultured cells were retarded in the S phase after plating, as judged by no increase of DNA labeling indices, no change in epithelial cell number, and the absence of mitosis (less than 0.01%). The proportion of cells expressing SI increased from 0 to 6-8% between 12 and 24 h, and reached 48% 48 h after plating on collagen-coated dishes. SI expression did not occur in cells plated on glass or plastic surfaces. Pulse labeling with [35S]methionine confirmed that de novo synthesis of SI occurred in cell cultures. Thus, additional cell cycling of committed cells occurring in vivo is not obligatory for the expression of SI. PMID:2736328

  19. Methionine protects against hyperthermia-induced cell injury in cultured bovine mammary epithelial cells.

    PubMed

    Han, Zhao-Yu; Mu, Tian; Yang, Zhen

    2015-01-01

    The aim of this study was to investigate the effects of methionine on cell proliferation, antioxidant activity, apoptosis, the expression levels of related genes (HSF-1, HSP70, Bax and Bcl-2) and the expression levels of protein (HSP70) in mammary epithelial cells, after heat treatment. Methionine (60 mg/L) increased the viability and attenuated morphological damage in hyperthermia-treated bovine mammary epithelial cells (BMECs). Additionally, methionine significantly reduced lactate dehydrogenase leakage, malondialdehyde formation, nitric oxide, and nitric oxide synthase activity. Superoxide dismutase, catalase, and glutathione peroxidase enzymatic activity was increased significantly in the presence of methionine. Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 h, and the expression level of the HSP70 gene and protein further increased with incubation at 42 °C for 30 min. Compared to the control, the expression of HSF-1 mRNA increased, and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress. Methionine also increased survival and decreased early apoptosis of hyperthermia-treated BMECs. Thus, methionine has cytoprotective effects on hyperthermia-induced damage in BMECs. PMID:25108357

  20. A NOVEL SYSTEM FOR THE CO-CULTURE OF EPIDIDYMAL EPITHELIAL CELLS AND SPERM FROM ADULT RATS

    EPA Science Inventory

    To study interactions which occur between the epididymal epithelial cells and sperm within the epididymis during sperm maturation, a specialized co-culture system capable of supporting the differentiated function of these cell types must be utilized. A multifaceted approach has b...

  1. Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells.

    PubMed

    Abul-Hassan, K; Walmsley, R; Tombran-Tink, J; Boulton, M

    2000-12-01

    The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated 'pcDNA3.1-Ty.prom.Luc'. The plasmid was co-transfected into RPE cells with a second mammalian expression plasmid (pRL-TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha-melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post-transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up-regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post-transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity. PMID:11153695

  2. Serotonin activates cell survival and apoptotic death responses in cultured epithelial thyroid cells.

    PubMed

    Cerulo, Giuliana; Tafuri, Simona; De Pasquale, Valeria; Rea, Silviana; Romano, Simona; Costagliola, Anna; Della Morte, Rossella; Avallone, Luigi; Pavone, Luigi Michele

    2014-10-01

    Anatomic and physiological interactions between central serotonergic system and thyroid gland are well established. However, the effects of locally available serotonin on the thyroid functions are poorly known. Here, we first demonstrate the expression of serotonin transporter SERT and 5-HT2A receptor subtype in rat thyroid epithelial cell line FRT both at mRNA and protein levels. In order to investigate the molecular mechanisms of serotonin action, FRT cells were exposed to increasing concentrations of the amine. Low concentrations of serotonin (up to 5 μM) enhanced FRT cell growth, and ERK1/2 and SMAD2/3 phosphorylation. Cell exposure to the selective 5-HT2A receptor agonist DOI recapitulated the effects of 5-HT on ERK1/2 phosphorylation. By contrast, administration of M100907, a specific 5-HT2A receptor inhibitor, prevented 5-HT induced ERK1/2 activation. On the other hand, high doses of serotonin (50 μM up to 1 mM) activated a caspase-3 mediated apoptosis of cells. Overall, our findings demonstrate that low levels of serotonin, interacting with 5-HT2A receptor, are able to activate proliferative signals in the thyroid epithelial cells, while high levels of serotonin cause pro-apoptotic responses, thus suggesting an active role of the amine in the thyroid functions and disorders. PMID:24997405

  3. Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis

    PubMed Central

    Moon, Clara; VanDussen, Kelli L.; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.

    2013-01-01

    There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining IgA transcytosis across Transwells. IgA transcytosis required induction of polymeric immunoglobulin receptor (pIgR) expression, which could be stimulated by a combination of LPS and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that TNFα, IL-1β, IL-17 and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that amongst these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. IFNγ however did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology. PMID:24220295

  4. The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains.

    PubMed

    Yue, Yujuan; Kaur, Amitinder; Lilja, Anders; Diamond, Don J; Walter, Mark R; Barry, Peter A

    2016-06-01

    Kidney epithelial cells are common targets for human and rhesus cytomegalovirus (HCMV and RhCMV) in vivo, and represent an important reservoir for long-term CMV shedding in urine. To better understand the role of kidney epithelial cells in primate CMV natural history, primary cultures of rhesus macaque kidney epithelial cells (MKE) were established and tested for infectivity by five RhCMV strains, including two wild-type strains (UCD52 and UCD59) and three strains containing different coding contents in UL/b'. The latter strains included 180.92 [containing an intact RhUL128-RhUL130-R hUL131 (RhUL128L) locus but deleted for the UL/b' RhUL148-rh167-loci], 68-1 (RhUL128L-defective and fibroblast-tropic) and BRh68-1.2 (the RhUL128L-repaired version of 68-1). As demonstrated by RhCMV cytopathic effect, plaque formation, growth kinetics and early virus entry, we showed that MKE were differentially susceptible to RhCMV infection, related to UL/b' coding contents of the different strains. UCD52 and UCD59 replicated vigorously in MKE, 68-1 replicated poorly, and 180.92 grew with intermediate kinetics. Reconstitution of RhUL128L in 68-1 (BRh68-1.2) restored its replication efficiency in MKE as compared to UCD52 and UCD59, consistent with the essential role of UL128L for HCMV epithelial tropism. Further analysis revealed that the UL/b' UL148-rh167-loci deletion in 180.92 impaired RhUL132 (rh160) expression. Given that 180.92 retains an intact RhUL128L, but genetically or functionally lacks genes from RhUL132 (rh160) to rh167 in UL/b', its attenuated infection efficiency indicated that, along with RhUL128L, an additional protein(s) encoded within the UL/b' RhUL132 (rh160)-rh167 region (potentially, RhUL132 and/or RhUL148) is indispensable for efficient replication in MKE. PMID:26974598

  5. Elevated non-esterified fatty acid concentrations hamper bovine oviductal epithelial cell physiology in three different in vitro culture systems.

    PubMed

    Jordaens, L; Arias-Alvarez, M; Pintelon, I; Thys, S; Valckx, S; Dezhkam, Y; Bols, P E J; Leroy, J L M R

    2015-10-01

    Elevated non-esterified fatty acids (NEFAs) have been recognized as an important link between lipolytic metabolic conditions and impaired fertility in high-yielding dairy cows. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesize that elevated NEFAs may contribute to the complex pathology of subfertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate direct NEFA effects on BOEC physiology in three different in vitro cell culture systems. Bovine oviductal epithelial cells (four replicates) were mechanically isolated, pooled, and cultured as conventional monolayers, as explants, and in a polarized cell culture system with Dulbecco's modified Eagle's medium/F12-based culture medium. Bovine oviductal epithelial cells were exposed to an NEFA mixture of oleic, stearic, and palmitic acids for 24 hours at both physiological and pathologic concentrations. A control (0 μM NEFA) and a solvent control (0 μM NEFA + 0.45% ethanol) group were implemented. Bovine oviductal epithelial cells physiology was assessed by means of cell number and viability, a sperm binding assay, transepithelial electric resistance (TER), and a wound-healing assay. Bovine oviductal epithelial cell morphology was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Bovine oviductal epithelial cell number was negatively affected by increasing NEFAs, however, cell viability was not. Sperm binding affinity significantly decreased with increasing NEFAs and tended (P = 0.051) to be more affected by the direction of NEFA exposure in the polarized cell culture system. The absolute TER increase after NEFA exposure in the control (110 ± 11 Ω.cm(2)) was significantly higher than that in all the other treatments and was also different depending on the exposure side. Bidirectional exposed monolayers were even

  6. Effect of different artificial tears against desiccation in cultured human epithelial cells

    PubMed Central

    Tost, Frank; Keiss, Ramona; Großjohann, Rico; Jürgens, Clemens; Giebel, Jürgen

    2012-01-01

    Summary Background A large number of artificial tears is widely used to treat dry eye symptoms. To test the efficacy of these drugs independent of individual parameters in vitro models are required. As described previously, we employed a reproducible in vitro cell culture system to evaluate the desiccation protection capability of some artificial tears. In the present paper data is presented of another set of pharmaceutical agents. Material/Methods Conjunctival epithelial cell line Chang 1-5c-4 (series 1) and the corneal cell line 2.040 pRSV-T (series 2) were cultured under standard conditions. Confluent cells were wetted for 20 min with artificial tears (Arufil® Uno, Arufil®, Lacrimal®, Lacophthal® sine, Siccaprotect®, Tears Again®, Vidisept® EDO, Vistil®, Wet Comod®) or PBS as a control. After exposure to a constant air flow for 0, 15, 30 and 45 minutes respectively, cells were incubated with the vital dye alamarBlue. Subsequently, absorption of the oxidised form of the dye was assessed using an ELISA-Reader. Results Cell best survival rates in series 1 after 15 min were found for Lacrimal® (0.89), Wet Comod® (0.84) compared to PBS (0.66) and in series 2 for Vidisept® EDO (0.57) and Lacrimal® (0.56) compared to PBS (0.01). After 45 min highest survival was seen in series 1 for Lacrimal® (0.46) and Lacophthal® sine (0.36) compared to PBS (0.33) and in series 2 for Lacrimal® (−0.06) and Arufil (−0.16) compared to PBS (−0.23). Conclusions Both cell lines tested showed different susceptibility towards desiccation and the artificial tears showed differences in preventing cells from desiccation. PMID:22534701

  7. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    SciTech Connect

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  8. Comparative Analysis of KnockOut™ Serum with Fetal Bovine Serum for the In Vitro Long-Term Culture of Human Limbal Epithelial Cells

    PubMed Central

    Liu, Zaoxia

    2016-01-01

    The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration. PMID:27446607

  9. Establishment of a Novel Lingual Organoid Culture System: Generation of Organoids Having Mature Keratinized Epithelium from Adult Epithelial Stem Cells

    NASA Astrophysics Data System (ADS)

    Hisha, Hiroko; Tanaka, Toshihiro; Kanno, Shohei; Tokuyama, Yoko; Komai, Yoshihiro; Ohe, Shuichi; Yanai, Hirotsugu; Omachi, Taichi; Ueno, Hiroo

    2013-11-01

    Despite the strong need for the establishment of a lingual epithelial cell culture system, a simple and convenient culture method has not yet been established. Here, we report the establishment of a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Histological analyses showed that the generated organoids had both a stratified squamous epithelial cell layer and a stratum corneum. Very recently, we showed via a multicolor lineage tracing method that Bmi1-positive stem cells exist at the base of the epithelial basal layer in the interpapillary pit. Using our new culture system, we found that organoids could be generated by single Bmi1-positive stem cells and that in the established organoids, multiple Bmi1-positive stem cells were generated at the outermost layer. Moreover, we observed that organoids harvested at an early point in culture could be engrafted and maturate in the tongue of recipient mice and that the organoids generated from carcinogen-treated mice had an abnormal morphology. Thus, this culture system presents valuable settings for studying not only the regulatory mechanisms of lingual epithelium but also lingual regeneration and carcinogenesis.

  10. EFFECTS OF ATRAZINE AND AN ATRAZINE METABOLITE MIXTURE ON DIFFERENTIATED MAMMARY EPITHELIAL CELL MILK PROTEIN PRODUCTION IN CULTURE

    EPA Science Inventory

    Effects of Atrazine and an Atrazine Metabolite Mixture on Differentiated Mammary Epithelial Cell Milk Protein Production in Culture

    E.P. Hines, R. Barbee, M. Blanton, M.S. Pooler, and S.E. Fenton. US EPA, ORD/NHEERL, RTD, RTP, NC, 27711, USA.

    Previous studies have ...

  11. Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

    PubMed Central

    Fain, G L; Farahbakhsh, N A

    1989-01-01

    1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials. Images Fig. 2 PMID:2621623

  12. Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche

    SciTech Connect

    Katano, Takahito; Ootani, Akifumi; Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Ebi, Masahide; Mori, Yoshinori; Kataoka, Hiromi; Kamiya, Takeshi; Toda, Shuji; Joh, Takashi

    2013-03-22

    Highlights: ► We established a 3D culture system to allow long-term culture of stomach cells. ► In this culture system, gastric epithelial cells grew for about 3 months. ► The cultured cells differentiated into multi-units of the stomach. ► This culture method should be useful for elucidating the cause of gastric diseases. -- Abstract: Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3 months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell–cell and epithelial–mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

  13. Effect of Aflatoxin B1 on Growth of Bovine Mammary Epithelial Cells in 3D and Monolayer Culture System

    PubMed Central

    Forouharmehr, Ali; Harkinezhad, Taher; Qasemi-Panahi, Babak

    2013-01-01

    Purpose: Many studies have been showed transfer of aflatoxins, toxins produced by Aspergillus flvaus and Aspergillus parasiticus fungi, into milk. These toxins are transferred into the milk through digestive system by eating contaminated food. Due to the toxicity of these materials, it seems that it has side effects on the growth of mammary cells. Therefore, the present work aimed to investigate possible toxic effects of aflatoxin B1 (AFB1) on bovine mammary epithelial cells in monolayer and three-dimensional cultures. Methods: Specimens of the mammary tissue of bovine were sized out in size 2×2 cm in slaughterhouse. After disinfection and washing in sterile PBS, primary cell culture was performed by enzymatic digestion of tissue with collagenase. When proper numbers of cells were achieved in monolayer culture, cells were seeded in a 24-well culture plate for three-dimensional (3D) culture in Matrigel matrix. After 21 days of 3D culture and reaching the required number of cells, the concentrations of 15, 25 and 35 µL of AFB1 were added to the culture in quadruplicate and incubated for 8 hours. Cellular cytotoxicity was examined using standard colorimetric assay and finally, any change in the morphology of the cells was studied by microscopic technique. Results: Microscopic investigations showed necrosis of the AFB1-exposed cells compared to the control cells. Also, bovine mammary epithelial cells were significantly affected by AFB1 in dose and time dependent manner in cell viability assays. Conclusion: According to the results, it seems that AFB1 can induce cytotoxicity and necrosis in bovine mammary epithelial cells. PMID:24312827

  14. Culture of fetal alveolar epithelial type II cells in serum-free medium.

    PubMed

    Fraslon, C; Rolland, G; Bourbon, J R; Rieutort, M; Valenza, C

    1991-11-01

    A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment. PMID:1748624

  15. Vortex or whorl formation of cultured human corneal epithelial cells induced by magnetic fields.

    PubMed

    Dua, H S; Singh, A; Gomes, J A; Laibson, P R; Donoso, L A; Tyagi, S

    1996-01-01

    The terms 'vortex keratopathy' and 'hurricane keratopathy' describe two similar conditions affecting the corneal surface. In the former, a vortex or whorl pattern is seen on the corneal surface and is due to the deposition of substances such as pigment, iron or drugs in the epithelial cells. In the latter, a similar pattern is presented by migrating epithelial cells but, unlike the former, the pattern is rendered more visible by fluorescein staining. Both represent the migratory pattern of normal epithelial cells which is otherwise not visible due to the slow rate of epithelial turnover and migration. The whorl pattern has a clockwise predisposition in the majority of cases and is hypothesised to be due to the influence of ocular electro-magnetic fields on the migrating epithelial cells. In this study we tested in vitro the effect of static magnetic fields on corneal epithelial cells. We were able to reproduce dramatic vortex or whorl patterns in response to magnetic fields, but without preferential migration towards the North or South Pole. PMID:8944095

  16. Establishment and characterization of immortalized gingival epithelial and fibroblastic cell lines for the development of organotypic cultures.

    PubMed

    Bao, Kai; Akguel, Baki; Bostanci, Nagihan

    2014-01-01

    In vitro studies using 3D co-cultures of gingival cells can resemble their in vivo counterparts much better than 2D models that typically only utilize monolayer cultures with short-living primary cells. However, the use of 3D gingival models is still limited through lack of appropriate cell lines. We aimed to establish immortalized cell line models of primary human gingival epithelium keratinocytes (HGEK) and gingival fibroblasts (GFB). Immortalized cell lines (HGEK-16 and GFB-16) were induced by E6 and E7 oncoproteins of human papillomavirus. In addition, 3D multilayered organotypic cultures were formed by embedding GFB-16 cells within a collagen (Col) matrix and seeding of HGEK-16 cells on the upper surfaces. Cell growth was analyzed in both immortalized cell lines and their parental primary cells. The expression levels of cell type-specific markers, i.e. cytokeratin (CK) 10, CK13, CK16, CK18, CK19 for HGEK-16 and Col I and Col II for GFB-16, were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Expansion of the primary cultures was impeded at early passages, while the transformed immortalized cell lines could be expanded for more than 30 passages. In 3D cultures, immortalized HGEK formed a multilayer of epithelial cells. qRT-PCR showed that cell-specific marker expression in the 3D cultures was qualitatively and quantitatively closer to that in human gingival tissue than to monolayer cultures. These results indicate that immortalized gingival fibroblastic and epithelial cell lines can successfully form organotypic multilayered cultures and, therefore, may be useful tools for studying gingival tissue in vitro. PMID:25471635

  17. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    PubMed Central

    González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

    2013-01-01

    Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

  18. Ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Nadine, Lusamba Kalonji; Ota, Chiharu; Kubo, Hiroshi; Nagatomi, Ryoichi

    2014-04-01

    The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection. PMID:23856970

  19. IN VIVO AND IN VITRO CHARACTERISTICS OF EARLY CARCINOGEN-INDUCED PREMALIGNANT PHENOTYPES IN CULTURED RAT TRACHEAL EPITHELIAL CELLS (JOURNAL VERSION)

    EPA Science Inventory

    The initial stages of neoplastic transformation in respiratory tract epithelial cells were defined and studied by characterizing a series of morphologically transformed cell colonies from carcinogen-exposed rat tracheal epithelial (TRE) cell cultures both in vivo and in vitro. RT...

  20. Chromosomal changes in cultured human epithelial cells transformed by low- and high-let radiation

    NASA Astrophysics Data System (ADS)

    Chui-Hsu Yang, Tracy; Craise, Laurie M.; Prioleau, John C.; Stampfer, Martha R.; Rhim, Johng S.

    1992-07-01

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

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

    NASA Technical Reports Server (NTRS)

    Craise, L. M.; Prioleau, J. C.; Stampfer, M. R.; Rhim, J. S.; Yang, TC-H (Principal Investigator)

    1992-01-01

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

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

    SciTech Connect

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

    1990-11-01

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

  3. Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions

    PubMed Central

    2011-01-01

    Background Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions. Results Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures. Conclusions This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma. PMID:21649893

  4. Tamoxifen Induces Expression of Immune Response-Related Genes in Cultured Normal Human Mammary Epithelial Cells

    PubMed Central

    Schild-Hay, Laura J.; Leil, Tarek A.; Divi, Rao L.; Olivero, Ofelia, A.; Weston, Ainsley; Poirier, Miriam C.

    2008-01-01

    Use of tamoxifen (TAM) is associated with a 50% reduction in breast cancer incidence and an increase in endometrial cancer incidence. Here, we documented TAM-induced gene expression changes in cultured normal human mammary epithelial cells (NHMEC strains numbered 5, 16 and 40), established from tissue taken at reduction mammoplasty from 3 individuals. Cells exposed to 0, 10 or 50 μM TAM for 48 hours were evaluated for (E)-α-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) adduct formation by TAM-DNA (DNA modified with dG-N2-TAM) chemiluminescence immunoassay (CIA), gene expression changes using NCI DNA-oligonucleotide microarray, and real time (RT)-PCR. At 48 hr, cells exposed to 10 μM and 50 μM TAM were 85.6% and 48.4% viable, respectively, and there were no measurable dG-N2-TAM adducts. For microarray, cells were exposed to 10 μM TAM and genes with expression changes of ≥ 3-fold were as follows: thirteen genes up-regulated and one down-related for strain 16; seventeen genes up-regulated for strain 5; and eleven genes up-regulated for strain 40. Interferon-inducible genes (IFITM1, IFIT1, IFNA1, MXI and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all 3 strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. RT-PCR revealed up-regulation of interferon α (IFNA1) and confirmed the TAM-induced up-regulation of the genes identified by microarray, with the exception of GIP3 and MX1, which were not up-regulated in strain 40. Induction of interferon-related genes in the three NHMEC strains suggests that, in addition to hormonal effects, TAM exposure may enhance immune response in normal breast tissue. PMID:19155303

  5. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mit...

  6. Effect of oestradiol on PAMP-mediated CCL20/MIP-3α production by mouse uterine epithelial cells in culture

    PubMed Central

    Soboll, Gisela; Crane-Godreau, Mardi A; Lyimo, Magdalena A; Wira, Charles R

    2006-01-01

    The present study was undertaken to establish whether mouse uterine epithelial cells produce CCL20/macrophage inflammatory protein 3α (CCL20/MIP-3α) and to determine whether secretion is under hormonal control and influenced by pathogen-associated molecular patterns (PAMPs). In the absence of PAMPs, polarized uterine epithelial cells grown to confluence on cell culture inserts constitutively secreted CCL20/MIP-3α with preferential accumulation into the apical compartment. When epithelial cells were treated with the Toll-like receptor (TLR) agonists Pam3Cys (TLR2/1), peptidoglycan (TLR2/6) or lipopolysaccharide (LPS; TLR4), CCL20/MIP-3α increased rapidly (4 hr) in both apical and basolateral secretions. Time–course studies indicated that responses to PAMPs added to the apical surface persisted for 12–72 hr. Stimulation with loxoribin (TLR7) and DNA CpG motif (TLR9) increased basolateral but not apical secretion of CCL20/MIP-3α. In contrast, the viral agonist Poly(I:C) (TLR3) had no effect on either apical or basolateral secretion. In other studies, we found that oestradiol added to the culture media decreased the constitutive release of CCL20/MIP-3α. Moreover, when added to the culture media along with LPS, oestradiol inhibited LPS-induced increases in CCL20/MIP-3α secretion into both the apical and basolateral compartments. In summary, these results indicate that CCL20/MIP-3α is produced in response to PAMPs. Since CCL20/MIP-3α is chemotactic for immature dendritic cells, B cells and memory T cells and has antimicrobial properties, these studies suggest that CCL20/MIP-3α production by epithelial cells, an important part of the innate immune defence in the female reproductive tract, is under hormonal control and is responsive to microbial challenge. PMID:16771853

  7. Subunit structure of calgranulins A and B obtained from sputum, plasma, granulocytes and cultured epithelial cells.

    PubMed

    Longbottom, D; Sallenave, J M; van Heyningen, V

    1992-04-01

    The calcium-binding proteins calgranulins A and B co-purified with an elastase-specific inhibitor after the affinity and cation-exchange chromatography of the perchloric acid-soluble fraction of pooled sputum collected from patients with chronic obstructive pulmonary disease (Sallenave, J.-M. and Ryle, A.P. (1991) Biol. Chem. Hoppe-Seyler 372, 13-21). The calgranulins were separated from the inhibitor by reverse-phase FPLC. Protein blot analysis of the calgranulin fraction in the absence of reducing agent revealed a band of 25 kDa corresponding to the disulphide-bonded heterodimerization of the two monomer components. Similar results were obtained from the immunoprecipitation and protein blot analysis of plasma, granulocytes and cultured epithelial cells. This implies that the calgranulins exist in the heterodimeric form in secretions in vivo. Their association with pancreatic elastase during the affinity chromatography stage of purification implicates them in the tissue destruction elicited by the inflammatory response in chronic obstructive pulmonary diseases. PMID:1562590

  8. Effect of D-valine and cytosine arabinoside on (/sup 3/H)thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    SciTech Connect

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by (/sup 3/H)thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.

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

    PubMed Central

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

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

  10. Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model.

    PubMed

    Toropainen, Elisa; Ranta, Veli-Pekka; Vellonen, Kati-Sisko; Palmgrén, Joni; Talvitie, Anu; Laavola, Mirka; Suhonen, Pekka; Hämäläinen, Kaisa Mari; Auriola, Seppo; Urtti, Arto

    2003-09-01

    A cell culture model of human corneal epithelium (HCE-model) was recently introduced [Invest. Ophthalmol. Vis. Sci. 42 (2001) 2942] as a tool for ocular drug permeation studies. In this study, passive permeability and esterase activity of the HCE-model were characterised. Immortalised human corneal epithelial cells were grown on collagen coated filters under air-lift. The sensitivity of transcellular permeability to lipophilicity was tested in studies using nine beta-blockers. The size selectivity of the paracellular route was investigated using 16 polyethylene glycol oligomers (PEG). An effusion-like approach was used to estimate porosity and pore sizes of the paracellular space in HCE membrane. Permeability and degradation of fluorescein diacetate to fluorescein in HCE-cells was used to probe the esterase activity of the HCE-model. Drug concentrations were analyzed using HPLC (beta-blockers), LC-MS (PEGs), and fluorometry (fluorescein). Permeabilities were compared to those in the excised rabbit cornea. Penetration of beta-blockers increased with lipophilicity according to a sigmoidal relationship. This was almost similar to the profile in excised cornea. No apical to basolateral directionality was seen in the permeation of beta-blockers. Paracellular permeability of the HCE-model was generally slightly higher than that of the excised rabbit cornea. The HCE-model has larger paracellular pores, but lower pore density than the excised cornea, but the overall paracellular space was fairly similar in both models. The HCE-model shows significant esterase activity (i.e. fluorescein diacetate was converted to free fluorescein). These data on permeability of 27 compounds demonstrate that the barrier of the HCE-model closely resembles that of the excised rabbit cornea. Therefore, the HCE-model is a promising alternative corneal substitute for ocular drug delivery studies. PMID:13678798

  11. CULTURE CONDITIONS AFFECT HUMAN AIRWAY EPITHELIAL CELL RESPONSE TO DIESEL PARTICLE EXPOSURE IN VITRO

    EPA Science Inventory

    Diesel exhaust particles (DEP) are a ubiquitous ambient air contaminant that may contribute to the health effects of particulate matter inhalation. In vitro studies have shown that DEP exposure induces pro-inflammatory proteins in human airway epithelial cells (HAEC) with varying...

  12. Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

    PubMed Central

    Lai, Jui-Yang; Ma, David Hui-Kang

    2013-01-01

    Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells. PMID:24204144

  13. In situ-forming click-crosslinked gelatin based hydrogels for 3D culture of thymic epithelial cells.

    PubMed

    Truong, Vinh X; Hun, Michael L; Li, Fanyi; Chidgey, Ann P; Forsythe, John S

    2016-07-21

    Hydrogels prepared from naturally derived gelatin can provide a suitable environment for cell attachment and growth, making them favourable materials in tissue engineering. However, physically crosslinked gelatin hydrogels are not stable under physiological conditions while chemical crosslinking of gelatin by radical polymerization may be harmful to cells. In this study, we attached the norbornene functional group to gelatin, which was subsequently crosslinked with a polyethylene glycol (PEG) linker via the nitrile oxide-norbornene click reaction. The rapid crosslinking process allows the hydrogel to be formed within minutes of mixing the polymer solutions under physiological conditions, allowing the gels to be used as injectable materials. The hydrogels properties including mechanical strength, swelling and degradation, can be tuned by changing either the ratio of the reacting groups or the total concentration of the polymer precursors. Murine embryonic fibroblastic cells cultured in soft gels (2 wt% of gelatin and 1 wt% of PEG linker) demonstrated high cell viability as well as similar phenotypic profiles (PDGFRα and MTS15) to Matrigel cultures over 5 days. Thymic epithelial cell and fibroblast co-cultures produced epithelial colonies in these gels following 7 days incubation. These studies demonstrate that gelatin based hydrogels, prepared using "click" crosslinking, provide a robust cell culture platform with retained benefits of the gelatin material, and are therefore suitable for use in various tissue engineering applications. PMID:27217071

  14. Effect of ionizing radiation on acinar morphogenesis of human prostatic epithelial cells under three-dimensional culture conditions.

    PubMed

    Wang, T; X, S Ma; Kong, D; Yi, H; Wang, X; Liang, B; Xu, H; He, M; Jia, L; Qased, A B; Yang, Y; Liu, X

    2012-01-01

    Homeostasis is maintained by the interplay of multiple factors that directly or indirectly regulate cell proliferation and cell death. Complex multiple interactions between cells and the extracellular matrix occur during acinar morphogenesis and changes in these might indicate carcinogenesis of cells from a normal to a malignant, invasive phenotype. In this study, the human prostatic epithelial cell line RWPE-1 was cultured under three-dimensional (3-D) culture conditions, and the effect of ionizing radiation on acinar morphogenesis and its association with autophagy were discussed. The results illustrated that formation of specific spheroid (acinar) structures was detectable under 3-D culture conditions. Radiation induced the disruption of acini in different cell models using either gene overexpression (Akt) or gene knock-down (Beclin 1 and ATG7). Introduction of Akt not only accelerated the growth of cells (i.e., caused the cells to manifest elongating and microspike-like structures that are obviously different from structures seen in wild-type RWPE-1 cells under two-dimensional conditions), but also changed their morphological characteristics under 3-D culture conditions. Knock-down of autophagy-related genes (Beclin 1 and ATG7) increased the radiosensitivity of cells under 3-D culture conditions, and cells died of non-apoptotic death after radiation. The results suggested that ionizing radiation may change the cell phenotype and the formation of acini. Additionally even the autophagy mechanism may play a role in these processes. PMID:22296497

  15. Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come.

    PubMed

    McLellan, Kirsty; Shields, Mike; Power, Ultan; Turner, Steve

    2015-12-01

    Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies. PMID:26178976

  16. Isolation and culture of adult epithelial stem cells from human skin.

    PubMed

    Guo, Zhiru; Draheim, Kyle; Lyle, Stephen

    2011-01-01

    The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue. Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo (1-3). Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress (4-5). Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue (6). For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles (7-9). We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality (10). Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias (11-13). Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies (14,15). Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)(16,17), the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and

  17. Protection against radiation-induced oxidative stress in cultured human epithelial cells by treatment with antioxidant agents

    SciTech Connect

    Wan, X. Steven; Ware, Jeffrey H.; Zhou, Zhaozong; Donahue, Jeremiah J.; Guan, Jun; Kennedy, Ann R. . E-mail: akennedy@mail.med.upenn.edu

    2006-04-01

    Purpose: To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells. Methods and Materials: The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, {alpha}-lipoic acid, L-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, {gamma}-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay. Results: The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure. Conclusion: The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.

  18. Azithromycin induces anti-viral effects in cultured bronchial epithelial cells from COPD patients

    PubMed Central

    Menzel, Mandy; Akbarshahi, Hamid; Bjermer, Leif; Uller, Lena

    2016-01-01

    Rhinovirus infection is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations and may contribute to the development into severe stages of COPD. The macrolide antibiotic azithromycin may exert anti-viral actions and has been reported to reduce exacerbations in COPD. However, little is known about its anti-viral actions on bronchial epithelial cells at clinically relevant concentrations. Primary bronchial epithelial cells from COPD donors and healthy individuals were treated continuously with azithromycin starting 24 h before infection with rhinovirus RV16. Expression of interferons, RIG-I like helicases, pro-inflammatory cytokines and viral load were analysed. Azithromycin transiently increased expression of IFNβ and IFNλ1 and RIG-I like helicases in un-infected COPD cells. Further, azithromycin augmented RV16-induced expression of interferons and RIG-I like helicases in COPD cells but not in healthy epithelial cells. Azithromycin also decreased viral load. However, it only modestly altered RV16-induced pro-inflammatory cytokine expression. Adding budesonide did not reduce interferon-inducing effects of azithromycin. Possibly by inducing expression of RIG-I like helicases, azithromycin increased rhinovirus-induced expression of interferons in COPD but not in healthy bronchial epithelium. These effects would reduce bronchial viral load, supporting azithromycin’s emerging role in prevention of exacerbations of COPD. PMID:27350308

  19. Azithromycin induces anti-viral effects in cultured bronchial epithelial cells from COPD patients.

    PubMed

    Menzel, Mandy; Akbarshahi, Hamid; Bjermer, Leif; Uller, Lena

    2016-01-01

    Rhinovirus infection is a major cause of chronic obstructive pulmonary disease (COPD) exacerbations and may contribute to the development into severe stages of COPD. The macrolide antibiotic azithromycin may exert anti-viral actions and has been reported to reduce exacerbations in COPD. However, little is known about its anti-viral actions on bronchial epithelial cells at clinically relevant concentrations. Primary bronchial epithelial cells from COPD donors and healthy individuals were treated continuously with azithromycin starting 24 h before infection with rhinovirus RV16. Expression of interferons, RIG-I like helicases, pro-inflammatory cytokines and viral load were analysed. Azithromycin transiently increased expression of IFNβ and IFNλ1 and RIG-I like helicases in un-infected COPD cells. Further, azithromycin augmented RV16-induced expression of interferons and RIG-I like helicases in COPD cells but not in healthy epithelial cells. Azithromycin also decreased viral load. However, it only modestly altered RV16-induced pro-inflammatory cytokine expression. Adding budesonide did not reduce interferon-inducing effects of azithromycin. Possibly by inducing expression of RIG-I like helicases, azithromycin increased rhinovirus-induced expression of interferons in COPD but not in healthy bronchial epithelium. These effects would reduce bronchial viral load, supporting azithromycin's emerging role in prevention of exacerbations of COPD. PMID:27350308

  20. GLUCOSE METABOLITE PATTERNS AS MARKERS OF FUNCTIONAL DIFFERENTIATION IN FRESHLY ISOLATED AND CULTURED MOUSE MAMMARY EPITHELIAL CELLS

    SciTech Connect

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

    1980-06-01

    In the mammary gland of nonruminant animals, glucose is utilized in a characteristic and unique way during lactation. We have measured the incorporation of glucose carbon from [U-{sup 14}C] glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice and demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate were important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells from pregnant mice have a pattern similar to freshly isolated cells from pregnant mice. The pattern of cells from lactating mice is different from that of the cells of origin, and resembles that of the cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  1. Evaluation of low-dosage environmental mutagens with a long-term, cultured epithelial cell line

    SciTech Connect

    Li, C.S.; Lin, R.H.

    1996-12-31

    Polycyclic or polynuclear aromatic hydrocarbons (PAHs) are a group of compounds consisting of at least two fused aromatic rings and are common environmental contaminants in soil, water, and air. Additionally, some PAHs are considered to be mutagenic, carcinogenic, and cytotoxic compounds. BaA (Benz(a)anthracene) and BaP (Benzo(a)pyrene) are the two major unsubstituted PAHs identified by the USEPA as priority pollutants. BaA is suspected to be a human carcinogen by the US Department of Health and Human Services, whereas BaP is regarded as an animal carcinogen. It is estimated that the emission concentrations of BaA and BaP from mainstream cigarette smoke are 20-70 ng per g cigarette and 20-40 ng per g cigarette, respectively, with an average sidestream to mainstream weight ratio of 3. In addition, BaA and BaP are also the main emissions from diesel particulate extracts with mean concentrations of 500 ppm and 40 ppm, respectively. From a recent report concerning PAHs in urban areas all over the world, the airborne concentration of BaP was typically found to be in the range of 1-20 ng/m{sup 3} in Europe and 1 ng/m{sup 3} in the U.S.A. For BaA, the concentrations commonly ranged from I to 50 ng/m{sup 3} in Europe and from 0.1 to 1 ng/m{sup 3} in the U.S.A. Therefore, the long term exposure to these two compounds may cause health effects. This study examines long-term cultured epithelial cell lines exposed to BaP or BaA, in vitro, and the frequency of HGPRT mutants. 19 refs., 3 figs.

  2. Propagation of normal human epithelial cell populations using an in vivo culture system. Description and applications.

    PubMed

    Klein-Szanto, A J; Terzaghi, M; Mirkin, L D; Martin, D; Shiba, M

    1982-08-01

    A new model using xenotransplanted human epithelia was developed for the study of toxic and carcinogenic effects of chemicals. Epithelial cells from the respiratory tract of 4 male and 3 female premature and fullterm fetuses were enzymatically removed and inoculated into deepithelialized rat tracheas. These were sealed at both ends and transplanted subcutaneously into nude mice. After 3-4 weeks, a normal mucociliary epithelium covered the tracheal lumen. At this stage the epithelial cells could be isolated again and transplanted into new denuded rat tracheas. This passaging could be repeated up to six times, each permitting an amplification factor of approximately 3. Tracheal transplants containing cells of human origin (in vivo Passages 2-4) were treated with 7,12-dimethylbenz(a)anthracene. Hyperplasias, squamous metaplasias, and dysplasias were seen 1-8 weeks after initiation of treatment, indicating that the responses of human and rodent epithelial cells to polycyclic aromatic hydrocarbons are similar. Initial experiments with skin and esophageal epithelia suggest that other covering epithelia could also be used in this fashion for evaluation of toxicants and carcinogens that are likely to come into contact with these tissues. PMID:6821529

  3. Expression of the Peptide Antibiotic Human β-Defensin 1 in Cultured Gingival Epithelial Cells and Gingival Tissue

    PubMed Central

    Krisanaprakornkit, Suttichai; Weinberg, Aaron; Perez, Christopher N.; Dale, Beverly A.

    1998-01-01

    Human β-defensin-1 (hBD-1) is a member of the family of small cationic antimicrobial peptides that have been identified in several mucosal epithelia. Because human gingival epithelium is a site that is constantly challenged by oral microorganisms, we examined the expression of hBD-1 in human gingival epithelial and fibroblast cell cultures and tissue samples. Cell cultures were challenged with cell wall extracts of Porphyromonas gingivalis or Fusobacterium nucleatum, Escherichia coli lipopolysaccharide, tumor necrosis factor alpha, or phorbol myristate acetate. hBD-1 mRNA was detected in unstimulated and stimulated cultures by reverse transcription (RT)-PCR using several primer sets specific for hBD-1. Gingival epithelial cells, but not gingival fibroblasts, expressed a product of the predicted size for hBD-1 mRNA. The sequence of the PCR product was identical to that of hBD-1. hBD-1 mRNA expression was not significantly modulated by any of the stimulants tested. Human gingival tissues from noninflamed and inflamed sites were also analyzed by RT-PCR. hBD-1 mRNA was expressed in all tissue samples. The relative expression of hBD-1 mRNA was similar in noninflamed and inflamed tissues obtained from each of four patients undergoing treatment for periodontitis. However, the relative expression of hBD-1 mRNA varied in gingival biopsies obtained from 15 different normal individuals, and the relative hBD-1 expression was unrelated to interleukin-8 expression. Our findings show the constitutive expression of hBD-1 mRNA in cultured epithelial cells and gingival tissues but not gingival fibroblasts. These findings suggest that expression of hBD-1 may play a role as part of the innate host defenses in maintaining normal gingival health. PMID:9712771

  4. Determination of apical membrane polarity in mammary epithelial cell cultures: The role of cell-cell, cell-substratum, and membrane-cytoskeleton interactions

    SciTech Connect

    Parry, G.; Beck, J.C.; Moss, L.; Bartley, J. ); Ojakian, G.K. )

    1990-06-01

    The membrane glycoprotein, PAS-O, is a major differentiation antigen on mammary epithelial cells and is located exclusively in the apical domain of the plasma membrane. The authors have used 734B cultured human mammary carcinoma cells as a model system to study the role of tight junctions, cell-substratum contacts, and submembranous cytoskeletal elements in restricting PAS-O to the apical membrane. Immunofluorescence and immunoelectronmicroscopy experiments demonstrated that while tight junctions demarcate PAS-O distribution in confluent cultures, apical polarity could be established at low culture densities when cells could not form tight junctions with neighboring cells. They suggest, then, that interactions between vitronectin and its receptor, are responsible for establishment of membrane domains in the absence of tight junctions. The role of cytoskeletal elements in restricting PAS-O distribution was examined by treating cultures with cytochalasin D, colchicine, or acrylamide. Cytochalasin D led to a redistribution of PAS0O while colchicine and acrylamide did not. They hypothesize that PAS-O is restricted to the apical membrane by interactions with a microfilament network and that the cytoskeletal organization is dependent upon cell-cell and cell-substratum interactions.

  5. Genetic Mechanisms Underlying the Pathogenicity of Cold-Stressed Salmonella enterica Serovar Typhimurium in Cultured Intestinal Epithelial Cells

    PubMed Central

    Shah, Jigna; Desai, Prerak T.

    2014-01-01

    Salmonella encounters various stresses in the environment and in the host during infection. The effects of cold (5°C, 48 h), peroxide (5 mM H2O2, 5 h) and acid stress (pH 4.0, 90 min) were tested on pathogenicity of Salmonella. Prior exposure of Salmonella to cold stress significantly (P < 0.05) increased adhesion and invasion of cultured intestinal epithelial (Caco-2) cells. This increased Salmonella-host cell association was also correlated with significant induction of several virulence-associated genes, implying an increased potential of cold-stressed Salmonella to cause an infection. In Caco-2 cells infected with cold-stressed Salmonella, genes involved in the electron transfer chain were significantly induced, but no simultaneous significant increase in expression of antioxidant genes that neutralize the effect of superoxide radicals or reactive oxygen species was observed. Increased production of caspase 9 and caspase 3/7 was confirmed during host cell infection with cold-stressed Salmonella. Further, a prophage gene, STM2699, induced in cold-stressed Salmonella and a spectrin gene, SPTAN1, induced in Salmonella-infected intestinal epithelial cells were found to have a significant contribution in increased adhesion and invasion of cold-stressed Salmonella in epithelial cells. PMID:25192993

  6. Long-term maintenance of taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line.

    PubMed

    Foliot, A; Glaise, D; Erlinger, S; Guguen-Guillouzo, C

    1985-01-01

    Taurocholate (TC) uptake by adult rat hepatocytes co-cultured with other rat liver epithelial cells (RLEC) was studied comparatively to hepatocytes in primary culture. Cells were cultured on Petri dishes for desired times prior to measuring their ability to transport TC. TC uptake was linear for 150 sec in both culture conditions. In hepatocytes cultured alone, the initial rate of TC uptake at an extracellular concentration of 100 microM was 0.19 +/- 0.02 nmole per min per 10(6) cells after 48 hr of culture and decreased by 75% after 4 to 6 days. In hepatocytes co-cultured with RLEC, the rate of uptake at 48 hr (0.31 +/- 0.01 nmole per min per 10(6) cells) was significantly higher than in hepatocytes cultured alone (p less than 0.01); in addition, TC uptake remained stable at an average rate of 0.17 +/- 0.01 nmole per min per 10(6) cells for up to 56 days. No detectable uptake was found in RLEC cultured alone. TC uptake exhibited both saturable (Vmax = 0.30 +/- 0.03 nmole per min per 10(6) cells and Km = 42.6 +/- 4.4 microM) and nonsaturable components. These kinetic parameters were similar to those previously reported in isolated hepatocytes and in short-term cultured hepatocytes. TC uptake exhibited sodium dependence and was significantly reduced when extracellular sodium was replaced by lithium and sucrose, or in the presence of 1 mM ouabain. After 18 days of co-culture, TC uptake had qualitatively the same characteristics as at 48 hr, with a saturable and a nonsaturable component.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3979952

  7. Two-dimensional culture of human pancreatic adenocarcinoma cells results in an irreversible transition from epithelial to mesenchymal phenotype

    PubMed Central

    Kang, Ya'an; Zhang, Ran; Suzuki, Rei; Li, Shao-qiang; Roife, David; Truty, Mark J.; Chatterjee, Deyali; Thomas, Ryan M.; Cardwell, James; Wang, Yu; Wang, Huamin; Katz, Matthew H.; Fleming, Jason B.

    2015-01-01

    Many commercially available cell lines have been in culture for ages, acquiring phenotypes that differ from the original cancers from which these cell lines were derived. Therefore, research on new cell lines could improve the success rates of translational research in cancer. We have developed methods for the isolation and culture of human pancreatic ductal adenocarcinoma (PDAC) cells from murine xenografts of human PDAC. We hypothesize that phenotypes of PDAC cells are modified by in vitro culture conditions over time and by in vivo implantation. Patient-derived xenografts were created in immunodeficient mice using surgically resected tumor specimens. These murine xenografts were then used to establish human PDAC cell lines in culture. Earlier (<5) passage and later (>20) passage cell lines were evaluated separately regarding proliferation, cell cycle, genetic mutations, invasiveness, chemosensitivity, tumorigenesis, epithelial-mesenchymal transition (EMT) status, and proteomics. Later passage cells accelerated their doubling time and colony formation, and were more concentrated in the G0/G1 phase and less in the G2/M checkpoint phase. Later passage cells were more sensitive to gemcitabine and 5-fluorouracil than earlier passage cells, but all four new cell lines were more chemo-resistant compared to commercial ATCC cell lines. EMT induction was observed when establishing and passaging cell lines in vitro and furthermore by growing them as subcutaneous tumors in vivo. This study demonstrates a novel approach to the establishment of PDAC cell lines and observes a process by which newly established cell lines undergo phenotypic changes during in vitro culture and in vivo tumorigenesis. This may help explain differences of treatment effects often observed between experiments conducted in vitro, in vivo, and in human clinical trials. PMID:25485535

  8. Glucocorticoid receptors in primary cultures of mouse mammary epithelial cells: characterization and modulation by prolactin and cortisol

    SciTech Connect

    Schneider, W.; Shyamala, G.

    1985-06-01

    Mammary epithelial cells isolated from midpregant mice and cultured on collagen gels contain soluble glucocorticoid receptors. The kinetics of binding of dexamethasone reveal a saturable binding site (dissociation constant (K /sub d/), approximately 1 nM), and the binding site obeys a steroid specificity characteristic of a glucocorticoid receptor. As with the receptor isolated from intact glands, the receptor from the cultured cells also requires the addition of dithiothreitol for maximal binding of dexamethasone. The receptors are maintained at in vivo levels (approximately 1.3 pmol/mg DNA) for at least a period of 10 days in culture. However, the presence of both cortisol and PRL is required for the maintenance of the receptors, and the effect of both these hormones is dose dependent.

  9. Trehalose-Based Eye Drops Preserve Viability and Functionality of Cultured Human Corneal Epithelial Cells during Desiccation

    PubMed Central

    Hill-Bator, Aneta; Misiuk-Hojło, Marta; Marycz, Krzysztof; Grzesiak, Jakub

    2014-01-01

    This paper presents the evaluation of cytoprotective ability of trehalose-based eye drops in comparison with commercially available preparations during the experimental desiccation of cultured human corneal epithelial cells. Cultured human corneal epithelial cells (hCEC) underwent incubation with 7 different, commercially available medicaments used commonly in dry eye syndrome treatment, followed by desiccation trial performed on air under the flow hood for 5, 15, 30, and 45 minutes. Cell viability was quantified by live/dead fluorescent assay, while the presence of apoptotic cells was estimated by immunofluorescent staining for active caspase 3 protein. The preservation of membrane functions was evaluated using neutral red staining, while the preservation of proper morphology and phenotype was determined by fluorescent staining for actin filaments, nuclei, and p63 protein. The trehalose-based eye drops showed the highest efficiency in prevention of cell death from desiccation; moreover, this preparation preserved the normal cellular morphology, functions of cell membrane, and proliferative activity more effectively than other tested medicaments. PMID:24995283

  10. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    SciTech Connect

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

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from (U-/sup 14/C)glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  11. Medium Calcium Concentration Determines Keratin Intermediate Filament Density and Distribution in Immortalized Cultured Thymic Epithelial Cells (TECs)

    NASA Astrophysics Data System (ADS)

    Sands, Sandra S.; Meek, William D.; Hayashi, Jun; Ketchum, Robert J.

    2005-08-01

    Isolation and culture of thymic epithelial cells (TECs) using conventional primary tissue culture techniques under conditions employing supplemented low calcium medium yielded an immortalized cell line derived from the LDA rat (Lewis [Rt1l] cross DA [Rt1a]) that could be manipulated in vitro. Thymi were harvested from 4 5-day-old neonates, enzymically digested using collagenase (1 mg/ml, 37°C, 1 h) and cultured in low calcium WAJC404A medium containing cholera toxin (20 ng/ml), dexamethasone (10 nM), epidermal growth factor (10 ng/ml), insulin (10 [mu]g/ml), transferrin (10 [mu]g/ml), 2% calf serum, 2.5% Dulbecco's Modified Eagle's Medium (DMEM), and 1% antibiotic/antimycotic. TECs cultured in low calcium displayed round to spindle-shaped morphology, distinct intercellular spaces (even at confluence), and dense reticular-like keratin patterns. In high calcium (0.188 mM), TECs formed cobblestone-like confluent monolayers that were resistant to trypsinization (0.05%) and displayed keratin intermediate filaments concentrated at desmosomal junctions between contiguous cells. Changes in cultured TEC morphology were quantified by an analysis of desmosome/membrane relationships in high and low calcium media. Desmosomes were significantly increased in the high calcium medium. These studies may have value when considering the growth conditions of cultured primary cell lines like TECs.

  12. A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases

    PubMed Central

    Heller, Janosch P.; Kwok, Jessica C. F.; Vecino, Elena; Martin, Keith R.; Fawcett, James W.

    2015-01-01

    Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases. PMID:26635529

  13. Enhanced pyrimidine dimer repair in cultured murine epithelial cells transfected with the denV gene of bacteriophage T4.

    PubMed

    Kusewitt, D F; Budge, C L; Ley, R D

    1994-04-01

    The patch size for excision repair of ultraviolet radiation (UV)-induced pyrimidine dimers was determined in cultured murine epithelial cells with normal and enhanced pyrimidine dimer repair capabilities. Cells with enhanced pyrimidine dimer repair were produced by transfecting 308 cells with the denV gene of bacteriophage T4; this gene encodes the enzyme endonuclease V. Pyrimidine dimer repair following exposure to UV from an FS-40 sunlamp was determined by micrococcal dimer-specific nuclease digestion and alkaline sucrose ultracentrifugation. Patch size ws estimated based on the photolytic lability of bromodeoxyuridine-substituted DNA. Excision repair of UV-induced pyrimidine dimers in denV-transfected 308 cells was enhanced two- to threefold. Production of mRNA from the denV gene in cell lines with enhanced repair was confirmed by RNA blotting. In control cells, the patch size for excision repair of DNA photoproducts was estimated to be 34 nucleotides per photoproduct removed; in denV-transfected cells, a smaller average patch size of 10-16 nucleotides per photoproduct removed was calculated. Thus, endonuclease V activity appears to alter not only the extent, but also the nature of excision repair in UV-exposed mammalian epithelial cells. PMID:8151125

  14. Low Power Laser Irradiation Stimulates the Proliferation of Adult Human Retinal Pigment Epithelial Cells in Culture

    PubMed Central

    Song, Qing; Uygun, Basak; Banerjee, Ipsita; Nahmias, Yaakov; Zhang, Quan; Berthiaume, François; Latina, Mark; Yarmush, Martin L.

    2015-01-01

    We investigated the effects of low power laser irradiation on the proliferation of retinal pigment epithelial (RPE) cells. Adult human RPE cells were artificially pigmented by preincubation with sepia melanin, and exposed to a single sublethal laser pulse (590 nm, 1 µs, <200 mJ/cm2). DNA synthesis, cell number, and growth factor activity in irradiated RPE cells were subsequently monitored. The effect of sublethal laser irradiation on the “wound” healing response of an RPE monolayer in an in vitro scratch assay was also investigated. Single pulsed laser irradiation increased DNA synthesis in pigmented RPE cells measured 6 h post-treatment. In the scratch assay, laser irradiation increased the rates of cell proliferation and wound closure. Conditioned medium, collected 48 h following laser treatment, increased cell proliferation of unirradiated cells. Irradiation increased RPE cell secretion of platelet-derived growth factor (PDGF)-B chain, and increased mRNA levels of several growth factors and their receptors, including PDGF, transforming growth factor-β1, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, as well as heat shock proteins. This demonstrates, for the first time, that low power single pulsed laser irradiation stimulates the proliferation of RPE cells, and upregulates growth factors that are mitogenic for RPE cells. PMID:26740823

  15. Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells.

    PubMed

    Zhou, M M; Wu, Y M; Liu, H Y; Liu, J X

    2015-04-01

    This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on αs1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium-F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide-bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl-phenylalanyl-phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide-bound Phe at 10% (11.7 μg/ml) significantly enhanced αs1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide-bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids. PMID:25199802

  16. Salivary α-amylase exhibits antiproliferative effects in primary cell cultures of rat mammary epithelial cells and human breast cancer cells

    PubMed Central

    2011-01-01

    Background Breast cancer is one of the most diagnosed cancers in females, frequently with fatal outcome, so that new strategies for modulating cell proliferation in the mammary tissue are urgently needed. There is some, as yet inconclusive evidence that α-amylase may constitute a novel candidate for affecting cellular growth. Methods The present investigation aimed to examine if salivary α-amylase, an enzyme well known for the metabolism of starch and recently introduced as a stress marker, is able to exert antiproliferative effects on the growth of mammary gland epithelial cells. For this purpose, primary epithelial cultures of breast tissue from two different inbred rat strains, Fischer 344 (F344) and Lewis, as well as breast tumor cells of human origin were used. Treatment with human salivary α-amylase was performed once daily for 2 days followed by cell counting (trypan blue assay) to determine alterations in cell numbers. Cell senescence after α-amylase treatment was assessed by β-galactosidase assay. Endogenous α-amylase was detected in cells from F344 and Lewis by immunofluorescence. Results Salivary α-amylase treatment in vitro significantly decreased the proliferation of primary cells from F344 and Lewis rats in a concentration-dependent manner. Noticeably, the sensitivity towards α-amylase was significantly higher in Lewis cells with stronger impact on cell growth after 5 and 50 U/ml compared to F344 cells. An antiproliferative effect of α-amylase was also determined in mammary tumor cells of human origin, but this effect varied depending on the donor, age, and type of the cells. Conclusions The results presented here indicate for the first time that salivary α-amylase affects cell growth in rat mammary epithelial cells and in breast tumor cells of human origin. Thus, α-amylase may be considered a novel, promising target for balancing cellular growth, which may provide an interesting tool for tumor prophylaxis and treatment. PMID:22027017

  17. Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS

    PubMed Central

    Paaske Utheim, Tor; Salvanos, Panagiotis; Aass Utheim, Øygunn; Ræder, Sten; Pasovic, Lara; Olstad, Ole Kristoffer; Fideliz de la Paz, Maria; Sehic, Amer

    2016-01-01

    The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets. PMID:26901233

  18. Transcriptome Analysis of Cultured Limbal Epithelial Cells on an Intact Amniotic Membrane following Hypothermic Storage in Optisol-GS.

    PubMed

    Utheim, Tor Paaske; Salvanos, Panagiotis; Utheim, Øygunn Aass; Ræder, Sten; Pasovic, Lara; Olstad, Ole Kristoffer; de la Paz, Maria Fideliz; Sehic, Amer

    2016-01-01

    The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets. PMID:26901233

  19. Lactobacillus acidophilus binds to MUC3 component of cultured intestinal epithelial cells with highest affinity.

    PubMed

    Das, Jugal Kishore; Mahapatra, Rajani Kanta; Patro, Shubhransu; Goswami, Chandan; Suar, Mrutyunjay

    2016-04-01

    Lactobacillus strains have been shown to adhere to the mucosal components of intestinal epithelial cells. However, established in vitro adhesion assays have several drawbacks in assessing the adhesion of new Lactobacillus strains. The present study aimed to compare the adhesion of four different Lactobacillus strains and select the most adherent microbe, based on in silico approach supported by in vitro results. The mucus-binding proteins in Lactobacillus acidophilus, L. plantarum, L. brevis and L. fermentum were identified and their capacities to interact with intestinal mucin were compared by molecular docking analysis. Lactobacillus acidophilus had the maximal affinity of binding to mucin with predicted free energy of -6.066 kcal mol(-1) Further, in vitro experimental assay of adhesion was performed to validate the in silico results. The adhesion of L. acidophilus to mucous secreting colon epithelial HT-29 MTX cells was highest at 12%, and it formed biofilm with maximum depth (Z = 84 μm). Lactobacillus acidophilus was determined to be the most adherent strain in the study. All the Lactobacillus strains tested in this study, displayed maximum affinity of binding to MUC3 component of mucus as compared to other gastrointestinal mucins. These findings may have importance in the design of probiotics and health care management. PMID:26946538

  20. Cancer Risk-Assessment of Radiation Damage in Ataxia Telangiectasia Heterozygous Human Breast Epithelial Cell Cultures

    NASA Technical Reports Server (NTRS)

    Applewhite, Lisa C.

    2002-01-01

    This paper describes the study of the markers of cellular changes that are found during the onset of carcinogenesis. Several of the biological factors are markers of stress response, oncoprotein expression, and differentiation factors. Oxidative stress response agents such as heat shock proteins (HSPs) protect cells from oxidative stresses such as ionizing radiation. The onocoprotein HER-2/neu, a specific breast cancer marker, indicates early onset of cancer. Additional structural and morphogenetic markers of differentiation were considered in order to determine initial cellular changes at the initial onset of cancer. As an additional consideration, all-trans retinoic acid (RA), a differentiation agent, was considered because of its known role in regulating normal differentiation and inhibiting tumor proliferation via specific nuclear receptors. This paper discusses study and results of the preliminary analyses of gamma irradiation of AT heterozygous human breast epithelial cells (WH). Comparisons are also made of the effects various RA concentrations post-irradiation.

  1. Cytotoxicity of antimicrobial photodynamic inactivation on epithelial cells when co-cultured with Candida albicans.

    PubMed

    Pellissari, Claudia Viviane Guimarães; Pavarina, Ana Claudia; Bagnato, Vanderlei Salvador; Mima, Ewerton Garcia de Oliveira; Vergani, Carlos Eduardo; Jorge, Janaina Habib

    2016-05-11

    This study assessed the cytotoxicity of antimicrobial Photodynamic Inactivation (aPDI), mediated by curcumin, using human keratinocytes co-cultured with Candida albicans. Cells and microorganisms were grown separately for 24 hours and then kept in contact for an additional 24 hours. After this period, aPDI was applied. The conditions tested were: P+L+ (experimental group aPDI); P-L+ (light emitting diode [LED] group); P+L- (curcumin group); and P-L- (cells in co-culture without curcumin nor LED). In addition, keratinocytes and C. albicans were grown separately, were not placed in the co-culture and did not receive aPDI (control group). Cell proliferation was assessed using Alamar Blue, MTT, XTT and CFU tests. Qualitative and quantitative analyses were performed. Analysis of variance (ANOVA) was applied to the survival percentages of cells compared to the control group (considered as 100% viability), complemented by multiple comparisons using Tukey's test. A 5% significance level was adopted. The results of this study showed no interference in the metabolism of the cells in co-culture, since no differences were observed between the control group (cultured cells by themselves) and the P-L- group (co-culture cells without aPDI). The aPDI group reached the highest reduction (p = 0.009), which was equivalent to 1.7 log10 when compared to the control group. The P+L-, P-L+, P-L- and control groups were not statistically different (ρ > 0.05). aPDI inhibited the growth of keratinocytes and C. albicans in all tests, so the therapy was considered slightly (inhibition between 25 and 50% compared to the control group) to moderately (inhibition between 50 and 75% compared to the control group) cytotoxic. PMID:27110908

  2. Nonviral in situ green fluorescent protein labeling and culture of primary, adult human hair follicle epithelial progenitor cells.

    PubMed

    Tiede, Stephan; Koop, Norbert; Kloepper, Jennifer E; Fässler, Reinhard; Paus, Ralf

    2009-11-01

    In this article we show that cloning of the human K15 promoter before a green fluorescence protein (GFP)/geneticin-resistance cassette and transfection of microdissected, organ-cultured adult human scalp hair follicles generates specific K15 promoter-driven GFP expression in their stem cell-rich bulge region. K15-GFP+ cells can be visualized in situ by GFP fluorescence and 2-photon laser scanning microscopy. Vital K15-GFP+ progenitor cells can then be selected by using the criteria of their green fluorescence, adhesion to collagen type IV and fibronectin, and geneticin resistance. Propagated K15-GFP+ cells express epithelial progenitor markers, show the expected differential gene expression profile of human bulge epithelium, and form holoclones. This application of nonretroviral, K15 promoter-driven, GFP labeling to adult human hair follicles facilitates the characterization and manipulation of human epithelial stem cells, both in situ and in vitro, and should be transferable to other complex human tissues. PMID:19750535

  3. High- and low-LET Radiation-induced Chromosome Aberrations in Human Epithelial Cells Cultured in 3-dimensional Matrices

    NASA Technical Reports Server (NTRS)

    Hada, M.; George K.; Cucinotta, F. A.; Wu, H.

    2008-01-01

    Energetic heavy ions pose a great health risk to astronauts who participate in extended ISS missions and will be an even greater concern for future manned lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied low- and high-LET radiation-induced chromosome aberrations in human epithelial cells cultured in 2-dimension (2D) using the multicolor banding fluorescence in situ hybridization (mBAND) technique. However, it has been realized that the biological response to radiation insult in a 2D in vitro cellular environment can differ significantly from the response in 3-dimension (3D) or at the actual tissue level. In this study, we cultured human epithelial cells in 3D to provide a more suitable model for human tissue. Human mammary epithelial cells (CH184B5F5/M10) were grown in Matrigel to form 3D structures, and exposed to Fe-ions at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or 137Cs-gamma radiation source at the University of Texas MD Anderson Cancer Center. After exposure, cells were allowed to repair for 16hr before dissociation and subcultured at low density in 2D. G2 and metaphase chromosomes in the first cell cycle were collected in the first cell cycle after irradiation using a chemical-induced premature chromosome condensation (PCC) technique, and chromosome aberrations were analyzed using mBAND technique. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). Our data indicate a significant difference in the

  4. Effect of Storage Temperature on Key Functions of Cultured Retinal Pigment Epithelial Cells

    PubMed Central

    Pasovic, Lara; Eidet, Jon Roger; Brusletto, Berit S.; Lyberg, Torstein; Utheim, Tor P.

    2015-01-01

    Purpose. Replacement of the diseased retinal pigment epithelium (RPE) with cells capable of performing the specialized functions of the RPE is the aim of cell replacement therapy for treatment of macular degenerative diseases. A storage method for RPE is likely to become a prerequisite for the establishment of such treatment. Herein, we analyze the effect of storage temperature on key functions of cultured RPE cells. Methods. Cultured ARPE-19 cells were stored in Minimum Essential Medium at 4°C, 16°C, and 37°C for seven days. Total RNA was isolated and the gene expression profile was determined using DNA microarrays. Comparison of the microarray expression values with qRT-PCR analysis of selected genes validated the results. Results. Expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4°C and 16°C. Cultures stored at 37°C displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. Conclusion. RPE cultures stored at 4°C and 16°C for one week are capable of maintaining the expression levels of genes important for key RPE functions close to control levels. PMID:26448872

  5. Phagosomal pH and glass fiber dissolution in cultured nasal epithelial cells and alveolar macrophages: a preliminary study.

    PubMed Central

    Johnson, N F

    1994-01-01

    The dissolution rate of glass fibers has been shown to be pH sensitive using in vitro lung fluid simulant models. The current study investigated whether there is a difference in phagosomal pH (ppH) between rat alveolar macrophages (AM) and rat nasal epithelial cells (RNEC) and whether such a difference would influence the dissolution of glass fibers. The ppH was measured in cultured AM and RNEC using flow cytometric, fluorescence-emission rationing techniques with fluorescein-labeled, amorphous silica particles. Glass fiber dissolution was determined in AM and RNEC cultured for 3 weeks with fast dissolving glass fibers (GF-A) or slow dissolving ones (GF-B). The mean diameters of GF-A were 2.7 microns and of GF-B, 2.6 microns, the average length of both fibers was approximately 22 to 25 microns. Dissolution was monitored by measuring the length and diameter of intracellular fibers and estimating the volume, assuming a cylindrical morphology. The ppH of AM was 5.2 to 5.8, and the ppH of RNEC was 7.0 to 7.5. The GF-A dissolved more slowly in RNEC than in AM, and no dissolution was evident in either cell type with GF-B. The volume loss with GF-A after a 3-week culture with AM was 66% compared to 45% for cultured RNEC. These results are different from those obtained using in vitro lung fluid-simulant models where dissolution is faster at higher pH. This difference suggests that dissolution rates of glass fibers in AM should not be applied to the dissolution of fibers in epithelial cells. Images Figure 1. a Figure 1. b Figure 2. a Figure 2. b Figure 3. a Figure 3. b PMID:7882965

  6. Selective growth of freshly isolated human breast epithelial cells cultured at low concentrations in the presence or absence of bone marrow cells.

    PubMed

    Emerman, J T; Stingl, J; Petersen, A; Shpall, E J; Eaves, C J

    1996-01-01

    In this study, we show that conditions previously found to promote the selective growth of human breast epithelial cells (HBEC) in serum-free primary cultures established from normal or malignant tissue can be extended to cultures initiated at low seeding densities (< 5000 cells/cm2). The epithelial nature of the cells produced was documented by their positive staining with antibodies specific for keratins 8, 14, and 18, and 2 antibodies that recognize epithelial-specific antigens (Ber-EP4 and HB8630). HBEC growth was not affected, either positively or negatively, by the use of a medium containing a combination of fetal calf and horse serum, which promotes the growth of many types of stromal cells and associated hematopoietic precursors, or by the inclusion in the initial cell suspension of marrow cells at HBEC to marrow cell ratios typical of bone marrow samples from patients with metastatic breast cancer. The presence of fibroblast feeders from a variety of sources enhanced the growth of HBEC to different degrees. In cultures initiated with low numbers of cells obtained from samples of breast carcinoma, HBEC growth was generally reduced by comparison to cultures of normal HBEC. With the detection methods used, it was not possible to determine the extent to which this decreased growth was due to a reduced frequency of malignant HBEC with in vitro precursor activity, or the presence of reduced numbers of residual normal HBEC precursors, or both. However, preliminary data indicate that this approach also allows the detection of some breast carcinoma cells with proliferative ability that are present in the marrow or pleural effusions of some breast cancer patients. These studies demonstrate the feasibility of detecting normal and malignant HBEC with growth potential when these are cultured at low density and/or as rare contaminants of marrow cell suspensions, and provide a starting point for their further characterization. PMID:8944333

  7. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

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

  8. Oncostatin M synergises with house dust mite proteases to induce the production of PGE2 from cultured lung epithelial cells

    PubMed Central

    Knight, Darryl A; Asokananthan, Nithiananthan; Watkins, D Neil; Misso, Neil L A; Thompson, Philip J; Stewart, Geoffrey A

    2000-01-01

    The release of PGE2 and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1β to stimulate PGE2 release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown.The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE2 and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay.Cells treated with a mixture of IL-1β, IFNγ and LPS for 48 h produced a 9 fold increase in PGE2 and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE2 production (22 fold, P<0.05). OSM also synergistically enhanced PGE2 production in response to a cysteine protease-enriched, but not serine protease-enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE2 or NO production, although it did induce the release of GM-CSF.These observations suggest that OSM is an important co-factor in the release of PGE2 and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM. PMID:11015296

  9. TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

    PubMed Central

    Schwingshackl, Andreas; Roan, Esra; Teng, Bin; Waters, Christopher M.

    2015-01-01

    Background TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements. Methods We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA. Results Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells. Conclusion Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the

  10. Human thyroid epithelial cells cultured in monolayers. II. Influence of serum on thyroglobulin and cAMP production.

    PubMed

    Rasmussen, A K; Kayser, L; Perrild, H; Brandt, M; Bech, K; Feldt-Rasmussen, U

    1996-02-01

    An in vitro system of secondary cultures of human thyroid follicular epithelial cells in monolayer is described. The 72-h influence of serum and six supplements (thyrotropin, insulin, somatostatin, transferrin, hydrocortisone, glycyl-histidyl-lysine acetate) on growth and function in presence of 3-isobutyl-L-methyl-xanthine (IBMX) was investigated. The function of the cells was evaluated by production of the second messenger adenylate cyclase (cAMP) and the end product thyroglobulin (Tg). Growth was measured as the 3H-thymidine uptake of the cells. Three days of TSH-depletion preceeded the experiments. In presence of IBMX TSH stimulated cAMP production, while stimulation of Tg was only present in some cultures. In absence of IBMX TSH always stimulated the Tg production. The stimulation was independent of the presence of the other five investigated nutritional factors in physiological concentrations. TSH in concentrations from 0.1-10 U/1 stimulated the 72ih 3H-thymidine uptake of the cells. The TSH-stimulated production of Tg and cAMP decreased significantly with increasing concentrations of fetal calf serum (0-10%), (tau = 0.49, P < 0.001, n = 6-29 and tau = 0.75, P < 0.001, n = 6-29, respectively). Thus, serum as a complex, variable and not fully characterized mixture of hormones and growth factors was crucial to the attachment of the cells to the substrate, but inhibited differentiated functions of the human thyroid cells. PMID:8647317

  11. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    PubMed

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems. PMID:25063497

  12. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function

    PubMed Central

    Graves, Christina L.; Harden, Scott W.; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J.; Wallet, Shannon M.

    2015-01-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. PMID:25193428

  13. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells

    PubMed Central

    Ryan, Jennifer S; Baldridge, William H; Kelly, Melanie E M

    1999-01-01

    We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5′-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 μM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at −1.5 ± 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. The outward current activated by ATP reversed at −68 ± 3 mV (equilibrium potential for potassium (EK) = −84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current. PMID:10545141

  14. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells.

    PubMed

    Ryan, J S; Baldridge, W H; Kelly, M E

    1999-11-01

    1. We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5'-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 microM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. 2. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at -1.5 +/- 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 3. The outward current activated by ATP reversed at -68 +/- 3 mV (equilibrium potential for potassium (EK) = -84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 4. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. 5. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current. PMID:10545141

  15. Indirect Co-Culture with Tendons or Tenocytes Can Program Amniotic Epithelial Cells towards Stepwise Tenogenic Differentiation

    PubMed Central

    Barboni, Barbara; Curini, Valentina; Russo, Valentina; Mauro, Annunziata; Di Giacinto, Oriana; Marchisio, Marco; Alfonsi, Melissa; Mattioli, Mauro

    2012-01-01

    Background Amniotic epithelial cells (AEC) have potential applications in cell-based therapy. Thus far their ability to differentiate into tenocytes has not been investigated although a cell source providing a large supply of tenocytes remains a priority target of regenerative medicine in order to respond to the poor self-repair capability of adult tendons. Starting from this premise, the present research has been designed firstly to verify whether the co-culture with adult primary tenocytes could be exploited in order to induce tenogenic differentiation in AEC, as previously demonstrated in mesenchymal stem cells. Since the co-culture systems inducing cell differentiation takes advantage of specific soluble paracrine factors released by tenocytes, the research has been then addressed to study whether the co-culture could be improved by making use of the different cell populations present within tendon explants or of the high regenerative properties of fetal derived cell/tissue. Methodology/Principal Findings Freshly isolated AEC, obtained from ovine fetuses at mid-gestation, were co-incubated with explanted tendons or primary tenocytes obtained from fetal or adult calcaneal tendons. The morphological and functional analysis indicated that AEC possessed tenogenic differentiation potential. However, only AEC exposed to fetal-derived cell/tissues developed in vitro tendon-like three dimensional structures with an expression profile of matrix (COL1 and THSB4) and mesenchymal/tendon related genes (TNM, OCN and SCXB) similar to that recorded in native ovine tendons. The tendon-like structures displayed high levels of organization as documented by the cell morphology, the newly deposited matrix enriched in COL1 and widespread expression of gap junction proteins (Connexin 32 and 43). Conclusions/Significance The co-culture system improves its efficiency in promoting AEC differentiation by exploiting the inductive tenogenic soluble factors released by fetal tendon cells or

  16. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ∼40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

  17. Carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) for corneal epithelium reconstruction: a histological study.

    PubMed

    Bardag-Gorce, Fawzia; Oliva, Joan; Wood, Andrew; Hoft, Richard; Pan, Derek; Thropay, Jacquelyn; Makalinao, Andrew; French, Samuel W; Niihara, Yutaka

    2015-04-01

    This study investigates the therapeutic effects of carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) transplantation for experimentally induced severe rabbit limbal stem cell deficiency (LSCD). Buccal biopsies were performed and CAOMECS were cultured and transplanted onto diseased corneas. Six-month follow-up examinations indicated that three out of four corneas with CAOMECS grafts showed a decrease in superficial vascularization, while almost all the sham corneas did not show a similar decrease. H&E staining of corneas showed that CAOMECS transplantation reduced blood vessel invasion of central cornea, reduced lymphocyte infiltration and fibrotic tissue formation. DeltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham corneas. PCNA and Ki-67 staining were much greater in the sham corneas than in the grafted and normal corneas. K3 and K13 staining demonstrated that CAOMECS transplanted corneas had much more K3- and less K13- positive cells compared to the sham corneas. Muc5AC was decreased in the central region of grafted corneas. Very little alpha-smooth muscle actin (aSMA) staining was detected in grafted corneas, while there was a greater amount of aSMA staining in sham corneas. Staining for anti-angiogenic factor TIMP -3 was also increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas. Our results indicate that CAOMECS grafts resulted in improved epithelialization of the corneal surface and decreased vascularization and fibrosis of the diseased corneas. PMID:25881998

  18. Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine

    PubMed Central

    Zhang, Shiqi; Ntasis, Emmanouil; Kabtni, Sarah; van den Born, Jaap; Navis, Gerjan; Bakker, Stephan J. L.; Krämer, Bernhard K.; Yard, Benito A.; Hauske, Sibylle J.

    2016-01-01

    Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity. PMID:26788523

  19. Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine.

    PubMed

    Zhang, Shiqi; Ntasis, Emmanouil; Kabtni, Sarah; van den Born, Jaap; Navis, Gerjan; Bakker, Stephan J L; Krämer, Bernhard K; Yard, Benito A; Hauske, Sibylle J

    2016-01-01

    Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity. PMID:26788523

  20. Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

    PubMed Central

    Chavez, Miquella G.; Hu, Jimmy; Seidel, Kerstin; Li, Chunying; Jheon, Andrew; Naveau, Adrien; Horst, Orapin; Klein, Ophir D.

    2014-01-01

    Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor. PMID:24834972

  1. Krüppel-Like Factor 4 Overexpression Initiates a Mesenchymal-to-Epithelial Transition and Redifferentiation of Human Pancreatic Cells following Expansion in Long Term Adherent Culture

    PubMed Central

    Docherty, Hilary M.; McGowan, Neil W. A.; Forbes, Shareen; Heremans, Yves; Forbes, Stuart J.; Heimberg, Harry; Casey, John; Docherty, Kevin

    2015-01-01

    A replenishable source of insulin-producing cells has the potential to cure type 1 diabetes. Attempts to culture and expand pancreatic β-cells in vitro have resulted in their transition from insulin-producing epithelial cells to mesenchymal stromal cells (MSCs) with high proliferative capacity but devoid of any hormone production. The aim of this study was to determine whether the transcription factor Krüppel-like factor 4 (KLF4), could induce a mesenchymal-to-epithelial transition (MET) of the cultured cells. Islet-enriched pancreatic cells, allowed to dedifferentiate and expand in adherent cell culture, were transduced with an adenovirus containing KLF4 (Ad-Klf4). Cells were subsequently analysed for changes in cell morphology by light microscopy, and for the presence of epithelial and pancreatic markers by immunocytochemistry and quantitative RT/PCR. Infection with Ad-Klf4 resulted in morphological changes, down-regulation of mesenchymal markers, and re-expression of both epithelial and pancreatic cell markers including insulin and transcription factors specific to β-cells. This effect was further enhanced by culturing cells in suspension. However, the effects of Ad-KLf4 were transient and this was shown to be due to increased apoptosis in Klf4-expressing cells. Klf4 has been recently identified as a pioneer factor with the ability to modulate the structure of chromatin and enhance reprogramming/transdifferentiation. Our results show that Klf4 may have a role in the redifferentiation of expanded pancreatic cells in culture, but before this can be achieved the off-target effects that result in increased apoptosis would need to be overcome. PMID:26457418

  2. 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression.

    PubMed

    Pinto, Sheena; Stark, Hans-Jürgen; Martin, Iris; Boukamp, Petra; Kyewski, Bruno

    2015-01-01

    Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance. Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80(lo), Aire-negative into CD80(hi), Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80(hi) mTECs. PMID:26275017

  3. Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement.

    PubMed

    Shahdadfar, Aboulghassem; Haug, Kristiane; Pathak, Meeta; Drolsum, Liv; Olstad, Ole Kristoffer; Johnsen, Erik O; Petrovski, Goran; Moe, Morten C; Nicolaissen, Bjørn

    2012-04-01

    In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement

  4. Epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma: Characterization in a 3D-cell culture model

    PubMed Central

    Gagliano, Nicoletta; Celesti, Giuseppe; Tacchini, Lorenza; Pluchino, Stefano; Sforza, Chiarella; Rasile, Marco; Valerio, Vincenza; Laghi, Luigi; Conte, Vincenzo; Procacci, Patrizia

    2016-01-01

    AIM: To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. METHODS: HPAF-II, HPAC, and PL45 PDAC cells were cultured in either 2D-monolayers or 3D-spheroids. Ultrastructure was analyzed by transmission electron microscopy. The expression of E-cadherin, β-catenin, N-cadherin, collagen type I (COL-I), vimentin, α-smooth muscle actin (αSMA), and podoplanin was assayed by confocal microscopy in cells cultured on 12-mm diameter round coverslips and in 3D-spheroids. Gene expression for E-cadherin, Snail, Slug, Twist, Zeb1, and Zeb2 was quantified by real-time PCR. E-cadherin protein level and its electrophoretic pattern were studied by Western blot in cell lysates obtained from cells grown in 2D-monolayers and 3D-spheroids. RESULTS: The E-cadherin/β-catenin complex was expressed in a similar way in plasma membrane cell boundaries in both 2D-monolayers and 3D-spheroids. E-cadherin increased in lysates obtained from 3D-spheroids, while cleavage fragments were more evident in 2D-monolayers. N-cadherin expression was observed in very few PDAC cells grown in 2D-monolayers, but was more evident in 3D-spheroids. Some cells expressing COL-I were observed in 3D-spheroids. Podoplanin, expressed in collectively migrating cells, and αSMA were similarly expressed in both experimental conditions. The concomitant maintenance of the E-cadherin/β-catenin complex at cell boundaries supports the hypothesis of a collective migration for these cells, which is consistent with podoplanin expression. CONCLUSION: We show that a 3D-cell culture model could provide deeper insight into understanding the biology of PDAC and allow for the detection of marked differences in the phenotype of PDAC cells grown in 3D-spheroids. PMID:27182158

  5. Challenge for 3D culture technology: Application in carcinogenesis studies with human airway epithelial cells.

    PubMed

    Emura, M; Aufderheide, M

    2016-05-01

    Lung cancer is still one of the major intractable diseases and we urgently need more efficient preventive and curative measures. Recent molecular studies have provided strong evidence that allows us to believe that classically well-known early airway lesions such as hyperplasia, metaplasia, dysplasia and carcinoma in situ are really precancerous lesions progressing toward cancer but not necessarily transient and reversible alteration. This suggests that adequate early control of the precancerous lesions may lead to improved prevention of lung cancer. This knowledge is encouraging in view of the imminent necessity for additional experimental systems to investigate the causal mechanisms of cancers directly in human cells and tissues. There are many questions with regard to various precancerous lesions of the airways. For example, should cells, before reaching a stage of invasive carcinoma, undergo all precancerous stages such as hyperplasia or metaplasia and dysplasia, or is there any shortcut to bypass one or more of the precancerous stages? For the study of such questions, the emerging 3-dimensional (3D) cell culture technology appears to provide an effective and valuable tool. Though a great challenge, it is expected that this in vitro technology will be rapidly and reliably improved to enable the cultures to be maintained in an in vivo-mimicking state of differentiation for much longer than a period of at best a few months, as is currently the case. With the help of a "causes recombination-Lox" (Cre-lox) technology, it has been possible to trace cells giving rise to specific lung tumor types. In this short review we have attempted to assess the future role of 3D technology in the study of lung carcinogenesis. PMID:26951634

  6. p57KIP2 expression and loss of heterozygosity during immortal conversion of cultured human mammary epithelial cells

    SciTech Connect

    Nijjar, Tarlochan; Wigington, Don; Garbe, James C.; Waha, Andreas; Stampfer, Martha R.; Yaswen, Paul

    1999-08-01

    The authors have uncovered a novel role for the cyclin-dependent kinase inhibitor, p57KIP2, during the immortalization of cultured human mammary epithelial cells (HMEC). HMEC immortalized following chemical carcinogen exposure initially expressed little or no telomerase activity, and their telomeres continued to shorten with passage. Cell populations whose mean terminal restriction fragment (TRF) length declined and exhibited slow heterogeneous growth, and contained many non-proliferative cells. These conditionally immortal HMEC cultures accumulated large quantities of p57 protein. With continued passage, the conditionally immortal cell populations very graduall2048nverted to a fully immortal phenotype of good uniform growth, expression of high levels of telomerase activity, and stabilization of telomere length. The fully immortal good growing HMEC did not accumulate p57 in G0 or during the cell cycle. DNA and RNA analysis of mass populations and individual subclones of conditionally immortal HMEC line 184A1 showed that continued growth of conditionally immortal cells with critically short telomeres was repeatedly accompanied by loss of the expressed p57 allele, and transient expression of the previously imprinted allele. Conditionally immortal 184A1 with mean TRF > 3 kb infected with retroviruses containing the p57 gene exhibited premature slow heterogeneous growth. Conversely, exogenous expression of hTERT, the catalytic subunit of telomerase, in 184A1 with mean TRF > 3 kb prevented both the slow heterogeneous growth phase and accumulation of p57 in cycling populations. These data indicate that in HMEC which have overcome replicative senescence, p57 may provide an additional barrier against indefinite proliferation. Overcoming p57 mediated growth inhibition in these cells may be crucial for acquisition of the unlimited growth potential thought to be critical for malignant progression.

  7. Interphase Chromosome Conformation and Chromatin-Chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Wong, Michael; Hada, Megumi; Wu, Honglu

    2015-01-01

    Microgravity has been shown to alter global gene expression patterns and protein levels both in cultured cells and animal models. It has been suggested that the packaging of chromatin fibers in the interphase nucleus is closely related to genome function, and the changes in transcriptional activity are tightly correlated with changes in chromatin folding. This study explores the changes of chromatin conformation and chromatin-chromatin interactions in the simulated microgravity environment, and investigates their correlation to the expression of genes located at different regions of the chromosome. To investigate the folding of chromatin in interphase under various culture conditions, human epithelial cells, fibroblasts, and lymphocytes were fixed in the G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome as separate colors. After images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multi-mega base pair scale. In order to determine the effects of microgravity on chromosome conformation and orientation, measures such as distance between homologous pairs, relative orientation of chromosome arms about a shared midpoint, and orientation of arms within individual chromosomes were all considered as potentially impacted by simulated microgravity conditions. The studies revealed non-random folding of chromatin in interphase, and suggested an association of interphase chromatin folding with radiation-induced chromosome aberration hotspots. Interestingly, the distributions of genes with expression changes over chromosome 3 in cells cultured under microgravity environment are apparently clustered on specific loci and chromosomes. This data provides important insights into how mammalian cells respond to microgravity at molecular level.

  8. Isospora suis in an epithelial cell culture system - an in vitro model for sexual development in coccidia.

    PubMed

    Worliczek, Hanna Lucia; Ruttkowski, Bärbel; Schwarz, Lukas; Witter, Kirsti; Tschulenk, Waltraud; Joachim, Anja

    2013-01-01

    Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia. PMID:23861983

  9. Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

    PubMed Central

    Worliczek, Hanna Lucia; Ruttkowski, Bärbel; Schwarz, Lukas; Witter, Kirsti; Tschulenk, Waltraud; Joachim, Anja

    2013-01-01

    Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isosporasuis, a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I. suis, including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I. suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia. PMID:23861983

  10. Plasma Polymer-Coated Contact Lenses for the Culture and Transfer of Corneal Epithelial Cells in the Treatment of Limbal Stem Cell Deficiency

    PubMed Central

    Brown, Karl David; Low, Suet; Mariappan, Indumathi; Abberton, Keren Maree; Short, Robert; Zhang, Hong; Maddileti, Savitri; Sangwan, Virender; Steele, David

    2014-01-01

    Extensive damage to the limbal region of the cornea leads to a severe form of corneal blindness termed as limbal stem cell deficiency (LSCD). Whereas most cases of corneal opacity can be treated with full thickness corneal transplants, LSCD requires stem cell transplantation for successful ocular surface reconstruction. Current treatments for LSCD using limbal stem cell transplantation involve the use of murine NIH 3T3 cells and human amniotic membranes as culture substrates, which pose the threat of transmission of animal-derived pathogens and donor tissue-derived cryptic infections. In this study, we aimed to produce surface modified therapeutic contact lenses for the culture and delivery of corneal epithelial cells for the treatment of LSCD. This approach avoids the possibility of suture-related complications and is completely synthetic. We used plasma polymerization to deposit acid functional groups onto the lenses at various concentrations. Each surface was tested for its suitability to promote corneal epithelial cell adhesion, proliferation, retention of stem cells, and differentiation and found that acid-based chemistries promoted better cell adhesion and proliferation. We also found that the lenses coated with a higher percentage of acid functional groups resulted in a higher number of cells transferred onto the corneal wound bed in rabbit models of LSCD. Immunohistochemistry of the recipient cornea confirmed the presence of autologous, transplanted 5-bromo-2′-deoxyuridine (BrdU)-labeled cells. Hematoxylin staining has also revealed the presence of a stratified epithelium at 26 days post-transplantation. This study provides the first evidence for in vivo transfer and survival of cells transplanted from a contact lens to the wounded corneal surface. It also proposes the possibility of using plasma polymer-coated contact lenses with high acid functional groups as substrates for the culture and transfer of limbal cells in the treatment of LSCD. PMID:24328453

  11. Analysis of TGF-β1- and drug-induced epithelial-mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3.

    PubMed

    Takano, Mikihisa; Yamamoto, Chieko; Yamaguchi, Koki; Kawami, Masashi; Yumoto, Ryoko

    2015-02-01

    In this study, we examined the induction of epithelial-mesenchymal transition (EMT) by transforming growth factor (TGF)-β1 and drugs in genetically engineered type II alveolar epithelial cell line RLE/Abca3. Treatment of RLE/Abca3 cells with TGF-β1 induced marked changes in cell morphology from epithelial-like to elongated fibroblast-like morphology. With these morphological changes, mRNA expression of epithelial markers such as cytokeratin 19 (CK19) decreased, while that of mesenchymal markers such as α-smooth muscle actin (α-SMA) increased. TGF-β1 treatment also decreased the mRNA expression of Abca3, a type II cell marker, and formation of lamellar body structures. Interestingly, the effect of TGF-β1 on Abca3 mRNA expression was observed in RLE/Abca3 cells, but not in wild-type RLE-6TN, A549, and H441 cells. Treatment of RLE/Abca3 cells with bleomycin (BLM) and methotrexate (MTX) induced similar morphological and mRNA expression changes. In addition, the increase in α-SMA and the decrease in Abca3 mRNA expression by these drugs were observed only in RLE/Abca3 cells. These findings suggest that, like TGF-β1, BLM and MTX induce EMT in RLE/Abca3 cells, and RLE/Abca3 cells would be a good model to study drug-induced EMT. The effect of pirfenidone, an antifibrotic and anti-inflammatory drug, on EMT induced by TGF-β1 was also discussed. PMID:25760538

  12. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells

    PubMed Central

    Volckens, John; Dailey, Lisa; Walters, Glenn; Devlin, Robert B.

    2010-01-01

    Exposure of cultured cells to particulate matter air pollution is usually accomplished by collecting particles on a solid matrix, extracting the particles from the matrix, suspending them in liquid, and applying the suspension to cells grown on plastic and submerged in medium. The objective of this work was to develop a more physiologically and environmentally relevant model of air pollutant deposition on cultures of human primary airway epithelial cells. We hypothesize that the toxicology of inhaled particulate matter depends strongly on both the particulate dispersion state and the mode of delivery to cells. Our exposure system employs a combination of unipolar charging and electrostatic force to deposit particles directly from the air onto cells grown at an air-liquid interface in a heated, humidified exposure chamber. Normal human bronchial epithelial cells exposed to concentrated, coarse ambient particulate matter in this system expressed increased levels of inflammatory biomarkers at 1 hour following exposure and relative to controls exposed to particle-free air. More importantly, these effects are seen at particulate loadings that are 1-2 orders of magnitude lower than levels applied using traditional in vitro systems. PMID:19603682

  13. Epithelial organization, cell polarity and tumorigenesis.

    PubMed

    McCaffrey, Luke Martin; Macara, Ian G

    2011-12-01

    Epithelial cells comprise the foundation for the majority of organs in the mammalian body, and are the source of approximately 90% of all human cancers. Characteristically, epithelial cells form intercellular adhesions, exhibit apical/basal polarity, and orient their mitotic spindles in the plane of the epithelial sheet. Defects in these attributes result in the tissue disorganization associated with cancer. Epithelia undergo self-renewal from stem cells, which might in some cases be the cell of origin for cancers. The PAR polarity proteins are master regulators of epithelial organization, and are closely linked to signaling pathways such as Hippo, which orchestrate proliferation and apoptosis to control organ size. 3D ex vivo culture systems can now faithfully recapitulate epithelial organ morphogenesis, providing a powerful approach to study both normal development and the initiating events in carcinogenesis. PMID:21782440

  14. Epithelial stem cells.

    PubMed

    Draheim, Kyle M; Lyle, Stephen

    2011-01-01

    It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process. PMID:21618097

  15. Characteristics of taurine transport in cultured renal epithelial cell lines: asymmetric polarity of proximal and distal cell lines.

    PubMed

    Jones, D P; Miller, L A; Budreau, A; Chesney, R W

    1992-01-01

    Taurine transport was determined in two continuous, renal epithelial cell lines: LLC-PK1 derived from the proximal tubule of the pig, and the Madin-Darby canine kidney cell (MDCK) from the distal tubule of the dog. In LLC-PK1, taurine transport is maximal at the apical surface, whereas in MDCK cells, transport is greatest at the basolateral surface. Transport is highly dependent on both sodium and chloride in the external medium, and is specific for beta-amino acids. The apical and basolateral surfaces of both cell lines show an adaptive response to extracellular taurine concentration, but only the basolateral surface of the MDCK cell responds to hyperosomolality by increased taurine accumulation. Thus, differential control of the beta-amino acid transport system by substrate and external tonicity exists. The role of the beta-amino acid transport system may differ according to the origin of the cell: in the proximal renal tubular cell, net transepithelial reabsorption of filtered taurine increases the body pool. By contrast, taurine accumulation by distal tubular cells may form a mechanism of cell volume regulation in response to osmotic stress. PMID:1509959

  16. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures

    PubMed Central

    Haller, D; Bode, C; Hammes, W; Pfeifer, A; Schiffrin, E; Blum, S

    2000-01-01

    BACKGROUND AND AIM—Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis. Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells. To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures.
METHODS—CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli. Expression of tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction.
RESULTS—Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1β, and TNF-α mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-α and IL-1β whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRNA in leucocyte sensitised CaCO-2 cells. TNF-α was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria.
CONCLUSION—The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of

  17. Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells.

    PubMed

    Valente, Maria João; Henrique, Rui; Vilas-Boas, Vânia; Silva, Renata; Bastos, Maria de Lourdes; Carvalho, Félix; Guedes de Pinho, Paula; Carvalho, Márcia

    2012-02-01

    Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1 mM) for 72 h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6 h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1 mM. PMID:21983858

  18. Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures.

    PubMed Central

    Grant, C C; Konkel, M E; Cieplak, W; Tompkins, L S

    1993-01-01

    Previous studies of Campylobacter jejuni have suggested that flagellin is an adhesin for epithelial cells and that motility is a virulence factor of this bacterium. The role of flagella in the interactions of C. jejuni with nonpolarized and polarized epithelial cells was examined with flagellar mutants. Flagellated, nonmotile (flaA flaB+ Mot-) and nonflagellated, nonmotile (flaA flaB Mot-) mutants of C. jejuni were constructed by in vivo homologous recombination and gene replacement techniques. Both classes of mutants were found to adhere to cells of human epithelial origin (INT 407) equally well; however, on the basis of the percentage of the inoculum internalized, internalization of the flaA flaB Mot- mutants was decreased by factors ranging from approximately 30 to 40 compared with the parent. The flaA flaB+ Mot- mutant was internalized by the INT 407 cells at levels six- to sevenfold higher than the flaA flaB Mot- mutants. Both classes of mutants, unlike the parent, were unable to translocate across polarized Caco-2 monolayers. These results indicate that flagella are not involved in C. jejuni adherence to epithelial cells but that they do play a role in internalization. Furthermore, the results suggest that either the motility of C. jejuni or the product of flaA is essential for the bacterium to cross polarized epithelial cell monolayers. Images PMID:8478066

  19. An In Vitro Culture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

    PubMed Central

    Janebodin, Kajohnkiart; Buranaphatthana, Worakanya; Ieronimakis, Nicholas; Hays, Aislinn L.; Reyes, Morayma

    2013-01-01

    Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used the Col1a1-GFP mouse model to characterize the salivary gland mesenchyme in vitro and in vivo. The Col1a1-GFP transgene was exclusively expressed in the salivary gland mesenchyme. Ex vivo culture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used this in vitro culture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7 and Fgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-β signaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors. PMID:23841093

  20. Senescent bronchial fibroblasts induced to senescence by Cr(VI) promote epithelial-mesenchymal transition when co-cultured with bronchial epithelial cells in the presence of Cr(VI).

    PubMed

    Val, Mariana Monteiro; Mendes, Luís André; Alarcão, Ana; Carvalho, Lina; Carreira, Isabel; Rodrigues, Carlos Fernando D; Alpoim, Maria Carmen

    2015-03-01

    Cellular senescence is a physiological process that serves as a powerful barrier for tumorigenesis. However, senescent cells can be deleterious for the tissue microenvironment. Such is the case of senescent fibroblasts that release several pro-tumorigenic factors that promote malignant transformation in the nearby epithelial cells. Occupational exposure to hexavalent chromium [Cr(VI)] compounds is a cause of respiratory cancers. Although Cr(VI) is known to induce senescence in human foreskin fibroblasts, the role of senescent fibroblasts in the Cr(VI)-induced malignant transformation of human bronchial epithelial cells was never assessed. Thus, to study the evolutionary dynamics generated by the interaction between human bronchial epithelial cells and senescent bronchial fibroblasts, the non-tumorigenic human bronchial epithelial BEAS-2B cells were co-cultured with Cr(VI)-induced senescent human bronchial fibroblasts for 4 weeks. Under the pressure of 0.5 µM Cr(VI), senescent fibroblasts promoted the acquisition of mesenchymal features on BEAS-2B cells, e.g. the fusiform shape and increased Vimentin expression, consistent with the occurrence of an epithelial-mesenchymal transition-like process. Features of transformed cells including larger nuclei, as well as nuclei with heterogeneous size, were also observed. Altogether the results obtained demonstrate that besides acting over the epithelium, Cr(VI) also affects bronchial fibroblasts driving them senescent. As a consequence, a paracrine communication loop is established with the above-placed epithelium prompting the epithelial cells for malignant transformation and thus facilitating the initial steps of tumorigenesis. PMID:25406472

  1. Primary cultures of female swine genital epithelial cells in vitro: a new approach for the study of hormonal modulation of Chlamydia infection.

    PubMed

    Guseva, Natalia V; Knight, Stephen T; Whittimore, Judy D; Wyrick, Priscilla B

    2003-08-01

    Previous studies have demonstrated that female reproductive hormones influence chlamydial infection both in vivo and in vitro. Due to the reduced availability of human genital tissues for research purposes, an alternative hormone-responsive model system was sought to study chlamydial pathogenesis. Mature female swine eliminated from breeding programs were selected as the animals of choice because of the similarity of a sexually transmitted disease syndrome and sequelae in swine to a disease syndrome and sequelae found in humans, because of the near identity of a natural infectious chlamydial isolate from swine to Chlamydia trachomatis serovar D from humans, and because a pig's epithelial cell physiology and the mean length of its estrous cycle are similar to those in humans. Epithelial cells from the cervix, uterus, and horns of the uterus were isolated, cultivated in vitro in Dulbecco's minimum essential medium-Hanks' F-12 (DMEM-F-12) medium with and without exogenous hormone supplementation, and analyzed for Chlamydia suis S-45 infectivity. The distribution of chlamydial inclusions in swine epithelial cells was uneven and was influenced by the genital tract site and hormone status. This study confirmed that, like primary human endometrial epithelial cells, estrogen-dominant swine epithelial cells are more susceptible to chlamydial infection than are progesterone-dominant cells. Further, the more differentiated luminal epithelial cells were more susceptible to infection than were glandular epithelial cells. Interestingly, chlamydial growth in mature luminal epithelia was morphologically more active than in glandular epithelia, where persistent chlamydial forms predominated. Attempts to reprogram epithelial cell physiology and thereby susceptibility to chlamydial infection by reverse-stage, exogenous hormonal supplementation were unsuccessful. Freshly isolated primary pig epithelial cells frozen at -80 degrees C in DMEM-F-12 medium with 10% dimethyl sulfoxide for

  2. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    PubMed

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  3. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  4. Cell kinetics of differentiation of Na/sup +/-dependent hexose transport in a cultured renal epithelial cell line

    SciTech Connect

    Cook, J.S.; Weiss, E.R.

    1985-01-01

    Fully differentiated cells of the renal proximal tubule have the capability of taking up hexoses across their apical borders by transport coupled to the Na/sup +/-electrochemical gradient. This property is also found in postconfluent cultures of the cloned cell line LLC-PK/sub 1/, a morphologically polarized line of renal cells. Postconfluent cells develop the Na/sup +/-dependent capacity to transport hexoses at their apical surface. This function is not observable during the growth phase of the cultures. To analyze the developmental process at the cellular level a method has been derived to separate transporting cells, expressing the differentiated function, from nontransporting cells. The method is based on the swelling of the cells accompanying the uptake of the nonmetabolizable glucose analog alpha methylglucoside. The swollen cells have a lower buoyant density than the undifferentiated cells and may be separated from them on density gradients. Analysis of the distribution of cells on such gradients shows that after the cells reach confluence the undifferentiated subpopulation is recruited onto the differentiation pathway with a rate constant of 0.2 per day, that 5 to 7 days are required for a cell to traverse this pathway to the fully differentiated state, and that once the maximum uptake capacity is achieved the cells do not develop further.

  5. Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism.

    PubMed

    Shandilya, Umesh K; Sharma, Ankita; Sodhi, Monika; Kapila, Neha; Kishore, Amit; Mohanty, Ashok; Kataria, Ranjit; Malakar, Dhruva; Mukesh, Manishi

    2016-02-01

    Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three-dimensional (3D) environment as important signals are lost when cells are cultured in two-dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1-casein (CSN1S1), alpha S2-casein (CSN1S2), beta-casein (CSN2), kappa-casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol-3-phosphate acyltransferase (GPAM), fatty acid-binding protein 3 (FABP3), and stearoyl-CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT-PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome-like structures were formed by day 5, whereas polarized acinus-like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10-day-old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism-related genes. Therefore, matrix-based 3D culture of MEC that recapitulate the structural and functional context of normal tissues

  6. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

    PubMed Central

    Bernet, M F; Brassart, D; Neeser, J R; Servin, A L

    1993-01-01

    Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains. Images PMID:8285709

  7. 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.

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

    PubMed

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

    2015-02-01

    In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms. PMID:25542757

  9. Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Vergara, Leoncio A; Wen, Julie W; Long, Dan; Rockx, Barry

    2016-05-01

    Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission. PMID:26932515

  10. Halofuginone Synergistically Enhances Anti-Proliferation of Rapamycin in T Cells and Reduces Cytotoxicity of Cyclosporine in Cultured Renal Tubular Epithelial Cells

    PubMed Central

    Chu, Tony L. H.; Guan, Qiunong; Nguan, Christopher Y. C.; Du, Caigan

    2015-01-01

    Both rapamycin (RAPA) and cyclosporin A (CsA) are commonly used for immunosuppression, however their adverse side effects limit their application. Thus, it is of interest to develop novel means to enhance or preserve the immunosuppressive activity of RAPA or CsA while reducing their toxicity. Halofuginone (HF) has been recently tested as a potential immunosuppressant. This study investigated the interaction of HF with RAPA or with CsA in cell cultures. Cell proliferation in cultures was determined using methylthiazol tetrazolium assay, and cell apoptosis assessed by flow cytometric analysis and Western blot. The drug-drug interaction was determined according to Loewe’s equation or Bliss independence. Here, we showed that addition of HF to anti-CD 3 antibody-stimulated splenocyte cultures induced synergistic suppression of T cell proliferation in the presence of RAPA, indicated by an interaction index (γ) value of < 1.0 between HF and RAPA, but not in those with CsA. The synergistic interaction of RAPA with HF in the suppression of T cell proliferation was also seen in a mixed lymphocyte reaction and Jurkat T cell growth, and was positively correlated with an increase in cell apoptosis, but not with proline depletion. In cultured kidney tubular epithelial cells, HF attenuated the cytotoxicity of CsA. In conclusion, these data indicate that HF synergistically enhances anti-T cell proliferation of RAPA and reduces the nephrotoxicity of CsA in vitro, suggesting the potential use of HF for enhancing anti-T cell proliferation of RAPA and reducing CsA-mediated nephrotoxicity. PMID:26671563

  11. Epithelial-mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions.

    PubMed

    Sueki, Akane; Matsuda, Kazuyuki; Iwashita, Chinami; Taira, Chiaki; Ishimine, Nau; Shigeto, Shohei; Kawasaki, Kenji; Sugano, Mitsutoshi; Yamamoto, Hiroshi; Honda, Takayuki

    2014-10-31

    Epithelial-mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O2) and hypoxic (2% O2) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis. PMID:25445593

  12. Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture

    SciTech Connect

    Hall, H.G.; Farson, D.A.; Bissel, M.J.

    1982-08-01

    Two cells lines--Madin-Darby canine kidney (MDCK) and normal murine mammary gland (NMuMG)--growing as monolayers on collagen gels were overlaid with another collagen gel. The cells responded to the overlay by undergoing reorganization resulting in the creation of lumina. MDCK cells formed lumina that coalesced to form large cavities comparable in size with a tubule. NMuMG cells formed clusters surrounding small lumina, which appeared similar to acini of glandular tissue. The characteristic arrangements, described here by light and electron microscopy, resembled the morphology of the tissues of cell line origin. MDCK cells, grown in the presence of serum, formed lumina whether or not serum was removed at the time of overlay, whereas NMuMG cells required either a nondialyzable component of serum or hormonal supplements in serum-free defined media. Lumen formation was delayed by MDCK cells in the presence of the glutamine analog 6-diazo-5-oxo-L-norleucine, but this compound did not affect NMuMG lumen formation. In both cell lines, lumen formation was unaffected by the absence of sulfate, the presence of an inhibitor of sulfate glycosaminoglycan synthesis, or an inhibitor of collagen synthesis. DNA synthesis accompanied lumen formation but was not required.

  13. Organotypic three-dimensional culture model of mesenchymal and epithelial cells to examine tissue fusion events.

    EPA Science Inventory

    Tissue fusion during early mammalian development requires coordination of multiple cell types, the extracellular matrix, and complex signaling pathways. Fusion events during processes including heart development, neural tube closure, and palatal fusion are dependent on signaling ...

  14. Characterization of Na+/H+ exchanger NHE8 in cultured renal epithelial cells.

    PubMed

    Zhang, Jianning; Bobulescu, Ion Alexandru; Goyal, Sunita; Aronson, Peter S; Baum, Michel G; Moe, Orson W

    2007-09-01

    NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger on the basis of its primary amino acid sequence. Functional characterization of native NHE8 in mammalian cells has not been possible to date. We screened a number of polarized renal cell lines for the plasma membrane Na+/H+ exchangers (NHE1, 2, 3, 4, and 8) and found only NHE1 and NHE8 transcripts in NRK cells by RT-PCR. NHE8 protein is expressed in the apical membrane of NRK cells as demonstrated by immunoblots, confocal fluorescent immunocytochemistry, and immunoelectron microscopy. NHE1, on the other hand, is expressed primarily in the basolateral membrane. Bilateral perfusion of NRK cells grown on permeable supports shows Na+/H+ exchange activity on both the apical and basolateral membranes. NHE8-specific small interfering RNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knockdown of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific small interfering RNA knocks down NHE1 protein expression without affecting NHE8 protein levels and reduces basolateral NHE activity without affecting apical NHE activity. NHE8 is the only apical membrane Na+/H+ exchanger in NRK cells. NHE8 activity is Na+ dependent, displaying a cooperative sigmoidal relationship, and is highly sensitive to 5-(N-ethyl-n-isopropyl)-amiloride (EIPA). NRK cells provide a useful system where NHE8 can be studied in its native environment. PMID:17581925

  15. 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.

  16. Specific entry of Helicobacter pylori into cultured gastric epithelial cells via a zipper-like mechanism.

    PubMed

    Kwok, Terry; Backert, Steffen; Schwarz, Heinz; Berger, Jürgen; Meyer, Thomas F

    2002-04-01

    Although Helicobacter pylori has generally been considered an extracellular pathogen, a number of in vitro infection experiments and biopsy examinations have shown that it is capable of occasionally entering mammalian host cells. Here, we characterized this entry process by using AGS cells as a host cell model. In gentamicin protection-invasion assays, the number of H. pylori colonies recovered was lower than that for Salmonella enterica serovar Typhimurium X22, Escherichia coli expressing InvA, and Yersinia enterocolitica YO:9 grown at 25 degrees C but higher than that for Neisseria gonorrhoeae VP1 and Y. enterocolitica YO:9 grown at 37 degrees C. At the ultrastructural level, the entry process was observed to occur via a zipper-like mechanism. Internalized H. pylori was bound in tight LAMP-1-containing vacuoles in close association with condensed filamentous actin and tyrosine phosphorylation signals. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, and calphostin C, an inhibitor of protein kinase C, both inhibited the entry of H. pylori in a sensitive and dose-dependent manner; however, the level of entry was enhanced by sodium vanadate, an inhibitor of tyrosine phosphatases and ATPases. Furthermore, the cytokine tumor necrosis factor alpha antagonized the entry of H. pylori into AGS cells. Collectively, these results demonstrate that the entry of H. pylori into AGS cells occurs via a zipper-like mechanism which involves various host signal transduction events. PMID:11895977

  17. In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells.

    PubMed

    Prichard, J F; Thibodeaux, J K; Pool, S H; Blakewood, E G; Menezo, Y; Godke, R A

    1992-04-01

    Early stage caprine embryos were incubated with goat oviduct and uterine cells to evaluate whether these cells could be used as a somatic cell culture system to enhance development through the developmental block at the 8- to 16-cell stage during in-vitro culture. Following gonadotrophin treatment and natural mating, 2- to 4-cell embryos were surgically recovered from donor females for in-vitro culture studies. In Experiment 1, embryos were equally and randomly allotted to culture treatments of either culture medium plus caprine oviduct cells or culture medium alone. In both treatment groups, embryos were incubated in Medium-199 with 10% fetal bovine serum, 0.25% lactalbumin and 1% antibiotic-antimycotic at 37 degrees C in a humidified atmosphere of 5% CO2 in air. In Experiment 2, similar embryos were cultured in the same medium with either caprine oviduct cells, caprine uterine cells or sequentially incubated with oviduct cells and then uterine cells during a corresponding incubation interval. The culture conditions in Experiment 2 were the same as in Experiment 1. Following 72 h in culture, (Experiment 1), significantly more embryos developed through the in-vitro developmental block into blastocysts and hatched blastocysts when cultured with oviduct cells compared with no embryos developing through the in-vitro block when incubated with medium alone. In Experiment 2, caprine embryos co-cultured with oviduct cells alone resulted in more embryos developing into blastocysts and hatched blastocysts compared with those co-cultured with uterine cells alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1522202

  18. Nrf2 activators modulate oxidative stress responses and bioenergetic profiles of human retinal epithelial cells cultured in normal or high glucose conditions.

    PubMed

    Foresti, Roberta; Bucolo, Claudio; Platania, Chiara Maria Bianca; Drago, Filippo; Dubois-Randé, Jean-Luc; Motterlini, Roberto

    2015-09-01

    Retinal pigment epithelial cells exert an important supporting role in the eye and develop adaptive responses to oxidative stress or high glucose levels, as observed during diabetes. Endogenous antioxidant defences are mainly regulated by Nrf2, a transcription factor that is activated by naturally-derived and electrophilic compounds. Here we investigated the effect of the Nrf2 activators dimethylfumarate (DMF) and carnosol on antioxidant pathways, oxygen consumption rate and wound healing in human retinal pigment epithelial cells (ARPE-19) cultured in medium containing normal (NG, 5mM) or high (HG, 25 mM) glucose levels. We also assessed wound healing using an in vivo corneal epithelial injury model. We found that Nrf2 nuclear translocation and heme oxygenase activity increased in ARPE cells treated with 10 μM DMF or carnosol irrespective of glucose culture conditions. However, HG rendered retinal cells more sensitive to regulators of glutathione synthesis or inhibition and caused a decrease of both cellular and mitochondrial reactive oxygen species. Culture in HG also reduced ATP production and mitochondrial function as measured with the Seahorse XF analyzer and electron microscopy analysis revealed morphologically damaged mitochondria. Acute treatment with DMF or carnosol did not restore mitochondrial function in HG cells; conversely, the compounds reduced cellular maximal respiratory and reserve capacity, which were completely prevented by N-acetylcysteine thus suggesting the involvement of thiols in this effect. Interestingly, the scratch assay showed that wound closure was faster in cells cultured in HG than NG and was accelerated by carnosol. This effect was reversed by an inhibitor of heme oxygenase activity. Moreover, topical application of carnosol to the cornea of diabetic rats significantly accelerated wound healing. In summary, these data indicate that culture of retinal epithelial cells in HG does not affect the activation of the Nrf2/heme oxygenase

  19. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

    PubMed Central

    Marcinkiewicz, Mariola M.; Baker, Sandy T.; Wu, Jichuan; Hubert, Terrence L.; Wolfson, Marla R.

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  20. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    PubMed

    Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  1. Effects of steroid hormones on differentiated glandular epithelial and stromal cells in a three dimensional cell culture model of the canine endometrium

    PubMed Central

    2013-01-01

    Background Oestrogens and progesterone have a significant impact on the endometrium during the canine oestrous cycle. Their receptors mediate plasma steroid hormone levels and are expressed in several endometrial cell types. Altered steroid receptor expression patterns are involved in serious uterine diseases; however the mechanisms of hormone action during pathogenesis in these tissues remain unclear. The development of 3D culture systems of canine endometrial cells provides an opportunity for the effects of steroid hormones to be quantitatively assessed in a more in vivo-like setting. The present study aimed to determine the effects of the steroid hormones 17β-estradiol (E) and progesterone (P) on the expression of the oestrogen and progesterone receptors (ER and PR), and on proliferative activity, in a 3D co-culture system of canine uterine origin, comprising differentiated endometrial glands, and stromal cells (SCs). Results Morphology, differentiation, and apical-basolateral polarity of cultured glandular epithelial cells (GECs) were comparable to those in native uterine tissue as assessed by immunohistochemistry using differentiation markers (β-catenin, laminin), lectin histochemistry, and transmission electron microscopy. Supplementation of our 3D-culture system with E (at 15, 30 and 100 pg/mL) resulted in constant levels of ER expression in GECs, but reduced expression levels in SCs. PR expression was reduced in both GECs and SCs following treatment with E. 3 ng/mL P resulted in increased ER expression in GECs, but a decrease in SCs. PR expression in GECs increased in all P-treated groups, whereas PRs in SCs decreased with the lowest and highest doses, but increased with the middle dose of treatment. Proliferative activity, assessed by Ki67 staining, remained below 1% in all assays and cell types. Conclusions The present study demonstrates the applicability of our 3D organotypic canine endometrium-derived culture system for cellular-level studies. 3D

  2. Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells: Induction of assembly on fibrillar type I collagen substrata

    SciTech Connect

    David, G.; van der Schueren, B.; van den Berghe, H. ); Nusgens, B.; Van Cauwenberge, D.; Lapiere, C. )

    1987-06-01

    Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable ({sup 3}H)hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the allover rates of collagen synthesis and degradation. The proportion of the ({sup 3}H)collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibers. Transformed cells on collagen produce and accumulate more ({sup 3}H)collage, yet are less effective in basement membrane formation than normal cells, indicting that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface.

  3. Cytotoxic effects and metabolism of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene in duodenal and ileal epithelial cell cultures. [Rats

    SciTech Connect

    Quaroni, A.; Isselbacher, K.J.

    1981-12-01

    Two epithelial cell lines have been established from the duodenum (IEC-17) and the ileum (IEC-18) of outbred germfree Crl:CD(SD)GN rats. They have a very similar morphology and ultrastruture, a normal rat diploid karyotype, comparable growth rates, and a similar set of surface antigens as detected with monoclonal antibodies specific for intestinal epithelial cell surface proteins in vivo. Both cell lines do not grow in soft agar and when injected into syngeneic animals do not form tumors. The toxic effects of benzo(a)pyrene (BP) and 7,12-dimethylbenz(a)anthracene (DMBA) were studied in these two cell lines. The IEC-18 cells were found to be much more sensitive to both chemicals than the IEC-17 cells. In paticular, at concentrations greater than 1 ..mu..g/ml, DMBA completely and irreversibly inhibited cell proliferaion in the IEC-18 cells, whereas it produced a less marked and reversible inhibition in the IEC-17 cells. The metabolism of BP and DMBA to water-soluble products was studied; BP was apparently metabolized at similar rates in the two cell lines, whereas a striking difference was observed with DMBA, which was metabolized at a rate 10-15 times greater in the IEC-18 cells than that in the IEC-17 cells. These results suggest that cultured cell lines derived from different portions of the intestinal tract have similar propertes in vitro, but they may have very different responses to chemical carcinogens.

  4. Intracrystalline Proteins Promote Dissolution of Urinary Calcium Oxalate Crystals in Cultured Renal Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Grover, Phulwinder K.; Thurgood, Lauren A.; Fleming, David E.; van Bronswijk, Wilhelm; Ryall, Rosemary L.

    2007-04-01

    We have proposed that internalized calcium oxalate (CaOx) crystals containing intracrystalline proteins would be vulnerable to intracellular dissolution. The aims of this study were (1) to measure non-uniform strain and crystallite size in CaOx monohydrate (COM) crystals containing increasing amounts of intracrystalline crystal matrix extract (CME) and (2) to compare the rates of crystal dissolution in Madin-Darby canine kidney (MDCKII) cells. CME was isolated by demineralization of COM crystals generated from human urine. Cold and 14C-oxalate-labelled COM crystals were precipitated from ultrafiltered urine containing CME at final concentrations of 0-5mg/L. Non-uniform strain and crystallite size were determined using synchrotron X-ray diffraction with Rietveld whole-pattern peak fitting and profile analysis, and the protein content of the crystals was analyzed using SDS-PAGE and Western blotting for prothrombin fragment 1. Radiolabeled crystals were added to MDCKII cells and dissolution was expressed as radioactive label released into the medium relative to that in the crystals at zero time. Non-uniform strain increased and crystallite size decreased proportionally with rising CME concentration, reaching saturation between approximately 1 and 5 mg/L, and demonstrating unequivocally the inclusion of increasing quantities of proteins in the crystals. This was confirmed by SDS-PAGE and Western blotting. Crystal dissolution also followed saturation kinetics. These findings were confirmed by field emission scanning electron microscopy (FESEM), which showed that the degree of crystal degradation increased relative to CME concentration. We conclude that intracrystalline proteins enhance intracellular dissolution of CaOx crystals and thus may provide a natural defense against stone pathogenesis.

  5. Cultured human airway epithelial cells (calu-3): a model of human respiratory function, structure, and inflammatory responses.

    PubMed

    Zhu, Yan; Chidekel, Aaron; Shaffer, Thomas H

    2010-01-01

    This article reviews the application of the human airway Calu-3 cell line as a respiratory model for studying the effects of gas concentrations, exposure time, biophysical stress, and biological agents on human airway epithelial cells. Calu-3 cells are grown to confluence at an air-liquid interface on permeable supports. To model human respiratory conditions and treatment modalities, monolayers are placed in an environmental chamber, and exposed to specific levels of oxygen or other therapeutic modalities such as positive pressure and medications to assess the effect of interventions on inflammatory mediators, immunologic proteins, and antibacterial outcomes. Monolayer integrity and permeability and cell histology and viability also measure cellular response to therapeutic interventions. Calu-3 cells exposed to graded oxygen concentrations demonstrate cell dysfunction and inflammation in a dose-dependent manner. Modeling positive airway pressure reveals that pressure may exert a greater injurious effect and cytokine response than oxygen. In experiments with pharmacological agents, Lucinactant is protective of Calu-3 cells compared with Beractant and control, and perfluorocarbons also protect against hyperoxia-induced airway epithelial cell injury. The Calu-3 cell preparation is a sensitive and efficient preclinical model to study human respiratory processes and diseases related to oxygen- and ventilator-induced lung injury. PMID:20948883

  6. Effect of selenium nanoparticles with different sizes in primary cultured intestinal epithelial cells of crucian carp, Carassius auratus gibelio.

    PubMed

    Wang, Yanbo; Yan, Xuxia; Fu, Linglin

    2013-01-01

    Nano-selenium (Se), with its high bioavailability and low toxicity, has attracted wide attention for its potential application in the prevention of oxidative damage in animal tissues. However, the effect of nano-Se of different sizes on the intestinal epithelial cells of the crucian carp (Carassius auratus gibelio) is poorly understood. Our study showed that different sizes and doses of nano-Se have varied effects on the cellular protein contents and the enzyme activities of secreted lactate dehydrogenase, intracellular sodium potassium adenosine triphosphatase, glutathione peroxidase, and superoxide dismutase. It was also indicated that nano-Se had a size-dependent effect on the primary intestinal epithelial cells of the crucian carp. Thus, these findings may bring us a step closer to understanding the size effect and the bioavailability of nano-Se on the intestinal tract of the crucian carp. PMID:24204137

  7. Effect of selenium nanoparticles with different sizes in primary cultured intestinal epithelial cells of crucian carp, Carassius auratus gibelio

    PubMed Central

    Wang, Yanbo; Yan, Xuxia; Fu, Linglin

    2013-01-01

    Nano-selenium (Se), with its high bioavailability and low toxicity, has attracted wide attention for its potential application in the prevention of oxidative damage in animal tissues. However, the effect of nano-Se of different sizes on the intestinal epithelial cells of the crucian carp (Carassius auratus gibelio) is poorly understood. Our study showed that different sizes and doses of nano-Se have varied effects on the cellular protein contents and the enzyme activities of secreted lactate dehydrogenase, intracellular sodium potassium adenosine triphosphatase, glutathione peroxidase, and superoxide dismutase. It was also indicated that nano-Se had a size-dependent effect on the primary intestinal epithelial cells of the crucian carp. Thus, these findings may bring us a step closer to understanding the size effect and the bioavailability of nano-Se on the intestinal tract of the crucian carp. PMID:24204137

  8. Genotoxic Effects of Low- and High-LET Radiation on Human Epithelial Cells Grown in 2-D Versus 3-D Culture

    NASA Technical Reports Server (NTRS)

    Patel, Z. S.; Cucinotta, F. A.; Huff, J. L.

    2011-01-01

    Risk estimation for radiation-induced cancer relies heavily on human epidemiology data obtained from terrestrial irradiation incidents from sources such as medical and occupational exposures as well as from the atomic bomb survivors. No such data exists for exposures to the types and doses of high-LET radiation that will be encountered during space travel; therefore, risk assessment for space radiation requires the use of data derived from cell culture and animal models. The use of experimental models that most accurately replicate the response of human tissues is critical for precision in risk projections. This work compares the genotoxic effects of radiation on normal human epithelial cells grown in standard 2-D monolayer culture compared to 3-D organotypic co-culture conditions. These 3-D organotypic models mimic the morphological features, differentiation markers, and growth characteristics of fully-differentiated normal human tissue and are reproducible using defined components. Cultures were irradiated with 2 Gy low-LET gamma rays or varying doses of high-LET particle radiation and genotoxic damage was measured using a modified cytokinesis block micronucleus assay. Our results revealed a 2-fold increase in residual damage in 2 Gy gamma irradiated cells grown under organotypic culture conditions compared to monolayer culture. Irradiation with high-LET particle radiation gave similar results, while background levels of damage were comparable under both scenarios. These observations may be related to the phenomenon of "multicellular resistance" where cancer cells grown as 3-D spheroids or in vivo exhibit an increased resistance to killing by chemotherapeutic agents compared to the same cells grown in 2-D culture. A variety of factors are likely involved in mediating this process, including increased cell-cell communication, microenvironment influences, and changes in cell cycle kinetics that may promote survival of damaged cells in 3-D culture that would

  9. 7,12-dimethylbenz(a)anthracene-induced DNA binding and repair synthesis in susceptible and nonsusceptible mammary epithelial cells in culture. [Rats

    SciTech Connect

    Tay, L.K.; Russo, J.

    1981-07-01

    The effect of age and parity on the binding of 7,12-dimethylbenz(a)anthracene (DMBA) to DNA and the repair of DMBA-damaged DNA have been demonstrated in logarithmic phase and confluent mammary epithelial cell cultures from young virgin (YV), old virgin (OV), and parous (P) noninbred and inbred Sprague-Dawley rats. Excision repair was determined by measuring, in the presence of hydroxyurea and 5-bromodeoxyuridine, tritiated thymidine incorporation into DNA during the repair process. These results suggest that age and parity not only lower the binding of DMBA to mammary epithelial cell DNA but also increase the efficiency of DNA repair processes, which may explain the lower susceptibility of OV and P rats to DMBA-induced mammary carcinogenesis.

  10. Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

    PubMed

    Zhang, Yuanyuan; George, Jasmine; Li, Yun; Olufade, Rebecca; Zhao, Xueying

    2015-01-01

    As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy

  11. Induced pluripotency of human prostatic epithelial cells.

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects. PMID:25387250

  13. Epithelial in vitro cell systems in carcinogenesis studies

    SciTech Connect

    Borek, C.

    1983-01-01

    The development of epithelial cells systems to study oncogenic transformation has presented a major challenge in the field of carcinogenesis. Because there exists in man a preponderance of carcinomas over sarcomas, the importance of studying oncogenic transformation in epithelial cells is of great relevance to human disease. The difficulty lies in the fact that different tissues contain epithelial cells with singular differentiated characteristics, which must be defined to assert the different nature of the cells being used. Liver cells in culture are a case in point. By careful maintenance and optimal culture conditions, one can maintain many of the differentiated characteristics of the cells for prolonged periods of time.

  14. Micropatterns of Matrigel for three-dimensional epithelial cultures.

    PubMed

    Sodunke, Temitope R; Turner, Keneshia K; Caldwell, Sarah A; McBride, Kevin W; Reginato, Mauricio J; Noh, Hongseok Moses

    2007-09-01

    Three-dimensional (3D) epithelial culture models are widely used to promote a physiologically relevant microenvironment for the study of normal and aberrant epithelial organization. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hindered by the lack of existing platforms for large-scale 3D cellular studies. Current 3D standard culture does not allow for single spheroid or 'acinus' analysis required for high-throughput systems. Here, we present general strategies for creating bulk micropatterns of Matrigel that can be used as a platform for 3D epithelial culture and cell-based assays at the single acinus level. Both buried and free-standing micropatterns of Matrigel were created using modified soft lithography techniques such as microtransfer molding (microTM) and dry lift-off technique. Surface modification of poly(dimethylsiloxane) (PDMS) with oxygen plasma followed by treatment with poly(2-hydroxy-ethylmethacrylate) (poly-HEMA) was sufficient to promote deformation-free release of Matrigel patterns. In addition, a novel dual-layer dry lift-off technique was developed to simultaneously generate patterns of Matrigel and poly-HEMA on a single substrate. We also demonstrate that the micropatterned Matrigel can support 3D culture originating from a single normal human mammary epithelial (MCF-10A) cell or a human breast cancer cell (MDA-MB-231) with comparable phenotypes to standard 3D culture techniques. Culture of normal MCF-10A cells on micropatterned Matrigel resulted in formation of structures with the characteristic apoptosis of centrally located cells and formation of hollow lumens. Moreover, the carcinoma cell line showed their characteristic formation of disorganized invasive cellular clusters, lacking the normal epithelial architecture on micropatterned Matrigel. Hence, micropatterned Matrigel can be used as a 3D epithelial cell-based platform for a wide variety of applications in epithelial

  15. Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions.

    PubMed

    Leelakanok, Nattawut; Fischer, Carol L; Bates, Amber M; Guthmiller, Janet M; Johnson, Georgia K; Salem, Aliasger K; Brogden, Kim A; Brogden, Nicole K

    2015-12-01

    Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro. PMID:26367466

  16. Surface-Modified Biodegradable Nanoparticles' Impact on Cytotoxicity and Inflammation Response on a Co-Culture of Lung Epithelial Cells and Human-Like Macrophages.

    PubMed

    Grabowski, Nadège; Hillaireau, Hervé; Vergnaud-Gauduchon, Juliette; Nicolas, Valérie; Tsapis, Nicolas; Kerdine-Römer, Saadia; Fattal, Elias

    2016-01-01

    The toxicity of polymeric biodegradable nanoparticles was evaluated on a co-culture made from direct contact of human lung alveolar epithelial cells (A459) and macrophages (differentiated THP-1 monocytes). The co-culture was characterized by its phenotype and by confocal laser scanning microscopy. Cytokine secretion induced by lipopolysaccharide was synergistically increased in the co-culture confirming cell-cell interactions. Poly(lactide-co-glycolide) (PLGA)-based nanoparticles of 200 nm were prepared in presence of hydrophilic polymers commonly used as stabilizers [poly(vinyl alcohol), chitosan and poloxamer 188] through their interaction with particle surface. Stabilizer-free PLGA nanoparticles and stabilizers alone were also evaluated as controls. Selective uptake kinetics of PLGA nanoparticles by cell subpopulations, as well as apoptosis/necrosis detection, was achieved using a specific label for each cell type, while cytokine secretions were quantified in culture supernatants. Both cell subpopulations took up PLGA nanoparticles with similar profiles, and induced only little cytotoxicity (mostly necrosis). A mild inflammatory response to stabilized nanoparticles was detected (compared to well-known inflammatory compounds), slightly higher than the one observed for stabilizer-free PLGA nanoparticles or stabilizing agents taken individually. These results demonstrate that although biodegradable nanoparticles can be considered as safe, they can internalize compounds such as the stabilizing agents which enhance their toxicity. PMID:27301179

  17. 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

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

    PubMed

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

    2016-03-01

    Infection with feline herpesvirus-1 (FHV-1) accounts for 50% of viral upper respiratory diseases in domestic cats and is a significant cause of ocular diseases. Despite the clinical significance and high prevalence of FHV-1 infection, currently available vaccines cannot completely protect cats from infection and lifelong latency. FHV-1 infects via the mucous membranes and replicates in respiratory epithelial cells, but very little is known about the early innate immunity at this site. To address questions about immunity to FHV-1, feline respiratory epithelial cells cultured at air-liquid interface (ALI-FRECs) were established by collecting respiratory tracts from 6 healthy cats after euthanasia. Cells were isolated, cultured and characterized histologically and immunologically before infection with FHV-1. The expression of Toll-like receptors (TLRs), cytokine and chemokine responses were measured by real time PCR. ALI-FRECs morphologically resembled the natural airways of cats with multilayered columnar epithelial cells and cilia. Immunological properties of the natural airways were maintained in ALI-FRECs, as evidenced by the expression of TLRs, cytokines, chemokines, interferons, beta-defensins, and other regulatory genes. Furthermore, ALI-FRECs were able to support infection and replication of FHV-1, as well as modulate transcriptional regulation of various immune genes in response to infection. IL-1β and TNFα were increased in ALI-FRECs by 24hpi, whereas expression levels of IFN-α and TLR9 were not increased until 36hpi. In contrast, TLR3, GM-CSF and TGF-1β expression was down-regulated at 36hpi. The data presented show the development of a system ideal for investigating the molecular pathogenesis and immunity of FHV-1 or other respiratory pathogens. PMID:26795546

  19. Novel Compstatin Family Peptides Inhibit Complement Activation by Drusen-Like Deposits in Human Retinal Pigmented Epithelial Cell Cultures

    PubMed Central

    Gorham, Ronald D.; Forest, David L.; Tamamis, Phanourios; de Victoria, Aliana López; Kraszni, Márta; Kieslich, Chris A.; Banna, Christopher D.; Bellows-Peterson, Meghan L.; Larive, Cynthia K.; Floudas, Christodoulos A.; Archontis, Georgios; Johnson, Lincoln V.; Morikis, Dimitrios

    2013-01-01

    We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment. PMID:23954241

  20. Modeling and Simulation of Mucus Flow in Human Bronchial Epithelial Cell Cultures - Part I: Idealized Axisymmetric Swirling Flow.

    PubMed

    Vasquez, Paula A; Jin, Yuan; Palmer, Erik; Hill, David; Forest, M Gregory

    2016-08-01

    A multi-mode nonlinear constitutive model for mucus is constructed directly from micro- and macro-rheology experimental data on cell culture mucus, and a numerical algorithm is developed for the culture geometry and idealized cilia driving conditions. This study investigates the roles that mucus rheology, wall effects, and HBE culture geometry play in the development of flow profiles and the shape of the air-mucus interface. Simulations show that viscoelasticity captures normal stress generation in shear leading to a peak in the air-mucus interface at the middle of the culture and a depression at the walls. Linear and nonlinear viscoelastic regimes can be observed in cultures by varying the hurricane radius and mean rotational velocity. The advection-diffusion of a drug concentration dropped at the surface of the mucus flow is simulated as a function of Peclet number. PMID:27494700

  1. Modeling and Simulation of Mucus Flow in Human Bronchial Epithelial Cell Cultures – Part I: Idealized Axisymmetric Swirling Flow

    PubMed Central

    Vasquez, Paula A.; Jin, Yuan; Palmer, Erik; Hill, David; Forest, M. Gregory

    2016-01-01

    A multi-mode nonlinear constitutive model for mucus is constructed directly from micro- and macro-rheology experimental data on cell culture mucus, and a numerical algorithm is developed for the culture geometry and idealized cilia driving conditions. This study investigates the roles that mucus rheology, wall effects, and HBE culture geometry play in the development of flow profiles and the shape of the air-mucus interface. Simulations show that viscoelasticity captures normal stress generation in shear leading to a peak in the air-mucus interface at the middle of the culture and a depression at the walls. Linear and nonlinear viscoelastic regimes can be observed in cultures by varying the hurricane radius and mean rotational velocity. The advection-diffusion of a drug concentration dropped at the surface of the mucus flow is simulated as a function of Peclet number. PMID:27494700

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

    PubMed

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

    2015-11-01

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

  3. Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

    PubMed Central

    Kuznetsov, G; Bush, K T; Zhang, P L; Nigam, S K

    1996-01-01

    The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment

  4. M-BAND analysis of chromosome aberration induced by Fe-ions in human epithelial cells cultured in 3-dimensional matrices

    NASA Astrophysics Data System (ADS)

    Hada, Megumi; Cucinotta, Francis A.; Wu, Honglu

    Energetic heavy ions pose a great health risk to astronauts in extended ISS and future lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied lowand high-LET radiationinduced chromosome aberrations in human epithelial cells cultured in 2-dimension (2D) using the multicolor banding fluorescence in situ hybridization (mBAND) technique. However, it has been realized that the biological response to radiation insult in a 2D cellular environment in vitro can differ significantly from the response in 3-dimension (3D) or at the actual tissue level. In this study, we cultured human epithelial cells in 3D to provide a more suitable model for human tissue. Human mammary epithelial cells (CH184B5F5/M10) were grown in Matrigel to form 3D structures, and exposed to Fe-ions at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or 137 Cs-gamma radiation source at the University of Texas MD Anderson Cancer Center. After exposure, cells were allowed to repair for 16hr before dissociation and subcultured at low density in 2D. G2 and metaphase chromosomes in the first cell cycle were collected using a chemical-induced premature chromosome condensation (PCC) technique, and chromosome aberrations were analyzed using mBAND technique. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). Our data indicate a significant difference of the chromosome aberration yield between 2D and 3D cell cultures for gamma exposures, but not for Fe ion exposures

  5. Formation of lipofuscin in cultured retinal pigment epithelial cells exposed to pre-oxidized photoreceptor outer segments.

    PubMed

    Wihlmark, U; Wrigstad, A; Roberg, K; Brunk, U T; Nilsson, S E

    1996-04-01

    Accumulation of lipofuscin in the retinal pigment epithelium (RPE) with increasing age may affect essential supportive functions for the photoreceptors. Earlier, we described a model system for the study of lipofuscinogenesis in RPE cell cultures and showed that mild oxidative stress enhances lipofuscin formation from phagocytized photoreceptor outer segments (POS). In the present study, bovine POS were photo-oxidized, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amorphous osmiophilic electron-dense mass. The formation of thiobarbituric acid reactive substances (TBARS), estimated during the irradiation process, indicated lipid peroxidation. Irradiated POS also showed a strong granular yellow autofluorescence. RPE cell cultures, kept at 21% ambient oxygen, were fed daily for 3, 5 or 7 days with either (i) UV-peroxidized POS, (ii) native POS or (iii) culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific autofluorescence compared to cells exposed to native POS after 3 days (p = 0.0056), 5 days (p = 0.0037) and 7 days (p = 0.0020), and to the non-exposed control cells (3 days: p = 0.005, 5 days: p = 0.0037, 7 days: p = 0.0094). The lipofuscin content of cells exposed to irradiated POS increased significantly between days 3 and 7 (p = 0.0335). Ultrastructural studies showed much more numerous and larger lipofuscin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previously peroxidized POS, thus artificially converted to lipofuscin and obviously not digestible by the lysosomal enzymes, accelerates the formation of severely lipofuscin-loaded cells. The results will be useful for further studies of possible harmful

  6. Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    The isolation of cancer epithelial cells from mouse mammary tumor is accomplished by digestion of the solid tumor. Red blood cells and other contaminates are removed using several washing techniques such that primary epithelial cells can further enriched. This procedure yields primary tumor cells that can be used for in vitro tissue culture, fluorescence-activated cell sorting (FACS) and a wide variety of other experiments (Lo et al., 2012).

  7. Interphase Chromosome Conformation and Chromatin-chromatin Interactions in Human Epithelial Cells Cultured Under Different Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Hada, Megumi; Wu, Honglu

    2014-01-01

    On a multi-mega base pair scale of the DNA, the arrangement of chromatin is non-random. In M10 epithelial cells, both telomere regions tend to be located towards the exterior of the chromosome domain, whereas the rest p-arm of the chromatin region towards the interior. In contrast, most of the q-arm of the chromatin is found in the peripheral of the domain. In lymphocytes, the p-arm chromatin regions towards the interior in close proximity with each other, whereas two q-arm regions are nearness in space. It indicates that G0 lymphocytes may lack secondary 3D chromatin folding. There chromatin folding patterns are consistent with our previous finding of non-random distribution of intra-chromosomal exchanges. In simulated microgravity conditions, the chromosome conformation may be altered and new regions in close proximity, especially to region 2 are suggested.

  8. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

    PubMed Central

    Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258

  9. 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.

  10. Failure of propagation of human norovirus in intestinal epithelial cells with microvilli grown in three-dimensional cultures

    PubMed Central

    Takanashi, Sayaka; Saif, Linda J.; Hughes, John H.; Meulia, Tea; Jung, Kwonil; Scheuer, Kelly A.; Wang, Qiuhong

    2013-01-01

    Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis. Establishment of a cell culture system for in vitro HuNoV growth remains challenging. Replication of HuNoVs in human intestinal cell lines (INT-407 and Caco-2) that differentiate to produce microvilli in rotation wall vessel (RWV) three-dimensional cultures has been reported (Straub et al., Emerg Infect Dis 13:396–403 2007, J Water Health 9:225–240 2011, and Water Sci Technol 67:863–868 2013). We used a similar RWV system, intestinal cell lines, and the same (Genogroup [G] I.1) plus additional (GII.4 and GII.12) HuNoV strains to test the system’s reproducibility and to expand the earlier findings. Apical microvilli were observed on the surface of both cell lines by light and electron microscopy. However, none of the cell types tested resulted in productive viral replication of any of the HuNoV strains, as confirmed by plateau or declining viral RNA titers in the supernatants and cell lysates of HuNoV-infected cells, determined by real-time reverse transcription PCR. These trends were the same when culture supplements were added that have been reported to be effective for replication of other fastidious enteric viruses in vitro. Additionally, by confocal microscopy and orthoslice analysis, viral capsid proteins were mainly observed above the actin filament signals, which suggested that the majority of viral antigens were on the cell surface. We conclude that even intestinal cells displaying microvilli were not sufficient to support HuNoV replication under the conditions tested. PMID:23974469

  11. Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. III. Growth conditions of human thymic epithelial cells and immunomodulatory activities in their culture supernatant.

    PubMed Central

    Schreiber, L; Eshel, I; Meilin, A; Sharabi, Y; Shoham, J

    1991-01-01

    We report here on a new approach to the cultivation of human thymic epithelial (HTE) cells, which apparently allows more faithful preservation of cell function. This approach, previously developed by us for mouse thymic epithelial (MTE) cells, is based on the use of culture plates coated with extracellular matrix (ECM), and on the use of serum-free, growth factor-supplemented medium. The nutritional requirements of HTE and MTE are somewhat different. Although both are critically dependent on ECM and insulin, they differ in their dependency on other growth factors: selenium and transferrin are much more important for HTE cells, whereas epidermal growth factor and hydrocortisone play a more essential role in MTE cultures. The epithelial nature of the cultured cells is indicated by positive staining with anti-keratin antibodies and by the presence of desmosomes and tonofilaments. The ultrastructural appearance of the cells further suggests high metabolic and secretory activities, not usually found in corresponding cell lines. The culture supernatant (CS) of HTE cells exhibited a strong enhancing effect on thymocyte response to Con A stimulation, as measured by cell proliferation and lymphokine production. The effect was observed on both human and mouse thymocytes, but was much stronger in the homologous combination. Thymic factors tested in parallel did not have such a differential effect. The dose-effect relationships were in the form of a bell-shaped curve, with fivefold enhancement of response at the peak and a measurable effect even with 1:1000 dilution, when human thymocytes were used. The responding thymocytes were those which do not bind peanut agglutinin and are resistant to hydrocortisone. The culture system described here may have advantages for the in vitro study of thymic stromal cell function. Images Figure 1 Figure 3 Figure 4 PMID:1783421

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

    PubMed

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

    2015-12-15

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance,we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity.We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥ 60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport,without affecting Cl- transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100-360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. PMID:26454031

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

    PubMed Central

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

    2016-01-01

    Inhalation of butter flavoring by workers in the microwave popcorn industry may result in “popcorn workers' lung.” In previous in vivo studies rats exposed for 6 h to vapor from the flavoring agents, diacetyl and 2,3-pentanedione, acquired flavoring concentration-dependent damage of the upper airway epithelium and airway hyporeactivity to inhaled methacholine. Because ion transport is essential for lung fluid balance, we hypothesized that alterations in ion transport may be an early manifestation of butter flavoring-induced toxicity. We developed a system to expose cultured human bronchial/tracheal epithelial cells (NHBEs) to flavoring vapors. NHBEs were exposed for 6 h to diacetyl or 2,3-pentanedione vapors (25 or ≥60 ppm) and the effects on short circuit current and transepithelial resistance (Rt) were measured. Immediately after exposure to 25 ppm both flavorings reduced Na+ transport, without affecting Cl− transport or Na+,K+-pump activity. Rt was unaffected. Na+ transport recovered 18 h after exposure. Concentrations (100–360 ppm) of diacetyl and 2,3-pentanedione reported earlier to give rise in vivo to epithelial damage, and 60 ppm, caused death of NHBEs 0 h post-exposure. Analysis of the basolateral medium indicated that NHBEs metabolize diacetyl and 2,3-pentanedione to acetoin and 2-hydroxy-3-pentanone, respectively. The results indicate that ion transport is inhibited transiently in airway epithelial cells by lower concentrations of the flavorings than those that result in morphological changes of the cells in vivo or in vitro. PMID:26454031

  14. Testing gene therapy vectors in human primary nasal epithelial cultures

    PubMed Central

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  15. Testing gene therapy vectors in human primary nasal epithelial cultures.

    PubMed

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  16. Cell invasion and survival of Shiga toxin-producing Escherichia coli within cultured human intestinal epithelial cells.

    PubMed

    Cordeiro, Fabiana; da Silva, Rita Ifuoe K; Vargas-Stampe, Thaís L Z; Cerqueira, Aloysio M F; Andrade, João R C

    2013-08-01

    Shiga toxin-producing Escherichia coli (STEC) cause severe human infections and their virulence abilities are not fully understood. Cattle are a key reservoir, and the terminal rectum is the principal site of bacterial carriage. Most STEC possess a pathogenicity island termed the locus of enterocyte effacement (LEE). Nonetheless, LEE-negative STEC have been associated with disease. We found that invasion of LEE-positive and LEE-negative strains was higher for human enterocytic cell lines and for undifferentiated Caco-2 cells. Intracellular bacteria could be detected as early as 5 min after infection and transmission electron microscopy showed bacteria within membrane-bound vacuoles. STEC invasion depended on actin microfilaments and protein kinases. Scanning electron microscopy revealed that bacterial entry was not associated with membrane ruffling. Absence of macropinocytosis or actin rearrangement at the entry points suggests a zipper-like entry mechanism. Disruption of the tight junction by EGTA enhanced invasion of Caco-2 monolayers, and bacterial invasion mostly proceeded through the basolateral pole of enterocytes. STEC persisted within Caco-2 cells for up to 96 h without cell death and bacterial viability increased after 48 h, suggesting intracellular multiplication. The relatively harmless intracellular localization of STEC can be an efficient strategy to prevent its elimination from the bovine intestinal tract. PMID:23704791

  17. Quantitative analysis of cultured thymic reticulo-epithelial cells labelled by different antibodies: a flow cytometric study.

    PubMed Central

    Fabien, N; Auger, C; Bonnard, M; Andreoni, C; Rigal, D; Monier, J C

    1989-01-01

    Quantitative measurements of cultured human and murine thymic, and human thymoma reticuloepithelial cells (REC), immunolabeled by different antibodies (Ab) (TE3, TE4, anti-HTLV p19(p19), lu5, K11 and Aks) and by thymic hormones (thymulin and thymosin alpha 1 (Ta1)) within these cells, were performed using a flow cytometric technique. The anti-keratin polyclonal Ab labeled nearly the whole human or murine population. The p19 monoclonal Ab (MoAb), specific for the subcortical/medullary thymic regions, labelled 37-77% of the human REC. The TE3 MoAb, specific for the cortical region, labelled 54-83% of the REC. These percentages suggest that the cultured thymic REC (TREC) had markers of both regions together and therefore that these markers are not absolutely specific to determine their subcortical/medullary or cortical thymic origin. For the three populations there were more cells containing Ta1 than thymulin. The overlap of the percentage of labelled cells suggests that the same cell could synthesize the two hormones and that these hormones could be localized within the TE3 positive cells. PMID:2649289

  18. 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

  19. Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures.

    PubMed

    Fischer, William A; Chason, Kelly D; Brighton, Missy; Jaspers, Ilona

    2014-03-26

    Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent

  20. Effects of Lutein on Hyperosmoticity-Induced Upregulation of IL-6 in Cultured Corneal Epithelial Cells and Its Relevant Signal Pathways

    PubMed Central

    Chao, Shih-Chun; Nien, Chan-Wei; Iacob, Codrin; Hu, Dan-Ning; Huang, Sheng-Chieh

    2016-01-01

    Dry eye is a common disorder characterized by deficiency of tear. Hyperosmoticity of tear stimulates inflammation and damage of ocular surface tissues and plays an essential role in the pathogenesis of dry eye. Cultured human corneal epithelial (CE) cells were used for the study of effects of lutein and hyperosmoticity on the secretion of IL-6 by CE cells. Cell viability of CE cells was not affected by lutein at 1–10 μM as determined by MTT assay. Hyperosmoticity significantly elevated the secretion of IL-6 by CE cells as measured by ELISA analysis. The constitutive secretion of IL-6 was not affected by lutein. Lutein significantly and dose-dependently inhibited hyperosmoticity-induced secretion of IL-6. Phosphorylated- (p)- p38 MAPK, p-JNK levels in cell lysates and NF-κB levels in cell nuclear extracts were increased by being exposed to hyperosmotic medium. JNK, p38, and NF-κB inhibitors decreased hyperosmoticity-induced secretion of IL-6. Lutein significantly inhibited hyperosmoticity-induced elevation of NF-κB, p38, and p-JNK levels. We demonstrated that lutein inhibited hyperosmoticity-induced secretion of IL-6 in CE cells through the deactivation of p38, JNK, and NF-κB pathways. Lutein may be a promising agent to be explored for the treatment of dry eye. PMID:27047687

  1. 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.

  2. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

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

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of 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. Furthermore, 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. PMID:15657080

  3. Apolipoprotein AI could be a significant determinant of epithelial integrity in rainbow trout gill cell cultures: a study in functional proteomics.

    PubMed

    Smith, Richard W; Wood, Chris M; Cash, Phil; Diao, Linda; Pärt, Peter

    2005-05-20

    The freshwater fish gill forms a barrier against an external hypotonic environment. By culturing rainbow trout gill cells on permeable supports, as intact epithelia, this study investigates barrier property mechanisms. Under symmetrical conditions the apical and basolateral epithelial surfaces contact cell culture media. Replacing apical media with water, to generate asymmetrical conditions (i.e. the situation encountered by the freshwater gill), rapidly increases transepithelial resistance (TER). Proteomic analysis revealed that this is associated with enhanced expression of pre-apolipoprotein AI (pre-apoAI). To test the physiological relevance, gill cells were treated with a dose of 50 microg ml(-1) human apolipoprotein (apoAI). This was found to elevate TER in those epithelia which displayed a lower TER prior to apoAI treatment. These results demonstrate the action of apoAI and provide evidence that the rainbow trout gill may be a site of apoAI synthesis. TER does not differentiate between the trans-cellular (via the cell membrane) and para-cellular (via intercellular tight junctions) pathways. However, despite the apoAI-induced changes in TER, para-cellular permeability (measured by polyethylene glycol efflux) remained unaltered suggesting apoAI specifically reduces trans-cellular permeability. This investigation combines proteomics with functional measurements to show how a proteome change may be associated with freshwater gill function. PMID:15848139

  4. M-BAND Analysis of Chromosome Aberration Induced by Fe-Ions in Human Epithelial Cells Cultured in 3-Dimensional Matrices

    NASA Technical Reports Server (NTRS)

    Hada, M.; Cucinotta, F. A.; Wu, H.

    2008-01-01

    Energetic heavy ions pose a great health risk to astronauts in extended ISS and future lunar and Mars missions. High-LET heavy ions are particularly effective in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, which can be utilized as a biomarker for radiation insults. Previously, we had studied low- and high-LET radiation-induced chromosome aberrations in human epithelial cells cultured in 2-dimension (2D) using the multicolor banding fluorescence in situ hybridization (mBAND) technique. However, it has been realized that the biological response to radiation insult in a 2D cellular environment in vitro can differ significantly from the response in 3-dimension (3D) or at the actual tissue level. In this study, we cultured human epithelial cells in 3D to provide a more suitable model for human tissue. Human mammary epithelia cells (CH184B5F5/M10) were grown in Matrigel to form 3D structures, and exposed to Fe-ions at NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory or 137Cs-gamma radiation source at the University of Texas MD Anderson Cancer Center. After exposure, cells were allowed to repair for 16hr before dissociation and subcultued at low density in 2D. G2 and metaphase chromosomes in the first cell cycle were collected using a chemical-induced premature chromosome condensation (PCC) technique, and chromosome aberrations were analyzed using mBAND technique. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of interchromosomal aberrations (translocation to unpainted chromosomes) and intrachromosomal aberrations (inversions and deletions within a single painted chromosome). Our data indicate a significant difference of the chromosome aberration yield between 2D and 3D cell cultures for gamma exposures, but not for Fe ion exposures

  5. Escherichia coli lipopolysaccharide upregulates the expression of both toll like receptor 4 and 2 (TLR4 and TLR2) in cultured bovine mammary epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine mammary epithelial cells contribute to the innate immune response to intramammary infection. Their ability to mount such a response is dependent upon mammary epithelial recognition of the invading pathogen by specialized receptors. Toll-like receptor 4 (TLR4) is one such receptor that recog...

  6. Aldehyde dehydrogenase inhibitors: alpha,beta-acetylenic N-substituted aminothiolesters are reversible growth inhibitors of normal epithelial but irreversible apoptogens for cancer epithelial cells from human prostate in culture.

    PubMed

    Quash, Gerard; Fournet, Guy; Courvoisier, Charlotte; Martinez, Rosa M; Chantepie, Jacqueline; Paret, Marie Julie; Pharaboz, Julie; Joly-Pharaboz, Marie Odile; Goré, Jacques; André, Jean; Reichert, Uwe

    2008-05-01

    The pharmacomodulation of the N atom of alpha,beta-acetylenic aminothiolesters or the replacement of the thiolester moiety by more electrophilic groups did not permit any clear rationale to be established for improving the selective growth-inhibitory activity of this family of compounds over that of the previously synthesized alpha,beta-acetylenic aminothiolesters DIMATE and MATE [G. Quash, G. Fournet, J. Chantepie, J. Goré, C. Ardiet, D. Ardail, Y. Michal, U. Reichert, Biochem Pharmacol 64 (2002) 1279-92]. Hence DIMATE and MATE were investigated more thoroughly for selectivity and growth-inhibitory activity using human prostate epithelial normal cells (HPENC) on the one hand and human prostate epithelial cancer cells (DU145) on the other. Unequivocal evidence was obtained showing that both compounds were reversible growth inhibitors of HPENC but irreversible growth inhibitors of DU145. Growth-inhibition of DU145 was due to the induction of early apoptosis as revealed by the flow cytometric analytical profile of inhibitor-treated cells, of the decrease in the redox potential and increase in superoxide anion content of their mitochondria. Of the two intracellular enzymes: aldehyde dehydrogenases 1 and 3 (ALDH1 and ALDH3) targeted by DIMATE and MATE, ALDH3 was inhibited to the same extent by both compounds whereas ALDH1 was less susceptible to inhibition by MATE. As the induction of ALDH3 by xenobiotics is hormone-dependent, MATE, the more selective of the two inhibitors, is a useful tool not only for examining the role of the ALDH3 isoform in hormone-sensitive and resistant prostate cancer cells in culture but also for investigating if it can inhibit the growth of xenografts of prostate cancer in immunodeficient mice. PMID:17692435

  7. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    SciTech Connect

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah; Cho, Jin Won; Lee, Joon H.

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  8. 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...

  9. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  10. ONCOGENE ALTERNATIONS IN IN VITRO TRANSFORMED RAT TRACHEAL EPITHELIAL CELLS

    EPA Science Inventory

    Ten derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 non-tumorigenic cell line transformed by treatment with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP) and/or 12-0-tetradecanoylphor...

  11. Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells.

    PubMed

    Talbot, Sébastien; Lin, James Chi-Jen; Lahjouji, Karim; Roy, Jean-Philippe; Sénécal, Jacques; Morin, André; Couture, Réjean

    2011-07-01

    Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O₂(●⁻)) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O₂(●⁻) level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O₂(●⁻) level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg⁹-BK, 10 μM; 30 min) increased O₂(●⁻)production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O₂(●⁻) levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases. PMID:21600945

  12. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

    PubMed Central

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

    2002-01-01

    The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by

  13. Polarized Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Monolayers Have Higher Resistance to Oxidative Stress-Induced Cell Death Than Nonpolarized Cultures

    PubMed Central

    Hsiung, Jamie; Zhu, Danhong

    2015-01-01

    Oxidative stress-mediated injury to the retinal pigment epithelium (RPE) is a major factor involved in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. Human embryonic stem cell (hESC)-derived RPE cells are currently being evaluated for their potential for cell therapy in AMD patients through subretinal injection of cells in suspension and subretinal placement as a polarized monolayer. To gain an understanding of how transplanted RPE cells will respond to the highly oxidatively stressed environment of an AMD patient eye, we compared the survival of polarized and nonpolarized RPE cultures following oxidative stress treatment. Polarized, nonpolarized/confluent, nonpolarized/subconfluent hESC-RPE cells were treated with H2O2. Terminal deoxynucleotidyl transferase dUTP nick end labeling stains revealed the highest amount of cell death in subconfluent hESC-RPE cells and little cell death in polarized hESC-RPE cells with H2O2 treatment. There were higher levels of proapoptotic factors (phosphorylated p38, phosphorylated c-Jun NH2-terminal kinase, Bax, and cleaved caspase 3 fragments) in treated nonpolarized RPE—particularly subconfluent cells—relative to polarized cells. On the other hand, polarized RPE cells had constitutively higher levels of cell survival and antiapoptotic signaling factors such as p-Akt and Bcl-2, as well as antioxidants superoxide dismutase 1 and catalase relative to nonpolarized cells, that possibly contributed to polarized cells’ higher tolerance to oxidative stress compared with nonpolarized RPE cells. Subconfluent cells were particularly sensitive to oxidative stress-induced apoptosis. These results suggest that implantation of polarized hESC-RPE monolayers for treating AMD patients with geographic atrophy should have better survival than injections of hESC-RPE cells in suspension. PMID:25411476

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

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

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

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

    PubMed

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

    2014-09-01

    Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself and also can be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1α (HIF-1α), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1α was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production. PMID:24980968

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

    PubMed Central

    2009-01-01

    Background 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. Results 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. Conclusion 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. PMID:19883504

  17. 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

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

    PubMed Central

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

    2016-01-01

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

  19. Apparatus for measuring the finite load-deformation behavior of a sheet of epithelial cells cultured on a mesoscopic freestanding elastomer membrane

    NASA Astrophysics Data System (ADS)

    Selby, John C.; Shannon, Mark A.

    2007-09-01

    Details are given for the design, calibration, and operation of an apparatus for measuring the finite load-deformation behavior of a sheet of living epithelial cells cultured on a mesoscopic freestanding elastomer membrane, 10μm thick and 5mm in diameter. Although similar in concept to bulge tests used to investigate the mechanical properties of micromachined thin films, cell-elastomer composite diaphragm inflation tests pose a unique set of experimental challenges. Composite diaphragm (CD) specimens are extremely compliant (E<50kPa), experience large displacements when subject to small inflation pressures (˜100Pa), and must be continuously immersed in a bath of liquid culture medium during the acquisition of load-deformation measurements. Given these considerations, we have constructed an inflation apparatus consisting of an air-piston-cylinder pump integrated with a modular specimen mounting fixture that constitutes a horizontally semi-infinite reservoir of liquid culture medium. In a deformation-controlled inflation test, pressurized air is used to inflate a CD specimen into the liquid reservoir with minimum disturbance of the liquid-air interface. Piston displacements and absolute pump chamber air pressures are utilized as feedback to cycle the displaced (or inflated) CD volume V in a 0.05Hz triangular or sinusoidal wave form (VMIN=0μl, VMAX⩽40μl) while simultaneously recording the inflation pressure acting at the fixed boundary of the specimen, p(r =a). Using a carefully prescribed six-cycle inflation test protocol, the apparatus is shown to be capable of measuring the [V,p(r=a)] inflation response of a cell-elastomer CD with random uncertainties estimated at ±0.45μl and ±2.5Pa, respectively.

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

    PubMed

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

    2010-12-01

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

  1. Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

    PubMed

    Gangalum, Rajendra K; Bhat, Ankur M; Kohan, Sirus A; Bhat, Suraj P

    2016-06-17

    Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes. PMID:27129211

  2. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    SciTech Connect

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  3. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    DOEpatents

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  4. Effect of freezing on lens epithelial cell growth.

    PubMed

    Fukaya, Y; Hara, T; Hara, T; Iwata, S

    1988-05-01

    The effect of freezing on the growth of rat lens epithelial cells was studied in vitro. We found that 80% of the lens epithelial cells died after freezing at -45 degrees C for two hours and that the surviving cells could grow with the addition of growth factors or when placed on a sheet of type 4 collagen, but not when placed on a plain plastic culture dish. These results suggest that the surviving cells are at the Go phase of the cell cycle and that type 4 collagen or growth factors can initiate cell division. PMID:3294380

  5. Molecular responses of rat tracheal epithelial cells to transmembrane pressure.

    PubMed

    Ressler, B; Lee, R T; Randell, S H; Drazen, J M; Kamm, R D

    2000-06-01

    Smooth muscle constriction in asthma causes the airway to buckle into a rosette pattern, folding the epithelium into deep crevasses. The epithelial cells in these folds are pushed up against each other and thereby experience compressive stresses. To study the epithelial cell response to compressive stress, we subjected primary cultures of rat tracheal epithelial cells to constant elevated pressures on their apical surface (i.e., a transmembrane pressure) and examined changes in the expression of genes that are important for extracellular matrix production and maintenance of smooth muscle activation. Northern blot analysis of RNA extracted from cells subjected to transmembrane pressure showed induction of early growth response-1 (Egr-1), endothelin-1, and transforming growth factor-beta1 in a pressure-dependent and time-dependent manner. Increases in Egr-1 protein were detected by immunohistochemistry. Our results demonstrate that airway epithelial cells respond rapidly to compressive stresses. Potential transduction mechanisms of transmembrane pressure were also investigated. PMID:10835333

  6. Generation of Mouse Lung Epithelial Cells

    PubMed Central

    Kasinski, Andrea L.; Slack, Frank J.

    2016-01-01

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172. While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

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

    PubMed Central

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

    2015-01-01

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

  8. Short communication: Urea induces T helper 2 (Th2) type environment at transcriptional level and prostaglandin E2 secretion in bovine oviduct epithelial cells in culture.

    PubMed

    Kowsar, R; Marey, M A; Shimizu, T; Miyamoto, A

    2016-07-01

    Excess dietary protein intake in early lactation dairy cows resulting in blood urea nitrogen of greater than 19 to 20mg/dL is associated with decreased fertility. Little is known about the local interference of urea in the normal immunological environment of the oviduct that provides optimal conditions for early reproductive events. A bovine oviduct epithelial cell (BOEC) culture was used to determine how urea influences immune environment. The BOEC monolayer was supplemented with low (20mg/dL) and high (40mg/dL) concentrations of urea together with ovarian steroids, estradiol (1ng/mL) and progesterone (1ng/mL), and LH (10ng/mL) at concentrations observed during the preovulatory period. The urea values used in this study were equivalent to 9.3 and 18.7mg/dL of blood urea nitrogen, which are typically common in lactating dairy cows with low or high protein intake, respectively. Stimulation of BOEC with 40mg/dL of urea induced gene expression of IL10 and IL4, epithelial-derived T helper type 2-driving (anti-inflammatory) cytokines as well as mPGES-1 expression and prostaglandin E2 (PGE2) secretion. However, urea concentrations of both 20 and 40mg/dL failed to alter the expression of IL1B and TNFA, Th1-driving cytokines, and the gene expression of TLR4. However, a concentration of 40mg/dL of urea stimulated α 1-acid glycoprotein expression, an acute phase protein. Data from this in vitro study suggest that urea, at least in part, contributes to influence the expression of some immune-related genes toward T helper type 2 type and prostaglandin E2 secretion, leading to disruption in local environment for fertilization and early embryonic development. PMID:27132094

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

    PubMed

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

    2007-03-01

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

  10. Differentiation of porcine mesenchymal stem cells into epithelial cells as a potential therapeutic application to facilitate epithelial regeneration.

    PubMed

    Kokubun, Kelsey; Pankajakshan, Divya; Kim, Min-Jung; Agrawal, Devendra K

    2016-02-01

    Epithelial denudation is one of the characteristics of chronic asthma. To restore its functions, the airway epithelium has to rapidly repair the injuries and regenerate its structure and integrity. Mesenchymal stem cells (MSCs) have the ability to differentiate into many cell lineages. However, the differentiation of MSCs into epithelial cells has not been fully studied. Here, we examined the differentiation of MSCs into epithelial cells using three different media compositions with various growth supplementations. The MSCs were isolated from porcine bone marrow by density gradient centrifugation. The isolated MSCs were CD11(-) CD34(-) CD45(-) CD44(+) CD90(+) and CD105(+) by immunostaining and flow cytometry. MSCs were stimulated with EpiGRO (Millipore), BEpiCM (ScienCell) and AECGM (PromoCell) media for 5 and 10 days, and epithelial differentiation was assessed by qPCR (keratin 14, 18 and EpCAM), fluorometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM), western blot analysis (pancytokeratin, EpCAM) and flow cytometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM). The functional marker MUC1 was also assessed after 10 days of air-liquid interface (ALI) culture in optimized media. Cells cultured in BEpiCM containing fibroblast growth factor and prostaglandin E2 showed the highest expression of the epithelial markers: CK7-8 (85.90%); CK-14-15-16-19 (10.14%); and EpCAM (64.61%). The cells also expressed functional marker MUC1 after ALI culture. The differentiated MSCs when cultured in BEpiCM medium ex vivo in a bioreactor on a decellularized trachea for 10 days retained the epithelial-like phenotype. In conclusion, porcine bone marrow-derived MSCs demonstrate commitment to the epithelial lineage and might be a potential therapy for facilitating the repair of denuded airway epithelium. PMID:23696537

  11. Direct particle-to-cell deposition of coarse ambient particulate matter increases the production of inflammatory mediators from cultured human airway epithelial cells

    EPA Science Inventory

    Exposure of cultured cells to particulate matter air pollution is usually accomplished by collecting particles on a solid matrix, extracting the particles from the matrix, suspending them in liquid, and applying the suspension to cells grown on plastic and submerged in medium. Th...

  12. 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...

  13. Polycyclic aromatic hydrocarbon coated onto Fe(2)O(3) particles: assessment of cellular membrane damage and antioxidant system disruption in human epithelial lung cells (L132) in culture.

    PubMed

    Garçon, G; Shirali, P; Garry, S; Fontaine, M; Zerimech, F; Martin, A; Hannothiaux, M H

    2000-09-30

    The aim of this study was to investigate the oxidative effects of Fe(2)O(3), benzo(a)pyrene (B(a)P) and pyrene, alone or in association (B(a)P or pyrene coated onto Fe(2)O(3) particles), in normal human embryonic lung epithelial cells (L132) in culture. We evaluated: (i) membrane integrity, through fatty acid release (stearic acid, oleic acid, linoleic and linolenic acids, homolinolenic acid, arachidonic acid) and malondialdehyde (MDA) production; and (ii) antioxidant status, through enzymatic and non-enzymatic antioxidant defenses (superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione status, beta-carotene). Fe(2)O(3) did not induce any change in L132 cells. In pyrene-treated cells, SOD induction (P<0. 05), glutathione oxidation (P<0.05) and beta-carotene consumption (P<0.001) may counteract free radicals (FR)-induced damage. However, in B(a)P-incubated cells, SOD inactivation (P<0.05), GR increases (P<0.05), glutathione oxidation (P<0.05) and beta-carotene decreases (P<0.001) showed high disruption of antioxidants, thereby allowing FR-induced damage (i.e. arachidonic acid release, P<0.01; MDA production, P<0.01). Our main finding was that both associations caused higher FR-induced damage (i.e. MDA production, P<0.001; SOD inactivation, P<0.01) than either chemical alone. Several mechanisms could account for this result: enhanced uptake of Fe(2)O(3) particles and/or greater availability of polycyclic aromatic hydrocarbons (PAHs). We hypothesized also that Fe(2)O(3) and polycyclic aromatic hydrocarbons are more deleterious by virtue of their associations being able to produce higher oxidative effects than either chemical alone. PMID:11033230

  14. Inflammatory and Oxidative Stress Responses of an Alveolar Epithelial Cell Line to Airborne Zinc Oxide Nanoparticles at the Air-Liquid Interface: A Comparison with Conventional, Submerged Cell-Culture Conditions

    PubMed Central

    Lenz, Anke-Gabriele; Karg, Erwin; Brendel, Ellen; Hinze-Heyn, Helga; Maier, Konrad L.; Eickelberg, Oliver; Stoeger, Tobias; Schmid, Otmar

    2013-01-01

    The biological effects of inhalable nanoparticles have been widely studied in vitro with pulmonary cells cultured under submerged and air-liquid interface (ALI) conditions. Submerged exposures are experimentally simpler, but ALI exposures are physiologically more realistic and hence potentially biologically more meaningful. In this study, we investigated the cellular response of human alveolar epithelial-like cells (A549) to airborne agglomerates of zinc oxide (ZnO) nanoparticles at the ALI, compared it to the response under submerged culture conditions, and provided a quantitative comparison with the literature data on different types of particles and cells. For ZnO nanoparticle doses of 0.7 and 2.5 μg ZnO/cm2 (or 0.09 and 0.33 cm2 ZnO/cm2), cell viability was not mitigated and no significant effects on the transcript levels of oxidative stress markers (HMOX1, SOD-2 and GCS) were observed. However, the transcript levels of proinflammatory markers (IL-8, IL-6, and GM-CSF) were induced to higher levels under ALI conditions. This is consistent with the literature data and it suggests that in vitro toxicity screening of nanoparticles with ALI cell culture systems may produce less false negative results than screening with submerged cell cultures. However, the database is currently too scarce to draw a definite conclusion on this issue. PMID:23484138

  15. Mitochondrial "movement" and lens optics following oxidative stress from UV-B irradiation: cultured bovine lenses and human retinal pigment epithelial cells (ARPE-19) as examples.

    PubMed

    Bantseev, Vladimir; Youn, Hyun-Yi

    2006-12-01

    Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta

  16. Development of Na/sup +/-dependent hexose transport in cultured renal epithelial cells (LLC-PK/sub 1/)

    SciTech Connect

    Weiss, E.R.; Amsler, K.; Dawson, W.D.; Cook, J.S.

    1984-01-01

    A number of factors were explored to analyze how they interact to yield the increasing transport capacity in differentiating cell populations. These factors include the number of functional transporters in the population, the distribution of these transporters among the individual cells, the Na/sup +/ chemical gradient, the transmembrane potential, the pathways and activities of these pathways for efflux of glucoside, and cell-cell coupling between accumulating and non-accumulating cells. 35 references, 9 figures, 2 tables. (ACR)

  17. 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.

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

    SciTech Connect

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

    1997-10-13

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

  19. DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles

    PubMed Central

    Danielsen, Pernille Høgh; Loft, Steffen; Møller, Peter

    2008-01-01

    Background Exposure to air pollution particles has been acknowledged to be associated with excess generation of oxidative damage to DNA in experimental model systems and humans. The use of standard reference material (SRM), such as SRM1650 and SRM2975, is advantageous because experiments can be reproduced independently, but exposure to such samples may not mimic the effects observed after exposure to authentic air pollution particles. This study was designed to compare the DNA oxidizing effects of authentic street particles with SRM1650 and SRM2975. The authentic street particles were collected at a traffic intensive road in Copenhagen, Denmark. Results All of the particles generated strand breaks and oxidized purines in A549 lung epithelial cells in a dose-dependent manner and there were no overt differences in their potency. The exposures also yielded dose-dependent increase of cytotoxicity (as lactate dehydrogenase release) and reduced colony forming ability with slightly stronger cytotoxicity of SRM1650 than of the other particles. In contrast, only the authentic street particles were able to generate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in calf thymus DNA, which might be due to the much higher level of transition metals. Conclusion Authentic street particles and SRMs differ in their ability to oxidize DNA in a cell-free environment, whereas cell culture experiments indicate that the particle preparations elicit a similar alteration of the level of DNA damage and small differences in cytotoxicity. Although it cannot be ruled out that SRMs and authentic street particles might elicit different effects in animal experimental models, this study indicates that on the cellular level, SRM1650 and SRM2975 are suitable surrogate samples for the study of authentic street particles. PMID:18397523

  20. Proinflammatory responses of a hTERT-transformed, immortalized line of cultured bovine mammary epithelial cells (BME)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Primary cultures BME were generated from healthy mammary glands as described (Vet Immunol Immunopath 101(3-4):191-202, 2004). Towards immortalization, BME from four cows were pooled and transfected with pCI neo-hEST2-HA , a human telomerase segment containing a neomycin/Geneticin resistance select...

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

    PubMed Central

    VanDussen, Kelli L; Marinshaw, Jeffrey M; Shaikh, Nurmohammad; Miyoshi, Hiroyuki; Moon, Clara; Tarr, Phillip I; Ciorba, Matthew A; Stappenbeck, Thaddeus S

    2014-01-01

    Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualized medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (i.e., barrier function and host-microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n = 65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within two weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarized monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusion This culture system will facilitate the study of inter-individual, functional studies of human intestinal epithelial cells, including host-microbial interactions. PMID:25007816

  6. Similarities and differences between free 211At and 125I- transport in porcine thyroid epithelial cells cultured in bicameral chambers.

    PubMed

    Lindencrona, U; Nilsson, M; Forssell-Aronsson, E

    2001-01-01

    The transport and accumulation of free 211At and 125I- were investigated in thyrocytes cultured as monolayers in bicameral chambers under the influence of thyroid-stimulating hormone, stable iodide, ouabain and perchlorate. The results indicate that there are similarities and differences in the transport mechanisms of free 211At and 125I-. These results will be valuable in the development of radiation protection when handling and using 211At-labeled radiopharmaceuticals, and for the potential use of free 211At in radiation therapy of poorly differentiated thyroid cancer. PMID:11182563

  7. Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

    PubMed

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

    2014-11-01

    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 Ca(2+) 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

  8. Plasticity of Airway Epithelial Cell Transcriptome in Response to Flagellin

    PubMed Central

    Clark, Joan G.; Kim, Kyoung-Hee; Basom, Ryan S.; Gharib, Sina A.

    2015-01-01

    Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional response by microarray and RNA-sequencing. ELISA and RT-PCR were used to validate changes in select candidates. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. We conclude that in vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium. PMID:25668187

  9. Plasticity of airway epithelial cell transcriptome in response to flagellin.

    PubMed

    Clark, Joan G; Kim, Kyoung-Hee; Basom, Ryan S; Gharib, Sina A

    2015-01-01

    Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional response by microarray and RNA-sequencing. ELISA and RT-PCR were used to validate changes in select candidates. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. We conclude that in vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium. PMID:25668187

  10. Symmetry breaking mechanism for epithelial cell polarization

    NASA Astrophysics Data System (ADS)

    Veglio, A.; Gamba, A.; Nicodemi, M.; Bussolino, F.; Serini, G.

    2009-09-01

    In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system, we provide evidence that epithelial cell polarization is a chemical phase-separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase-separation scenario is likely common to many processes of cell polarity formation.

  11. 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. PMID:27189858

  12. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    PubMed Central

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

  13. Cell line-dependent cytotoxicity of poly(isobutylcyanoacrylate) nanoparticles coated with chitosan and thiolated chitosan: Insights from cultured human epithelial HeLa, Caco2/TC7 and HT-29/MTX cells.

    PubMed

    Pradines, Bénédicte; Lievin-Le Moal, Vanessa; Vauthier, Christine; Ponchel, Gilles; Loiseau, Philippe M; Bouchemal, Kawthar

    2015-08-01

    Nanoparticles composed of poly(isobutylcyanoacrylate) core coated with a mixture of chitosan and thiolated chitosan have already shown promising results in terms of mucoadhesion and permeation enhancement properties of pharmaceutical active drugs delivered via mucosal routes. In the present work, the cytotoxicity of these nanoparticles was first investigated using direct contact assay on undifferentiated human cervix epithelial HeLa cells. The results showed strong toxicity in HeLa cells for the two investigated concentrations 25 and 50 μg/mL. The cytotoxic effect was mainly attributed to the poly(isobutylcyanoacrylate) core since no significant differences in nanoparticle cytotoxicity were reported when nanoparticle shell composition was modified by adding chitosan or thiolated chitosan. In contrast, lower nanoparticle toxicity was reported using human fully-differentiated enterocyte-like Caco-2/TC7, and fully-differentiated mucus-secreting HT-29/MTX cells forming monolayer in culture mimicking an intestinal epithelial barrier. This study demonstrated that the toxicity of poly(isobutylcyanoacrylate) nanoparticles is highly cell line-dependent. PMID:26051544

  14. Proliferation of rhesus ovarian surface epithelial cells in culture: Lack of mitogenic response to steroid or gonadotropic hormones

    SciTech Connect

    Wright, Jay W.; Toth-Fejel, Suellen; Stouffer, Richard L.; Rodland, Karin D.

    2002-06-30

    Ovarian cancer is the most lethal gynecological cancer and approximately 90% of ovarian cancers derive from the ovarian surface epithelium (OSE), yet the biology of the OSE is poorly understood. Factors associated with increased risk of non-hereditary ovarian cancer include the formation of inclusion cysts, effects of reproductive hormones cytokeratin, vimentin, N-cadherin, E-cadherin, estrogen receptor-a, and progesterone receptor. We show that these cells activate MAP Kinase and proliferate in response to extracellular calcium, as do human and rat OSE. In contrast, the gonadotropic hormones FSH (4-400 IU/L), LH (8.5-850 IU/l), and hCG (10-1000 IU/l) fail to stimulate proliferation. We find that concentrations of progesterone and estrogen normally present in follicles just prior to ovulation ( ~1000 ng/ml) significantly decrease the number of mitotically active RhOSE cells as determined by PCNA labelling, total cell count, and 3H-thymidine uptake, while lower steroid concentrations have no effect.

  15. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing

    PubMed Central

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency. PMID:26673160

  16. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    PubMed

    Sun, Chi-Chin; Chiu, Hsiao-Ting; Lin, Yi-Fang; Lee, Kuo-Ying; Pang, Jong-Hwei Su

    2015-01-01

    Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency. PMID:26673160

  17. Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

    PubMed

    Pennington, Britney O; Clegg, Dennis O; Melkoumian, Zara K; Hikita, Sherry T

    2015-02-01

    Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies. PMID:25593208

  18. Small molecule and RNAi induced phenotype transition of expanded and primary colonic epithelial cells

    PubMed Central

    Sharbati, Jutta; Hanisch, Carlos; Pieper, Robert; Einspanier, Ralf; Sharbati, Soroush

    2015-01-01

    Recent progress in mammalian intestinal epithelial cell culture led to novel concepts of tissue modeling. Especially the development of phenotypically stable cell lines from individual animals enables an investigation of distinct intestinal loci and disease states. We here report primary and prolonged culture of normal porcine epithelial cells from colon for cell line development. In addition, a novel primary three-dimensional intestinal culture system is presented, which generated organoids composed of a highly polarized epithelial layer lining a core of subepithelial tissue. Cellular characterization of monolayer cell lines revealed epithelial identity and pointed to a proliferative crypt cell phenotype. We evaluated both RNAi and chemical approaches to induce epithelial differentiation in generated cell lines by targeting promoters of epithelial to mesenchymal transition (EMT). By in silico prediction and ectopic expression, miR-147b was proven to be a potent trigger of intestinal epithelial cell differentiation. Our results outline an approach to generate phenotypically stable cell lines expanded from primary colonic epithelial cultures and demonstrate the relevance of miR-147b and chemical inhibitors for promoting epithelial differentiation features. PMID:26223582

  19. Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells.

    PubMed

    Jeng, Kuo-Shyang; Jeng, Chi-Juei; Jeng, Wen-Juei; Sheen, I-Shyan; Li, Shih-Yun; Hung, Zih-Hang; Hsiau, Hsin-I; Yu, Ming-Che; Chang, Chiung-Fang

    2016-03-01

    Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy. PMID:26647726

  20. Comparing acute toxicity of gunshot particles, from firing conventional and lead-free ammunition, in pulmonary epithelial cell cultures.

    PubMed

    Bergström, Ulrika; Ekstrand-Hammarström, Barbro; Hägglund, Lars; Wingfors, Håkan

    2015-01-01

    Numerous studies demonstrated that the use of lead (Pb)-containing ammunition is associated with mainly chronic health problems and also is a burden on the environment and wildlife. Recently, a number of reports showed evidence of undesirable acute health effects related to the use of newly developed Pb-free small-caliber ammunition. In this study, particles from leaded and Pb-free ammunition were collected in liquid collection medium, in a highly controlled chamber, while firing a pistol (9 mm) or a rifle (7.62 × 51 mm). The emitted particles were typically smaller than 4 μm, with the great majority in even smaller size ranges, as shown by gravimetrical analysis and a multistage impactor. Chemical analysis revealed significant differences in content and concentration of several metals in the particles. After administration of the liquids to alveolar and bronchial in vitro cell systems, particles were taken up by the cells; the Pb-free particles displayed higher cytotoxicity (EC50 = 2 μg/cm(2)) than particles from Pb ammunition. High correlation factors (>0.9) were found between cell death and content of copper and zinc. Particles from both Pb-containing and Pb-free ammunition were able to induce oxidative stress and the proinflammatory marker interleukin (IL)-8 in both in vitro systems. These results support previous findings that indicate an association between gunshot emissions and metal fume fever. This study demonstrates the usefulness of combining chemical data with biological in vitro responses in assessing acute toxicological effects from emissions from firing both Pb and Pb-free ammunition. PMID:26039682

  1. Cytotoxicity of the HpmA hemolysin and urease of Proteus mirabilis and Proteus vulgaris against cultured human renal proximal tubular epithelial cells.

    PubMed

    Mobley, H L; Chippendale, G R; Swihart, K G; Welch, R A

    1991-06-01

    Proteus mirabilis, a common agent of nosocomially acquired and catheter-associated bacteriuria, can cause acute pyelonephritis. In ascending infections, bacteria colonize the bladder and ascend the ureters to the proximal tubules of the kidney. We postulate that Proteus species uses the HpmA hemolysin and urease to elicit tissue damage that allows entry of these bacteria into the kidney. To study this interaction, strains of Proteus mirabilis and P. vulgaris and their isogenic hemolysin-negative (hpmA) or isogenic urease-negative (ureC) constructs were overlaid onto cultures of human renal proximal tubular epithelial cells (HRPTEC) isolated from kidneys obtained by immediate autopsy. Cytotoxicity was measured by release of soluble lactate dehydrogenase (LDH). Two strains of P. mirabilis inoculated at 10(6) CFU caused a release of 80% of total LDH after 6 h, whereas pyelonephritogenic hemolytic Escherichia coli CFT073 released only 25% at 6 h (P less than 0.012). Ten P. mirabilis isolates and five P. vulgaris isolates were all hemolytic and cytotoxic and produced urease which was induced by urea. The HpmA hemolysin is apparently responsible for the majority of cytotoxicity in vitro since the hemolysin-negative (hpmA) mutants of P. mirabilis and P. vulgaris were significantly less cytotoxic than wild-type strains. P. mirabilis WPM111 (hemolysin negative) was used to test the effect of urease-catalyzed urea hydrolysis on HRPTEC viability. In the presence of 50 mM urea, WPM111 caused the release of 42% of LDH versus 1% at 6 h in the absence of substrate (P = 0.003). We conclude that the HpmA hemolysin of Proteus species acts as a potent cytotoxin against HRPTEC. In addition, urease apparently contributes to this process when substrate urea is available. PMID:2037363

  2. 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

  3. Porphyromonas gingivalis invades oral epithelial cells in vitro.

    PubMed

    Sandros, J; Papapanou, P; Dahlén, G

    1993-05-01

    The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells. PMID:8388449

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

    PubMed

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

    2016-02-01

    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. PMID:26859350

  5. Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced Pluripotent Stem Cells

    PubMed Central

    Yoshida, Satoru; Yasuda, Miyuki; Miyashita, Hideyuki; Ogawa, Yoko; Yoshida, Tetsu; Matsuzaki, Yumi; Tsubota, Kazuo; Okano, Hideyuki; Shimmura, Shigeto

    2011-01-01

    Background Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. Methodology/Principal Findings We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. Conclusions/Significance These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets. PMID:22174914

  6. Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

    PubMed Central

    Zhong, Aimei; Wang, Guohua; Yang, Jie; Xu, Qijun; Yuan, Quan; Yang, Yanqing; Xia, Yun; Guo, Ke; Horch, Raymund E; Sun, Jiaming

    2014-01-01

    True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. PMID:24720804

  7. 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. PMID:26367485

  8. Advances in cell culture

    SciTech Connect

    Maramorosch, K. )

    1987-01-01

    This book presents papers on advances in cell culture. Topics covered include: Genetic changes in the influenza viruses during growth in cultured cells; The biochemistry and genetics of mosquito cells in culture; and Tree tissue culture applications.

  9. 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

  10. 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. PMID

  11. 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. PMID:25239531

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

  13. Macrophages promote benzopyrene-induced tumor transformation of human bronchial epithelial cells by activation of NF-κB and STAT3 signaling in a bionic airway chip culture and in animal models

    PubMed Central

    Sun, Zhao; Wang, Lei; Guo, Zhe; Zhao, Yang; Gao, Zhancheng; Wang, Qi

    2015-01-01

    We investigated the role of macrophages in promoting benzopyrene (BaP)-induced malignant transformation of human bronchial epithelial cells using a BaP-induced tumor transformation model with a bionic airway chip in vitro and in animal models. The bionic airway chip culture data showed that macrophages promoted BaP-induced malignant transformation of human bronchial epithelial cells, which was mediated by nuclear factor (NF)-κB and STAT3 pathways to induce cell proliferation, colony formation in chip culture, and tumorigenicity in nude mice. Blockage of interleukin (IL)-6 or tumor necrosis factor (TNF)-α signaling or inhibition of NF-κB, STAT3, or cyclinD1 expression abrogated the effect of macrophages on malignant transformation in the bionic airway chip culture. In vivo, macrophages promoted lung tumorigenesis in a carcinogen-induced animal model. Similarly, blockage of NF-κB, STAT3, or cyclinD1 using siRNA transfection decreased the carcinogen-induced tumorigenesis in rats. We demonstrated that macrophages are critical in promoting lung tumorigenesis and that the macrophage-initiated TNF-α/NF-κB/cyclinD1 and IL-6/STAT3/cyclinD1 pathways are primarily responsible for promoting lung tumorigenesis. PMID:25823926

  14. CYTOTOXICITY OF CHEMICAL CARCINOGENS TOWARDS HUMAN BRONCHIAL EPITHELIAL CELLS EVALUATED IN A CLONAL ASSAY

    EPA Science Inventory

    Survival of human bronchial epithelial cells after administration of four chemical carcinogens was measured in a clonal assay. Human bronchial epithelial cells were obtained from outgrowths of explanted tissue pieces. Serum-free medium was used for both explant culture and clonal...

  15. Intestinal anti-inflammatory activity of Sasa quelpaertensis leaf extract by suppressing lipopolysaccharide-stimulated inflammatory mediators in intestinal epithelial Caco-2 cells co-cultured with RAW 264.7 macrophage cells

    PubMed Central

    Kim, Kyung-Mi; Kim, Yoo-Sun; Lim, Ji Ye; Min, Soo Jin; Ko, Hee-Chul; Kim, Se-Jae

    2015-01-01

    BACKGROUND/OBJECTIVES Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, involves chronic inflammation of the gastrointestinal tract. Previously, Sasa quelpaertensis leaves have been shown to mediate anti-inflammation and anti-cancer effects, although it remains unclear whether Sasa leaves are able to attenuate inflammation-related intestinal diseases. Therefore, the aim of this study was to investigate the anti-inflammatory effects of Sasa quelpaertensis leaf extract (SQE) using an in vitro co-culture model of the intestinal epithelial environment. MATERIALS/METHODS An in vitro co-culture system was established that consisted of intestinal epithelial Caco-2 cells and RAW 264.7 macrophages. Treatment with lipopolysaccharide (LPS) was used to induce inflammation. RESULTS Treatment with SQE significantly suppressed the secretion of LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, and IL-1β in co-cultured RAW 264.7 macrophages. In addition, expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α were down-regulated in response to inhibition of IκBα phosphorylation by SQE. Compared with two bioactive compounds that have previously been identified in SQE, tricin and P-coumaric acid, SQE exhibited the most effective anti-inflammatory properties. CONCLUSIONS SQE exhibited intestinal anti-inflammatory activity by inhibiting various inflammatory mediators mediated through nuclear transcription factor kappa-B (NF-kB) activation. Thus, SQE has the potential to ameliorate inflammation-related diseases, including IBD, by limiting excessive production of pro-inflammatory mediators. PMID:25671061

  16. Bleomycin (BLM) Induces Epithelial-to-Mesenchymal Transition in Cultured A549 Cells via the TGF-β/Smad Signaling Pathway

    PubMed Central

    Chen, Kui-Jun; Li, Qing; Wen, Cang-mei; Duan, Zhao-Xia; Zhang, Jie yuan; Xu, Chuan; Wang, Jian-Min

    2016-01-01

    The epithelial-to-mesenchymal transition (EMT) is a crucial cellular event in wound healing, tissue repair, and cancer progression in adult tissues, with the interactions with numerous signals. In this study, we aimed to determine whether bleomycin (BLM), an agent that causes pulmonary fibrosis, induces the EMT of the alveolar epithelial cell line A549 and investigated the possible mechanisms. We examined the EMT involved changes in cell morphology, isoform switching of the fibroblast growth factor receptor 2 (FGFR2) by alternative splicing, and expression of the phenotypic markers including E-cadherin, vimentin, and α-SMA using RT-PCR, Western blotting, and immunofluorescence assays. A TGF-β/Smad inhibitor was used to determine whether coculture with BLM would inhibit the EMT of A549 cells. The results showed that BLM induced the EMT of A549 cells possibly via the TGF-β/Smad signaling pathway, evident from the decrease in the expression of E-cadherin and increase in the expression on vimentin. PMID:27471572

  17. Human Papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

    PubMed Central

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

    2012-01-01

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone. PMID:22284893

  18. Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

    SciTech Connect

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

    2012-03-30

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

  19. 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

  20. 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

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

    PubMed

    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

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

    PubMed

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

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. PMID:25546438

  3. Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells

    PubMed Central

    Han, Yiping W.; Shi, Wenyuan; Huang, George T.-J.; Kinder Haake, Susan; Park, No-Hee; Kuramitsu, Howard; Genco, Robert J.

    2000-01-01

    Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections. PMID:10816455

  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. PMID:26649819

  6. Survivin expression is associated with lens epithelial cell proliferation and fiber cell differentiation

    PubMed Central

    Mansergh, Fiona C.; Boulton, Michael E.; Gunhaga, Lena

    2012-01-01

    Purpose Survivin (Birc5) is the smallest member of the inhibitor of apoptosis (IAP) protein family, which regulates the cell cycle/apoptosis balance. The purpose of this study was to examine Survivin expression in the embryonic chick lens, in chick lens epithelial cell cultures, and in the postnatal mouse lens. Methods Survivin expression was examined using a combination of quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. To correlate Survivin expression with the timing of proliferation, we determined the profile of cell proliferation in the developing lens using the cell cycle marker proliferating cell nuclear antigen (PCNA) in quantitative western blotting and immunocytochemistry studies. We also examined the expression of PCNA and the extent of denucleation using terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labeling (TUNEL) of lentoids (lens fiber-like cells) during chick lens epithelial cell differentiation in vitro. Results At embryonic day (ED) 4, Survivin immunostaining was present in two pools in lens epithelial cells and fiber cells: cytoplasmic and nuclear. The nuclear staining became more pronounced as the lens epithelial cells differentiated into lens fiber cells. At ED12, Survivin staining was observed in lens fiber cell nuclei containing marginalized chromatin, indicative of early denucleation events. Using western blotting, Survivin expression peaked at ED6, diminishing thereafter. This profile of expression correlated with the events in chick lens epithelial cell cultures: i) increased Survivin expression was associated with an increase in PCNA staining up to day 6 of culture and ii) downregulation of Survivin expression at day 8 of culture was coincident with a dramatic decrease in PCNA staining and an increase in TdT-mediated biotin-dUTP nick-end labeling in lentoids. In early postnatal mouse lenses, Survivin and PCNA were highly expressed and decreased thereafter during

  7. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  8. Epithelial Cell Shedding and Barrier Function

    PubMed Central

    Williams, J. M.; Duckworth, C. A.; Burkitt, M. D.; Watson, A. J. M.; Campbell, B. J.

    2015-01-01

    The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. PMID:25428410

  9. 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...

  10. Epithelial Cell Regulation of Allergic Diseases.

    PubMed

    Gour, Naina; Lajoie, Stephane

    2016-09-01

    Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental stimuli. Why only certain individuals mount inappropriate type 2 immune responses to these otherwise harmless allergens has remained an unanswered question. Mounting evidence suggests that the epithelium, by sensing its environment, is the central regulator of allergic diseases. Once considered to be a passive barrier to allergens, epithelial cells at mucosal surfaces are now considered to be the cornerstone of the allergic diathesis. Beyond their function as maintaining barrier at mucosal surfaces, mucosal epithelial cells through the secretion of mediators like IL-25, IL-33, and TSLP control the fate of downstream allergic immune responses. In this review, we will discuss the advances in recent years regarding the process of allergen recognition and secretion of soluble mediators by epithelial cells that shape the development of the allergic response. PMID:27534656

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

    EPA Science Inventory

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

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

    PubMed

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

    2016-03-01

    A major cause of respiratory failure during influenza A virus (IAV) infection is damage to the epithelial-endothelial barrier of the pulmonary alveolus. Damage to this barrier results in flooding of the alveolar lumen with proteinaceous oedema fluid, erythrocytes and inflammatory cells. To date, the exact roles of pulmonary epithelial and endothelial cells in this process remain unclear.Here, we used an in vitro co-culture model to understand how IAV damages the pulmonary epithelial-endothelial barrier. Human epithelial cells were seeded on the upper half of a transwell membrane while human endothelial cells were seeded on the lower half. These cells were then grown in co-culture and IAV was added to the upper chamber.We showed that the addition of IAV (H1N1 and H5N1 subtypes) resulted in significant barrier damage. Interestingly, we found that, while endothelial cells mounted a pro-inflammatory/pro-coagulant response to a viral infection in the adjacent epithelial cells, damage to the alveolar epithelial-endothelial barrier occurred independently of endothelial cells. Rather, barrier damage was associated with disruption of tight junctions amongst epithelial cells, and specifically with loss of tight junction protein claudin-4.Taken together, these data suggest that maintaining epithelial cell integrity is key in reducing pulmonary oedema during IAV infection. PMID:26743480

  13. Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues.

    PubMed

    Chen, W T; Greve, J M; Gottlieb, D I; Singer, S J

    1985-06-01

    A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions. PMID:3889142

  14. Immunolocalization of thymosin alpha 1, thymopoietin and thymulin in mouse thymic epithelial cells at different stages of culture: a light and electron microscopic study.

    PubMed Central

    Fabien, N; Auger, C; Monier, J C

    1988-01-01

    The secretory evolution of the thymic hormones (thymulin, thymosin alpha 1 and thymopoietin) in cultured thymic reticuloepithelial cells (TREC) was studied by immunocytochemical techniques using monoclonal anti-thymulin or anti-thymosin alpha 1 and polyclonal anti-thymopoietin antibodies (Ab). The culture of TREC was performed with a medium where L-valine was replaced by D-valine, thus ensuring rapid and selective development of these cells. The number of thymulin, thymosin alpha 1 or thymopoietin-containing cells increased progressively from Day 6 to Day 12 of the culture. The localization of the three thymic hormones within the TREC also varied according to the age of the culture. By light microscopy the staining of the three hormones was localized in some cytoplasmic granules at the beginning of the culture and at Day 90, while at Day 12 it was throughout the cytoplasm. In electron microscopy these localizations corresponded respectively to vacuoles of different sizes and to cytosol. All these results show that the synthesis and excretion of thymulin, thymosin alpha 1 and thymopoietin evolve during the development of TREC in culture. Images Figure 1 Figure 2 PMID:3284819

  15. Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

    PubMed

    Pickard, Adam; Cichon, Ann-Christin; Barry, Anna; Kieran, Declan; Patel, Daksha; Hamilton, Peter; Salto-Tellez, Manuel; James, Jacqueline; McCance, Dennis J

    2012-07-18

    Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. PMID:22643222

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

    PubMed

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

    2012-08-01

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

  17. Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

    PubMed

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2014-09-12

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  18. Control of cell mechanics by RhoA and calcium fluxes during epithelial scattering.

    PubMed

    Haws, Hillary J; McNeil, Melissa A; Hansen, Marc D H

    2016-01-01

    Epithelial tissues use adherens junctions to maintain tight interactions and coordinate cellular activities. Adherens junctions are remodeled during epithelial morphogenesis, including instances of epithelial-mesenchymal transition, or EMT, wherein individual cells detach from the tissue and migrate as individual cells. EMT has been recapitulated by growth factor induction of epithelial scattering in cell culture. In culture systems, cells undergo a highly reproducible series of cell morphology changes, most notably cell spreading followed by cellular compaction and cell migration. These morphology changes are accompanied by striking actin rearrangements. The current evidence suggests that global changes in actomyosin-based cellular contractility, first a loss of contractility during spreading and its activation during cell compaction, are the main drivers of epithelial scattering. In this review, we focus on how spreading and contractility might be controlled during epithelial scattering. While we propose a central role for RhoA, which is well known to control cellular contractility in multiple systems and whose role in epithelial scattering is well accepted, we suggest potential roles for additional cellular systems whose role in epithelial cell biology has been less well documented. In particular, we propose critical roles for vesicle recycling, calcium channels, and calcium-dependent kinases. PMID:27583192

  19. Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds.

    PubMed

    Shojaie, Sharareh; Lee, Joyce; Wang, Jinxia; Ackerley, Cameron; Post, Martin

    2016-01-01

    Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis. PMID:27214388

  20. Primary in vitro culture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium and Aspergillus fumigatus interactions.

    PubMed

    Khoufache, Khaled; Cabaret, Odile; Farrugia, Cécile; Rivollet, Danièle; Alliot, Annie; Allaire, Eric; Cordonnier, Catherine; Bretagne, Stéphane; Botterel, Françoise

    2010-12-01

    Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus. PMID:20608777

  1. An in vitro model of epithelial cell growth stimulation in the rodent mammary gland.

    PubMed

    Ehmann, U K; DeVries, J T; Chen, M S C; Adamos, A A; Guzman, R C; Omary, M B

    2003-08-01

    Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFalpha and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro-in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another. PMID:12950387

  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. Preparation and culture of rat lens epithelial explants for studying terminal differentiation.

    PubMed

    Zelenka, Peggy S; Gao, Chun Y; Saravanamuthu, Senthil S

    2009-01-01

    The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body. Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens. Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2. Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers. The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100 ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied. Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques. Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways. PMID:19773734

  4. 3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion

    PubMed Central

    Nguyen-Ngoc, Kim-Vy; Shamir, Eliah R.; Huebner, Robert J.; Beck, Jennifer N.; Cheung, Kevin J.; Ewald, Andrew J.

    2016-01-01

    Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called “organoids,” to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems. PMID:25245692

  5. 3D culture assays of murine mammary branching morphogenesis and epithelial invasion.

    PubMed

    Nguyen-Ngoc, Kim-Vy; Shamir, Eliah R; Huebner, Robert J; Beck, Jennifer N; Cheung, Kevin J; Ewald, Andrew J

    2015-01-01

    Epithelia are fundamental tissues that line cavities, glands, and outer body surfaces. We use three-dimensional (3D) embedded culture of primary murine mammary epithelial ducts, called "organoids," to recapitulate in days in culture epithelial programs that occur over weeks deep within the body. Modulating the composition of the extracellular matrix (ECM) allows us to model cell- and tissue-level behaviors observed in normal development, such as branching morphogenesis, and in cancer, such as invasion and dissemination. Here, we describe a collection of protocols for 3D culture of mammary organoids in different ECMs and for immunofluorescence staining of 3D culture samples and mammary gland tissue sections. We illustrate expected phenotypic outcomes of each assay and provide troubleshooting tips for commonly encountered technical problems. PMID:25245692

  6. Respiratory epithelial cells orchestrate pulmonary innate immunity.

    PubMed

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

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

  7. Differentiated kidney epithelial cells repair injured proximal tubule.

    PubMed

    Kusaba, Tetsuro; Lalli, Matthew; Kramann, Rafael; Kobayashi, Akio; Humphreys, Benjamin D

    2014-01-28

    Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER(T2) cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair. PMID:24127583

  8. Notch Signaling in Meibomian Gland Epithelial Cell Differentiation

    PubMed Central

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

    2016-01-01

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

  9. Characterization of a unique technique for culturing primary adult human epithelial progenitor/“stem cells”

    PubMed Central

    2012-01-01

    Background Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that rapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty acid free, low calcium medium that is replaced every 24 hrs. Methods Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of tissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely confluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested every 24 hrs. over a 7–10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask to form another confluent monolayer over 2–4 days. This new culture, in turn produced additional cell suspensions that when serially passed expand the cell strain over 2–3 months, without the use of enzymes to split the cultures. The cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size and glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte differentiation, and characterized by immunohistochemistry. Results The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used approximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells (8–10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and 15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The ePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing). Conclusions Using this primary culture technique, large quantities of epithelial cells can

  10. Zinc modulates cytokine-induced lung epithelial cell barrier permeability.

    PubMed

    Bao, Shenying; Knoell, Daren L

    2006-12-01

    Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury. PMID:16844947

  11. Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris.

    PubMed

    Sweat JMDunigan, D D; Wright, S D

    2001-06-01

    The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 x 10(3) cells/cm2, and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells. PMID:11515973

  12. 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. PMID:26477404

  13. Mechanism of initial attachment of corneal epithelial cells to polymeric surfaces.

    PubMed

    Steele, J G; Johnson, G; Griesser, H J; Underwood, P A

    1997-12-01

    The initial attachment of cultured bovine corneal epithelial cells and stromal fibroblasts to two oxygen-containing synthetic polymers was studied. Cultured epithelial cells and stromal fibroblasts were seeded onto two oxygen-containing surfaces: 'tissue culture' polystyrene (TCPS) and a polymer film deposited by RF plasma deposition using a methylmethacrylate monomer (MMA/FEP). To establish the mechanism of cell attachment, the effect of the selective removal of the vitronectin and fibronectin from the serum used in the culture medium was tested. The attachment of cultured epithelial cells during the first 90 min of culture was reduced by 40% (TCPS)-80% (MMA/FEP) as a result of removing vitronectin from the medium. Attachment of these cells to TCPS was reduced by 85-95% when the serum was depleted of both fibronectin and vitronectin. However, depletion of fibronectin reduced cell attachment to TCPS by 20%, whilst on MMA/FEP cell attachment was equivalent, or higher, than that for intact serum. The attachment of cultured corneal stromal fibroblasts was similarly dependent on vitronectin but less dependent on fibronectin. Therefore, for the attachment of both cultured epithelial cells and fibroblasts to oxygen-containing surfaces in the presence of serum, there is a high requirement for serum vitronectin but a lesser requirement for fibronectin. The effects of the establishment of corneal epithelial cells in culture and the site of origin of the cells, were determined. Primary isolates of epithelial cells isolated from the limbal, central or peripheral regions of the cornea were less dependent on vitronectin for initial attachment to TCPS than were these cells after several passages in culture. Furthermore, the primary isolates were dramatically less responsive to vitronectin than the cultured cells. These results indicate that the mechanism of attachment of corneal epithelial cells to TCPS varies with the culture experience of the cells. Cells that are culture

  14. 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

  15. Comparison of functional limbal epithelial stem cell isolation methods.

    PubMed

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Dziasko, Marc; Galindo, Sara; Rey, Esther; Herreras, José M; Corrales, Rosa M; Daniels, Julie T; Calonge, Margarita

    2016-05-01

    The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either

  16. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

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

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

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

    PubMed Central

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

    2015-01-01

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

  18. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  19. AGE-RAGE interaction in the TGFβ2-mediated epithelial to mesenchymal transition of human lens epithelial cells.

    PubMed

    Raghavan, Cibin T; Nagaraj, Ram H

    2016-08-01

    Basement membrane (BM) proteins accumulate chemical modifications with age. One such modification is glycation, which results in the formation of advanced glycation endproducts (AGEs). In a previous study, we reported that AGEs in the human lens capsule (BM) promote the TGFβ2-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells, which we proposed as a mechanism for posterior capsule opacification (PCO) or secondary cataract formation. In this study, we investigated the role of a receptor for AGEs (RAGE) in the TGFβ2-mediated EMT in a human lens epithelial cell line (FHL124). RAGE was present in FHL124 cells, and its levels were unaltered in cells cultured on either native or AGE-modified BM or upon treatment with TGFβ2. RAGE overexpression significantly enhanced the TGFβ2-mediated EMT responses in cells cultured on AGE-modified BM compared with the unmodified matrix. In contrast, treatment of cells with a RAGE antibody or EN-RAGE (an endogenous ligand for RAGE) resulted in a significant reduction in the TGFβ2-mediated EMT response. This was accompanied by a reduction in TGFβ2-mediated Smad signaling and ROS generation. These results imply that the interaction of matrix AGEs with RAGE plays a role in the TGFβ2-mediated EMT of lens epithelial cells and suggest that the blockade of RAGE could be a strategy to prevent PCO and other age-associated fibrosis. PMID:27263094

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

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

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

  1. Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection

    PubMed Central

    Wu, Y-Y; Lin, C-W; Cheng, K-S; Lin, C; Wang, Y-M; Lin, I-T; Chou, Y-H; Hsu, P-N

    2010-01-01

    B7-H1 [programmed death-ligand-1 (PD-L1)] is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD-L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD-L1 expression, regulation and function during Helicobacter pylori infection. PD-L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co-culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD-L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon-γ or tumour necrosis factor-α. Moreover, PD-L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD-L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach. PMID:20646001

  2. Guiding Epithelial Cell Phenotypes with Engineered Integrin-Specific Recombinant Fibronectin Fragments

    PubMed Central

    Brown, Ashley C.; Rowe, Jessica A.

    2011-01-01

    The extracellular matrix (ECM) provides important cues for directing cell phenotype. Cells interact with underlying ECM through cell-surface receptors known as integrins, which bind to specific sequences on their ligands. During tissue development, repair, and regeneration of epithelial tissues, cells must interact with an interstitial fibronectin (Fn)-rich matrix, which has been shown to direct a more migratory/repair phenotype, presumably through interaction with Fn’s cell binding domain comprised of both synergy Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) sequences. We hypothesized that the Fn synergy site is critical to the regulation of epithelial cell phenotype by directing integrin specificity. Epithelial cells were cultured on Fn fragments displaying stabilized synergy and RGD (FnIII9’10), or RGD alone (FnIII10) and cell phenotype analyzed by cytoskeleton changes, epithelial cell–cell contacts, changes in gene expression of epithelial and mesenchymal markers, and wound healing assay. Data indicate that epithelial cells engage RGD only with αv integrins and display a significant shift toward a mesenchymal phenotype due, in part, to enhanced transforming growth factor-β activation and/or signaling compared with cells on the synergy containing FnIII9’10. These studies demonstrate the importance of synergy in regulating epithelial cell phenotype relevant to tissue engineering as well as the utility of engineered integrin-specific ECM fragments in guiding cell phenotype. PMID:20695776

  3. Silk Fibroin as a Biomaterial Substrate for Corneal Epithelial Cell Sheet Generation

    PubMed Central

    Liu, Jingbo; Lawrence, Brian D.; Liu, Aihong; Schwab, Ivan R.; Oliveira, Lauro A.; Rosenblatt, Mark I.

    2012-01-01

    Purpose. To evaluate a silk fibroin (SF) biomaterial as a substrate for corneal epithelial cell proliferation, differentiation, and stratification in vitro compared with denuded human amniotic membrane (AM). Methods. Primary human and rabbit corneal epithelial cells and immortalized human corneal limbal epithelial cells were cultured on the SF and denuded AM, respectively. The biological cell behavior, including the morphology, proliferation, differentiation, and stratification, on the two substrates was compared and analyzed. Results. Corneal epithelial cells can adhere and proliferate on the SF and denuded AM with a cobblestone appearance, abundant microvilli on the surface, and wide connection with the adjacent cells. MTT assay showed that cell proliferation on denuded AM was statistically higher than that on SF at 24 and 72 hours after plating (P = 0.001 and 0.0005, respectively). Expression of ΔNp63a and keratin 3/12 was detected in primary cell cultures on the two substrates with no statistical difference. When cultured at the air-liquid interface for 7 days, cells on SF could form a comparable stratified graft with a 2- to 3-cell layering, which compared similarly to AM cultures. Conclusions. SF, a novel biomaterial, could support corneal epithelial cells to proliferate, differentiate, and stratify, retaining the normal characteristic epithelium phenotype. Compared with AM, its unique features, including the transparency, ease of handling, and transfer, and inherent freedom from disease transmission, make it a promising substrate for corneal wound repair and tissue-engineering purposes. PMID:22661480

  4. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  5. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  6. Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD

    PubMed Central

    Deslee, Gaetan; Dury, Sandra; Perotin, Jeanne M; Al Alam, Denise; Vitry, Fabien; Boxio, Rachel; Gangloff, Sophie C; Guenounou, Moncef; Lebargy, François; Belaaouaj, Abderrazzaq

    2007-01-01

    Background Chronic obstructive pulmonary disease (COPD) is characterized by abnormal lung inflammation that exceeds the protective response. Various culture models using epithelial cell lines or primary cells have been used to investigate the contribution of bronchial epithelium in the exaggerated inflammation of COPD. However, these models do not mimic in vivo situations for several reasons (e.g, transformed epithelial cells, protease-mediated dissociation of primary cells, etc.). To circumvent these concerns, we developed a new epithelial cell culture model. Methods Using non transformed non dissociated bronchial epithelium obtained by bronchial brushings from COPD and non-COPD smokers, we developed a 3-dimensional culture model, bronchial epithelial spheroids (BES). BES were analyzed by videomicroscopy, light microscopy, immunofluorescence, and transmission electron microscopy. We also compared the inflammatory responses of COPD and non-COPD BES. In our study, we chose to stimulate BES with lipopolycaccharide (LPS) and measured the release of the pro-inflammatory mediators interleukin-8 (IL-8) and leukotriene B4 (LTB4) and the anti-inflammatory mediator prostaglandin E2 (PGE2). Results BES obtained from both COPD and non-COPD patients were characterized by a polarized bronchial epithelium with tight junctions and ciliary beating, composed of basal cells, secretory cells and ciliated cells. The ciliary beat frequency of ciliated cells was not significantly different between the two groups. Of interest, BES retained their characteristic features in culture up to 8 days. BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS. Interestingly, LPS induced a higher release of IL-8, but not PGE2 and LTB4 in COPD BES (p < 0.001) which correlated with lung function changes. Conclusion This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium. More

  7. Deletion of Lkb1 in Renal Tubular Epithelial Cells Leads to CKD by Altering Metabolism.

    PubMed

    Han, Seung Hyeok; Malaga-Dieguez, Laura; Chinga, Frank; Kang, Hyun Mi; Tao, Jianling; Reidy, Kimberly; Susztak, Katalin

    2016-02-01

    Renal tubule epithelial cells are high-energy demanding polarized epithelial cells. Liver kinase B1 (LKB1) is a key regulator of polarity, proliferation, and cell metabolism in epithelial cells, but the function of LKB1 in the kidney is unclear. Our unbiased gene expression studies of human control and CKD kidney samples identified lower expression of LKB1 and regulatory proteins in CKD. Mice with distal tubule epithelial-specific Lkb1 deletion (Ksp-Cre/Lkb1(flox/flox)) exhibited progressive kidney disease characterized by flattened dedifferentiated tubule epithelial cells, interstitial matrix accumulation, and dilated cystic-appearing tubules. Expression of epithelial polarity markers β-catenin and E-cadherin was not altered even at later stages. However, expression levels of key regulators of metabolism, AMP-activated protein kinase (Ampk), peroxisome proliferative activated receptor gamma coactivator 1-α (Ppargc1a), and Ppara, were significantly lower than those in controls and correlated with fibrosis development. Loss of Lkb1 in cultured epithelial cells resulted in energy depletion, apoptosis, less fatty acid oxidation and glycolysis, and a profibrotic phenotype. Treatment of Lkb1-deficient cells with an AMP-activated protein kinase (AMPK) agonist (A769662) or a peroxisome proliferative activated receptor alpha agonist (fenofibrate) restored the fatty oxidation defect and reduced apoptosis. In conclusion, we show that loss of LKB1 in renal tubular epithelial cells has an important role in kidney disease development by influencing intracellular metabolism. PMID:26054542

  8. Anti-Adhesive Activity of Cranberry Phenolic Compounds and Their Microbial-Derived Metabolites against Uropathogenic Escherichia coli in Bladder Epithelial Cell Cultures

    PubMed Central

    de Llano, Dolores González; Esteban-Fernández, Adelaida; Sánchez-Patán, Fernando; Martín-Álvarez, Pedro J.; Moreno-Arribas, Mª Victoria; Bartolomé, Begoña

    2015-01-01

    Cranberry consumption has shown prophylactic effects against urinary tract infections (UTI), although the mechanisms involved are not completely understood. In this paper, cranberry phenolic compounds and their potential microbial-derived metabolites (such as simple phenols and benzoic, phenylacetic and phenylpropionic acids) were tested for their capacity to inhibit the adherence of uropathogenic Escherichia coli (UPEC) ATCC®53503™ to T24 epithelial bladder cells. Catechol, benzoic acid, vanillic acid, phenylacetic acid and 3,4-dihydroxyphenylacetic acid showed anti-adhesive activity against UPEC in a concentration-dependent manner from 100–500 µM, whereas procyanidin A2, widely reported as an inhibitor of UPEC adherence on uroepithelium, was only statistically significant (p < 0.05) at 500 µM (51.3% inhibition). The results proved for the first time the anti-adhesive activity of some cranberry-derived phenolic metabolites against UPEC in vitro, suggesting that their presence in the urine could reduce bacterial colonization and progression of UTI. PMID:26023719

  9. Anti-Adhesive Activity of Cranberry Phenolic Compounds and Their Microbial-Derived Metabolites against Uropathogenic Escherichia coli in Bladder Epithelial Cell Cultures.

    PubMed

    de Llano, Dolores González; Esteban-Fernández, Adelaida; Sánchez-Patán, Fernando; Martínlvarez, Pedro J; Moreno-Arribas, Maria Victoria; Bartolomé, Begoña

    2015-01-01

    Cranberry consumption has shown prophylactic effects against urinary tract infections (UTI), although the mechanisms involved are not completely understood. In this paper, cranberry phenolic compounds and their potential microbial-derived metabolites (such as simple phenols and benzoic, phenylacetic and phenylpropionic acids) were tested for their capacity to inhibit the adherence of uropathogenic Escherichia coli (UPEC) ATCC®53503™ to T24 epithelial bladder cells. Catechol, benzoic acid, vanillic acid, phenylacetic acid and 3,4-dihydroxyphenylacetic acid showed anti-adhesive activity against UPEC in a concentration-dependent manner from 100-500 µM, whereas procyanidin A2, widely reported as an inhibitor of UPEC adherence on uroepithelium, was only statistically significant (p < 0.05) at 500 µM (51.3% inhibition). The results proved for the first time the anti-adhesive activity of some cranberry-derived phenolic metabolites against UPEC in vitro, suggesting that their presence in the urine could reduce bacterial colonization and progression of UTI. PMID:26023719

  10. Roles of Wnt/{beta}-catenin signaling in epithelial differentiation of mesenchymal stem cells

    SciTech Connect

    Wang, Yajing; Sun, Zhaorui; Qiu, Xuefeng; Li, Yan; Qin, Jizheng; Han, Xiaodong

    2009-12-25

    Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/{beta}-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/{beta}-catenin signaling were determined, suggested down-regulation of Wnt/{beta}-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3{alpha} can inhibit the epithelial differentiation of MSCs. A loss of {beta}-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated {beta}-catenin expression and subsequently decreased {beta}-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/{beta}-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.

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

    SciTech Connect

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

    1994-02-01

    Sulfur dioxide (SO[sub 2]) may act as a cocarcinogen with benzo[a]pyrene (BaP) in the respiratory tract. We have modeled this effect by examining the interactions of 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with sulfite, the physiological form of SO[sub 2], in a murine respiratory epithelial cell line (C10). We exposed C10 cells to [[sup 3]H]-anti-BPDE and determined the effects of 1 and 10 mM sulfite on the uptake and subcellular localization of labeled products. Autoradiographic analysis showed that sulfite doubled the nuclear localization of anti-BPDE-derived materials was not affected by sulfite during the first 60 min, but nuclear localization continued to increase in the sulfite-containing incubations throughout the 4-hr incubation period. Little increase in nuclear localization of anti-BPDE-derived material was noted in the incubations without sulfite after 60 min. Subcellular fractionation was performed to determine the amount of label associated with cytosolic and nuclear fractions and to determine covalent binding to protein and DNA. Sulfite produced a modest increase in the amount of [[sup 3]H]-anti-BPDE-derived products bount to protein; however, binding to nuclear DNA increase by more than 200% with 10 mM sulfite. Analysis of the supernatents from the cytosolic and nuclear fractions of cells exposed to anti-BPDE and sulfite demonstrated the presence of 7r,8t9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10c- sulfonate (BPT-10-sulfonate). [[sup 3]H]-BPT-10-sulfonate was unable to enter C10 cells, suggesting that it is formed intracellularly. Once formed, this compound may be unable to leave the cell. The observed intracellular formation of BPT-10-sulfonate, a more stable DNA-modifying BaP derivative than BPDE and one which probably cannot leave the cell, could be responsible for this extended time course of nuclear localization and DNA modification.

  12. Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells.

    PubMed

    Coloff, Jonathan L; Murphy, J Patrick; Braun, Craig R; Harris, Isaac S; Shelton, Laura M; Kami, Kenjiro; Gygi, Steven P; Selfors, Laura M; Brugge, Joan S

    2016-05-10

    Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation. PMID:27133130

  13. Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells.

    PubMed

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

    2013-02-01

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

  14. DNA typing of epithelial cells after strangulation.

    PubMed

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  15. Selection of carcinogen-altered rat tracheal epithelial cells preexposed to 7,12-dimethylbenz(a)anthracene by their loss of a need for pyruvate to survive in culture

    SciTech Connect

    Marchok, A.C.; Huang, S.F.; Martin, D.H.

    1984-01-01

    Normal rat tracheal epithelial cells require exogenous pyruvate to survive in culture while carcinogen-altered cells do not. The aims of our study were, (i) to utilize this loss of requirement for pyruvate by carcinogen-altered cells as a way of selecting out and quantitating the numbers of altered cell populations induced in tracheas exposed to 200 ..mu..g 7,12-dimethylbenz(a)-anthracene (DMBA)-beeswax pellets for different lengths of time, (ii) to determine the constancy of these numbers at increasing time intervals after the exposure, (iii) to look for distinctive growth properties in cell populations that may be related to the exposure conditions. Results show that there were 1.8/altered or selected cell populations (SPC) trachea 2 months after the 2 weeks exposure, while 5.0 and 7.2 SPC were obtained from the 4 weeks and continuously exposed tracheas, respectively, at this time. The numbers of SPC did not increase with time after exposure indicating that the maximal number of initiation sites was fixed. Between 2 and 9 months the number of subculturable SPC increased from 67 to 88% in the 2 week exposed group, while 93-100% of the SPC were subculturable after the longer DMBA exposures. Only the SPC from the 2 week DMBA-2 month group were distinguished by smaller numbers of cells surviving the selection medium and by a slower average growth rate compared to all the other experimental conditions. 29 references, 3 figures, 2 tables.

  16. IL-4 attenuates pulmonary epithelial cell-mediated suppression of T cell priming.

    PubMed

    Albrecht, Melanie; Arnhold, Markus; Lingner, Sandra; Mahapatra, Subhashree; Bruder, Dunja; Hansen, Gesine; Dittrich, Anna-Maria

    2012-01-01

    We have previously shown that Th2-polarized airway inflammation facilitates sensitization towards new, protein antigens. In this context, we could demonstrate that IL-4 needs to act on cells of the hematopoetic and the structural compartment in order to facilitate sensitization towards new antigens. We thus aimed to elucidate possible mechanisms of action of IL-4 on structural cells choosing to analyze pulmonary epithelial cells as an important part of the lung's structural system. We used a co-culture system of DC- or APC-dependent in vitro priming of T cells, co-cultivated on a layer of cells of a murine pulmonary epithelial cell line (LA-4) pretreated with or without IL-4. Effects on T cell priming were analyzed via CFSE-dilution and flow cytometric assessment of activation status. Pulmonary epithelial cells suppressed T cell proliferation in vitro but this effect was attenuated by pre-treatment of the epithelial cells with IL-4. Transwell experiments suggest that epithelial-mediated suppression of T cell activation is mostly cell-contact dependent and leads to attenuation in an early naive T cell phenotype. Secretion of soluble factors like TARC, TSLP, GM-CSF and CCL20 by epithelial cells did not change after IL-4 treatment. However, analysis of co-stimulatory expression on pulmonary epithelial cells revealed that pre-treatment of epithelial cells with IL-4 changed expression GITR-L, suggesting a possible mechanism for the effects observed. Our studies provide new insight into the role of IL-4 during the early phases of pulmonary sensitization: The inhibitory activity of pulmonary epithelial cells in homeostasis is reversed in the presence of IL-4, which is secreted in the context of Th2-dominated allergic airway inflammation. This mechanism might serve to explain facilitated sensitization in the clinical context of polysensitization where due to a pre-existing sensitization increased levels of IL-4 in the airways might facilitate T cell priming towards new

  17. Mycoplasma genitalium promotes epithelial crossing and peripheral blood mononuclear cell infection by HIV-1

    PubMed Central

    Das, Kishore; De la Garza, Georgina; Siwak, Edward B.; Scofield, Virginia L.; Dhandayuthapani, Subramanian

    2014-01-01

    Summary Background Mycoplasma genitalium co-infection in HIV-infected individuals has been reported to increase the shedding of HIV in the urogenital region of females. To better understand this relationship, we investigated the influence of M. genitalium on the transmission and replication of HIV using an in vitro model. Methods The Transwell co-culture system was employed to assess the crossing of an endocervical cell barrier by HIV-1. Immunocytochemistry and confocal microscopy were used to assess the distribution of the nectin-1 molecule on M. genitalium-infected epithelial cells of the End1/E6E7 endocervical cell line, grown as monolayers in the insert wells. Peripheral blood mononuclear cells (PBMC) were cultured in the bottom wells to assess the effects of M. genitalium, passing through the semipermeable culturing membrane, on subsequent HIV infection of susceptible target cells. Results Infection of the endocervical cells with the adhesion-positive M. genitalium G37 strain (wild-type) significantly elevated the passage of HIV across the epithelial cell barrier relative to HIV transfer across endocervical cells infected with the adhesion-negative M. genitalium JB1 strain. Immunostaining of the M. genitalium-G37-infected epithelial cells disclosed capping and internalization of the junctional regulatory protein nectin-1, in association with reduced transepithelial resistance (TER) in the cell monolayer. When PBMC were cultured beneath insert wells containing M. genitalium-G37-infected epithelial cell monolayers, we observed significantly enhanced infectivity and replication of HIV added afterward to the cultures. Conclusions M. genitalium influences events on both sides of a cultured mucosal epithelial monolayer: (1) by infecting the epithelial cells and reducing the integrity of the barrier itself, and (2) by activating HIV target cells below it, thereby promoting HIV infection and progeny virus production. PMID:24661929

  18. Interaction exists between matriptase inhibitors and intestinal epithelial cells.

    PubMed

    Pászti-Gere, Erzsebet; Barna, Réka Fanni; Ujhelyi, Gabriella; Steinmetzer, Torsten

    2016-10-01

    The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro. PMID:26118419

  19. Generation of airway epithelial cells with native characteristics from mouse induced pluripotent stem cells.

    PubMed

    Yoshie, Susumu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Ikeda, Masakazu; Nomoto, Yukio; Wada, Ikuo; Omori, Koichi

    2016-05-01

    Airway epithelial cells derived from induced pluripotent stem (iPS) cells are expected to be a useful source for the regeneration of airway epithelium. Our preliminary study of embryoid body (EB) formation and the air-liquid interface (ALI) method suggested that mouse iPS cells can differentiate into airway epithelial cells. However, whether the cells generated from mouse iPS cells had the character and phenotype of native airway epithelial cells remained uninvestigated. In this study, we generated airway epithelial cells from EBs by culturing them under serum-free conditions supplemented with Activin and bFGF and by the ALI method and characterized the iPS cell-derived airway epithelial cells in terms of their gene expression, immunoreactivity, morphology, and function. Analysis by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) revealed that the expression of the undifferentiated cell marker Nanog decreased time-dependently after the induction of differentiation, whereas definitive endoderm markers Foxa2 and Cxcr4 were transiently up-regulated. Thereafter, the expression of airway epithelium markers such as Tubb4a, Muc5ac, and Krt5 was detected by RT-PCR and immunostaining. The formation of tight junctions was also confirmed by immunostaining and permeability assay. Analysis by hematoxylin and eosin staining and scanning electron microscopy indicated that the cells generated from mouse iPS cells formed airway-epithelium-like tissue and had cilia, the movement of which was visualized and observed to be synchronized. These results demonstrate that the airway epithelial cells generated by our method have native characteristics and open new perspectives for the regeneration of injured airway epithelium. PMID:26590823

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

    PubMed Central

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

    2015-01-01

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

  1. Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation.

    PubMed

    Feng, Wenqiang; Guo, Juanjuan; Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

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

  2. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In

  3. Hypoxia affects in vitro proliferation and differentiation of mouse corneal epithelial progenitor cell.

    PubMed

    Dong, Nuo; Qin, Wenjuan; Xue, Yuhua; Li, Cheng; Liu, Zuguo

    2013-08-01

    This study was to investigate the proliferation and differentiation of mouse corneal epithelial progenitor cell in hypoxic airlift culture. Mouse corneal epithelial progenitor cell line progenitor cells were cultured under airlift with normoxic and hypoxic conditions for various durations up to 2 wk. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of mouse corneal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The mechanism by which hypoxia prevents abnormal differentiation may involve downregulation of Wnt signaling pathways, which were inhibited in cells cultured with hypoxic airlift technique. In conclusion, hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal epithelial cells by blocking Wnt/β-catenin signaling pathway. PMID:23739874

  4. Rho-associated protein kinase inhibition enhances airway epithelial Basal-cell proliferation and lentivirus transduction.

    PubMed

    Horani, Amjad; Nath, Aditya; Wasserman, Mollie G; Huang, Tao; Brody, Steven L

    2013-09-01

    The identification of factors that regulate airway epithelial cell proliferation and differentiation are essential for understanding the pathophysiology of airway diseases. Rho-associated protein kinases (ROCKs) are downstream effector proteins of RhoA GTPase that direct the functions of cell cytoskeletal proteins. ROCK inhibition with Y27632 has been shown to enhance the survival and cloning of human embryonic stem cells and pluripotent cells in other tissues. We hypothesized that Y27632 treatment exerts a similar effect on airway epithelial basal cells, which function as airway epithelial progenitor cells. Treatment with Y27632 enhanced basal-cell proliferation in cultured human tracheobronchial and mouse tracheal epithelial cells. ROCK inhibition accelerated the maturation of basal cells, characterized by a diminution of the cell size associated with cell compaction and the expression of E-cadherin at cell-cell junctions. Transient treatment of cultured basal cells with Y27632 did not affect subsequent ciliated or mucous cell differentiation under air-liquid interface conditions, and allowed for the initial use of lower numbers of human or mouse primary airway epithelial cells than otherwise possible. Moreover, the use of Y27632 during lentivirus-mediated transduction significantly improved posttransduction efficiency and the selection of a transduced cell population, as determined by reporter gene expression. These findings suggest an important role for ROCKs in the regulation of proliferation and maturation of epithelial basal cells, and demonstrate that the inhibition of ROCK pathways using Y27632 provides an adjunctive tool for the in vitro genetic manipulation of airway epithelial cells by lentivirus vectors. PMID:23713995

  5. Hyperoxia alters the mechanical properties of alveolar epithelial cells.

    PubMed

    Roan, Esra; Wilhelm, Kristina; Bada, Alex; Makena, Patrudu S; Gorantla, Vijay K; Sinclair, Scott E; Waters, Christopher M

    2012-06-15

    Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability. PMID:22467640

  6. 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

  7. 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. PMID:26627458

  8. 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

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

    SciTech Connect

    Jin, Hua; Shen, Shuijie; Chen, Xiaoyan; Zhong, Dafang; Zheng, Jiang

    2012-06-15

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

  10. Using organotypic (raft) epithelial tissue cultures for the biosynthesis and isolation of infectious human papillomaviruses

    PubMed Central

    Ozbun, Michelle A.; Patterson, Nicole A.

    2014-01-01

    Papillomaviruses have a strict tropism for epithelial cells and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro wherein virion morphogenesis occurs under cooperative viral and cellular cues requires the cultivation of epithelium. Presented in the first section of this unit is a protocol for growing differentiating epithelial tissues, whose structure and function mimics many important morphological and biochemical aspects of normal skin. The technique, pioneered by A