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Sample records for epithelial cell model

  1. From cells to tissue: A continuum model of epithelial mechanics

    Science.gov (United States)

    Ishihara, Shuji; Marcq, Philippe; Sugimura, Kaoru

    2017-08-01

    A two-dimensional continuum model of epithelial tissue mechanics was formulated using cellular-level mechanical ingredients and cell morphogenetic processes, including cellular shape changes and cellular rearrangements. This model incorporates stress and deformation tensors, which can be compared with experimental data. Focusing on the interplay between cell shape changes and cell rearrangements, we elucidated dynamical behavior underlying passive relaxation, active contraction-elongation, and tissue shear flow, including a mechanism for contraction-elongation, whereby tissue flows perpendicularly to the axis of cell elongation. This study provides an integrated scheme for the understanding of the orchestration of morphogenetic processes in individual cells to achieve epithelial tissue morphogenesis.

  2. Biomechanics of epithelial cell islands analyzed by modeling and experimentation

    CERN Document Server

    Coburn, Luke; Noppe, Adrian; Caldwell, Benjamin J; Moussa, Elliott; Yap, Chloe; Priya, Rashmi; Lobaskin, Vladimir; Roberts, Anthony P; Yap, Alpha S; Neufeld, Zoltan; Gomez, Guillermo A

    2016-01-01

    We generated a new computational approach to analyze the biomechanics of epithelial cell islands that combines both vertex and contact-inhibition-of-locomotion models to include both cell-cell and cell-substrate adhesion. Examination of the distribution of cell protrusions (adhesion to the substrate) in the model predicted high order profiles of cell organization that agree with those previously seen experimentally. Cells acquired an asymmetric distribution of protrusions (and traction forces) that decreased when moving from the edge to the island center. Our in silico analysis also showed that tension on cell-cell junctions (and monolayer stress) is not homogeneous across the island. Instead it is higher at the island center and scales up with island size, which we confirmed experimentally using laser ablation assays and immunofluorescence. Moreover, our approach has the minimal elements necessary to reproduce mechanical crosstalk between both cell-cell and cell substrate adhesion systems. We found that an i...

  3. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

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

  4. Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation.

    Directory of Open Access Journals (Sweden)

    Wenqiang Feng

    Full Text Available 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(aP 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.

  5. Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation.

    Science.gov (United States)

    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.

  6. Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.

    Science.gov (United States)

    Hynds, Robert E; Giangreco, Adam

    2013-03-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. Copyright © 2012 AlphaMed Press.

  7. Modeling nanoparticle–alveolar epithelial cell interactions under breathing conditions using captive bubble surfactometry

    OpenAIRE

    Schürch, David; Vanhecke, Dimitri; Clift, Martin J D; Raemy, David; de Aberasturi, Dorleta Jimenez; Parak, Wolfgang J.; Gehr, Peter; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

    2014-01-01

    Many advances have been made in recent years in cell culture models of the epithelial barrier of the lung from simple monolayers to complex 3-D systems employing different cell types. However, the vast majority of these models still present a static air–liquid interface which is unrealistic given the dynamic nature of breathing. We present here a method where epithelial lung cells are integrated into a system, the captive bubble surfactometer, which allows the cyclical compression and expansi...

  8. Choroid plexus epithelial monolayers ? a cell culture model from porcine brain

    OpenAIRE

    Reichel Valeska; Baehr Carsten; Fricker Gert

    2006-01-01

    Abstract Background The goal of the present study was to develop an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF) and vice versa. Methods Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the ch...

  9. Active Vertex Model for cell-resolution description of epithelial tissue mechanics.

    Science.gov (United States)

    Barton, Daniel L; Henkes, Silke; Weijer, Cornelis J; Sknepnek, Rastko

    2017-06-01

    We introduce an Active Vertex Model (AVM) for cell-resolution studies of the mechanics of confluent epithelial tissues consisting of tens of thousands of cells, with a level of detail inaccessible to similar methods. The AVM combines the Vertex Model for confluent epithelial tissues with active matter dynamics. This introduces a natural description of the cell motion and accounts for motion patterns observed on multiple scales. Furthermore, cell contacts are generated dynamically from positions of cell centres. This not only enables efficient numerical implementation, but provides a natural description of the T1 transition events responsible for local tissue rearrangements. The AVM also includes cell alignment, cell-specific mechanical properties, cell growth, division and apoptosis. In addition, the AVM introduces a flexible, dynamically changing boundary of the epithelial sheet allowing for studies of phenomena such as the fingering instability or wound healing. We illustrate these capabilities with a number of case studies.

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

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    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

  11. Three-dimensional Mammary Epithelial Cell Morphogenesis Model for Analysis of TGFß Signaling.

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    Rashidian, Juliet; Luo, Kunxin

    2016-01-01

    Culturing mammary epithelial cells in laminin-rich extracellular matrices (three dimensional or 3D culture) offers significant advantages over that in the conventional two-dimensional (2D) tissue culture system in that it takes into considetation the impact of extracellular matrix (ECM) microenvironment on the proliferation, survival, and differentiation of mammary epithelial cells. When grown in the 3D culture, untransformed mammary epithelial cells undergo morphogenesis to form a multicellular and polarized acini-like structure that functionally mimics the differentiated alveoli in the pregnancy mammary gland. This process is subjected to regulation by many growth factors and cytokines. The transforming growth factor-ß (TGFß) is a multipotent cytokine that regulates multiple aspects of development and tumorigenesis. In addition to its effects on epithelial cell proliferation, survival, and differentiation, it is also a potent regulator of the cell-matrix interaction. Thus, the 3D culture model may recapitulate the complex in vivo epithelial cell microenvironment and allow us to fully evaluate the role of TGFß signaling in multiple aspects of normal and cancerous cell behavior. In this chapter we provide detailed protocols for growing mammary epithelial cells in the 3D Matrigel for analysis of signaling pathways.

  12. Auxin induces cell proliferation in an experimental model of mammalian renal tubular epithelial cells.

    Science.gov (United States)

    Cernaro, Valeria; Medici, Maria Antonietta; Leonello, Giuseppa; Buemi, Antoine; Kohnke, Franz Heinrich; Villari, Antonino; Santoro, Domenico; Buemi, Michele

    2015-06-01

    Indole-3-acetic acid is the main auxin produced by plants and plays a key role in the plant growth and development. This hormone is also present in humans where it is considered as a uremic toxin deriving from tryptophan metabolism. However, beyond this peculiar aspect, the involvement of auxin in human pathophysiology has not been further investigated. Since it is a growth hormone, we evaluated its proliferative properties in an in vitro model of mammalian renal tubular epithelial cells. We employed an experimental model of renal tubular epithelial cells belonging to the LLC-PK1 cell line that is derived from the kidney of healthy male pig. Growth effects of auxin against LLC-PK1 cell lines were determined by a rapid colorimetric assay. Increasing concentrations of auxin (to give a final concentration from 1 to 1000 ng/mL) were added and microplates were incubated for 72 h. Each auxin concentration was assayed in four wells and repeated four times. Cell proliferation significantly increased, compared to control cells, 72 h after addition of auxin to cultured LLC-PK1 cells. Statistically significant values were observed when 100 ng/mL (p auxin influences cell growth not only in plants, where its role is well documented, but also in mammalian cell lines. This observation opens new scenarios in the field of tissue regeneration and may stimulate a novel line of research aiming at investigating whether this hormone really influences human physiology and pathophysiology and in particular, kidney regeneration.

  13. Establishment of an In Vitro Intestinal Epithelial Cell Culture Model of Avian Origin.

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    Kaiser, Annette; Willer, Thomas; Steinberg, Pablo; Rautenschlein, Silke

    2017-06-01

    The role of intestinal epithelial cells (IECs) in the physiology of the gastrointestinal tract (GIT) of chickens and pathogenesis of various diseases in chickens is still poorly understood. IECs line the GIT and represent the border between the unsterile environment and the sterile internal tissues. Bacterial, viral, fungal, or parasitic pathogens are able to invade or pass IECs under certain circumstances and cause various diseases. Pathogen-host interactions in the chicken gut are poorly understood because of the lack of suitable in vitro and ex vivo models. In this context, there is a need to optimize the cell isolation and culture conditions to be able to provide reproducible IEC cultures with defined epithelial characteristics. We compared different mechanical IEC isolation protocols and cell culture media and established a reproducible primary intestinal epithelial cell culture model from specific-pathogen-free layer-type chickens. By using isolated crypts from the duodenum of 5- to 12-wk-old birds to create the starting material, we were able to culture replicating cells between 7 and 10 days. Cells built an almost closed monolayer and showed epithelial-like characteristics, such as the expression of cytokeratin and epithelial cadherin. The primary IEC cultures described in this study represent a suitable model with which to investigate in vitro pathogen-host interactions relevant to the chicken gut.

  14. Explicit targeting of transformed cells by VSV in ovarian epithelial tumor-bearing Wv mouse models.

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    Capo-chichi, Callinice D; Yeasky, Toni M; Heiber, Joshua F; Wang, Ying; Barber, Glen N; Xu, Xiang-Xi

    2010-02-01

    Current treatment options for epithelial ovarian cancer are limited and therapeutic development for recurrent and drug-resistant ovarian cancer is an urgent agenda. We investigated the potential use of genetically engineered Vesicular Stomatitis Virus (VSV) to treat ovarian cancer patients who fail to respond to available therapies. Specifically, we examined the toxicity to hosts and specificity of targeting ovarian tumors using a Wv ovarian tumor model. We first tested recombinant VSV for oncolytic activity in a panel of human ovarian epithelial cancer, immortalized, and primary ovarian surface epithelial cells in culture. Then, we tested VSV oncolytic therapy using the immune competent Wv mice that develop tubular adenomas, benign tumor lesions derived from ovarian surface epithelial cells. The expression of GFP encoded by the recombinant VSV genome was detected in about 5% of primary ovarian surface epithelial cells (3 lines) up to 30 days without significantly altering the growth pattern of the cells, suggesting the lack of toxicity to the normal ovarian surface epithelial cells. However, VSV-GFP was detected in the majority (around 90%) of cells that are either "immortalized" by SV40 antigen expression or cancer lines. Some variation in killing time courses was observed, but all the transformed cell lines were killed within 3 days. We found that regardless of the inoculation route (intra bursal, IP, or IV), VSV specifically infected and replicated in the in situ ovarian tumors in the Wv mice without significant activity in any other organs and tissues, and showed no detectable toxicity. The epithelial tumor lesions were greatly reduced in VSV-targeted ovarian tumors in the Wv mice. VSV oncolytic activity depends on a cell autonomous property distinguishing primary and transformed cells. The efficient oncolytic activity of VSV for the "immortalized" non-tumorigenic ovarian surface epithelial cells suggests that the selective specificity extends from pre

  15. Modeling nanoparticle-alveolar epithelial cell interactions under breathing conditions using captive bubble surfactometry.

    Science.gov (United States)

    Schürch, David; Vanhecke, Dimitri; Clift, Martin J D; Raemy, David; de Aberasturi, Dorleta Jimenez; Parak, Wolfgang J; Gehr, Peter; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

    2014-05-06

    Many advances have been made in recent years in cell culture models of the epithelial barrier of the lung from simple monolayers to complex 3-D systems employing different cell types. However, the vast majority of these models still present a static air-liquid interface which is unrealistic given the dynamic nature of breathing. We present here a method where epithelial lung cells are integrated into a system, the captive bubble surfactometer, which allows the cyclical compression and expansion of the surfactant film at the air-liquid interface, thus modeling the dynamics of breathing. We found that cellular uptake of deposited gold nanoparticles was significantly increased under the dynamic (breathing) conditions of compression and expansion as compared to static conditions. The method could be very useful for studying nanoparticle-alveolar lung cell interactions under breathing conditions for applications in nanomedicine and toxicology.

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

    Science.gov (United States)

    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.

  17. Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Liara M Gonzalez

    Full Text Available 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.

  18. Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

    OpenAIRE

    Nakles, Rebecca E.; Millman, Sarah L.; Cabrera, M. Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S.; Schroeder, Timm; Furth, Priscilla A.

    2013-01-01

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without...

  19. Metabolic cleavage of cell-penetrating peptides in contact with epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg

    2004-01-01

    We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47...... models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C......-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting...

  20. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

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    Miessen Katrin

    2012-03-01

    Full Text Available Abstract Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  1. Evaluation of MCF10A as a Reliable Model for Normal Human Mammary Epithelial Cells.

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    Ying Qu

    Full Text Available Breast cancer is the most common cancer in women and a leading cause of cancer-related deaths for women worldwide. Various cell models have been developed to study breast cancer tumorigenesis, metastasis, and drug sensitivity. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. However, there is limited knowledge about whether MCF10A cells reliably represent normal human mammary cells. MCF10A cells were grown in monolayer, suspension (mammosphere culture, three-dimensional (3D "on-top" Matrigel, 3D "cell-embedded" Matrigel, or mixed Matrigel/collagen I gel. Suspension culture was performed with the MammoCult medium and low-attachment culture plates. Cells grown in 3D culture were fixed and subjected to either immunofluorescence staining or embedding and sectioning followed by immunohistochemistry and immunofluorescence staining. Cells or slides were stained for protein markers commonly used to identify mammary progenitor and epithelial cells. MCF10A cells expressed markers representing luminal, basal, and progenitor phenotypes in two-dimensional (2D culture. When grown in suspension culture, MCF10A cells showed low mammosphere-forming ability. Cells in mammospheres and 3D culture expressed both luminal and basal markers. Surprisingly, the acinar structure formed by MCF10A cells in 3D culture was positive for both basal markers and the milk proteins β-casein and α-lactalbumin. MCF10A cells exhibit a unique differentiated phenotype in 3D culture which may not exist or be rare in normal human breast tissue. Our results raise a question as to whether the commonly used MCF10A cell line is a suitable model for human mammary cell studies.

  2. Construction of predictive promoter models on the example of antibacterial response of human epithelial cells

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    Wingender Edgar

    2005-01-01

    Full Text Available Abstract Background Binding of a bacteria to a eukaryotic cell triggers a complex network of interactions in and between both cells. P. aeruginosa is a pathogen that causes acute and chronic lung infections by interacting with the pulmonary epithelial cells. We use this example for examining the ways of triggering the response of the eukaryotic cell(s, leading us to a better understanding of the details of the inflammatory process in general. Results Considering a set of genes co-expressed during the antibacterial response of human lung epithelial cells, we constructed a promoter model for the search of additional target genes potentially involved in the same cell response. The model construction is based on the consideration of pair-wise combinations of transcription factor binding sites (TFBS. It has been shown that the antibacterial response of human epithelial cells is triggered by at least two distinct pathways. We therefore supposed that there are two subsets of promoters activated by each of them. Optimally, they should be "complementary" in the sense of appearing in complementary subsets of the (+-training set. We developed the concept of complementary pairs, i.e., two mutually exclusive pairs of TFBS, each of which should be found in one of the two complementary subsets. Conclusions We suggest a simple, but exhaustive method for searching for TFBS pairs which characterize the whole (+-training set, as well as for complementary pairs. Applying this method, we came up with a promoter model of antibacterial response genes that consists of one TFBS pair which should be found in the whole training set and four complementary pairs. We applied this model to screening of 13,000 upstream regions of human genes and identified 430 new target genes which are potentially involved in antibacterial defense mechanisms.

  3. Effect calcusol to reduce the calcium crystal retention in kidney epithelial cells model of nephrolothiasis

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    Ahmad Soni

    2014-12-01

    Full Text Available Kidney stones is a disease that characterized by a disturbance in the bladder. The main constituent of kidney stones namely Calcium Oxalate Monohydrate (COM crystals. The presence of a COM crystal adhesion to renal tubular cells, will initiate the internalization which will further lead to the formation of crystals retention in the kidney. In Indonesia, there are many herbal products are considered able to cope the complaints due to the kidney stone disease. One of the herbal product is Calcusol „¢, which is the main constituent of those herbal product was the leaf extract of tempuyung. This study observed the effectiveness of Calcusol „¢ in reducing crystals retention that was formed in kidney epithelial cells model of nephrolithiasis. The result showed that Calcusol „¢ is able to reduce the average number of calcium crystals retention in the renal epithelial cells. It indicate that Calcusol „¢ has the ability to reduce crystals retention that already formed in renal epithelial cells. Furthermore, the results of this study are expected to be one of the considerations for further research on the potential of overcoming Calcusol „¢ in kidney stone disease

  4. Gastric Epithelial Stem Cells

    Science.gov (United States)

    MILLS, JASON C.; SHIVDASANI, RAMESH A.

    2013-01-01

    Advances in our understanding of stem cells in the gastrointestinal tract include the identification of molecular markers of stem and early progenitor cells in the small intestine. Although gastric epithelial stem cells have been localized, little is known about their molecular biology. Recent reports describe the use of inducible Cre recombinase activity to indelibly label candidate stem cells and their progeny in the distal stomach, (ie, the antrum and pylorus). No such lineage labeling of epithelial stem cells has been reported in the gastric body (corpus). Among stem cells in the alimentary canal, those of the adult corpus are unique in that they lie close to the lumen and increase proliferation following loss of a single mature progeny lineage, the acid-secreting parietal cell. They are also unique in that they neither depend on Wnt signaling nor express the surface marker Lgr5. Because pathogenesis of gastric adenocarcinoma has been associated with abnormal patterns of gastric differentiation and with chronic tissue injury, there has been much research on the response of stomach epithelial stem cells to inflammation. Chronic inflammation, as induced by infection with Helicobacter pylori, affects differentiation and promotes metaplasias. Several studies have identified cellular and molecular mechanisms in spasmolytic polypeptide–expressing (pseudopyloric) metaplasia. Researchers have also begun to identify signaling pathways and events that take place during embryonic development that eventually establish the adult stem cells to maintain the specific features and functions of the stomach mucosa. We review the cytologic, molecular, functional, and developmental properties of gastric epithelial stem cells. PMID:21144849

  5. Transport and uptake effects of marine complex lipid liposomes in small intestinal epithelial cell models.

    Science.gov (United States)

    Du, Lei; Yang, Yu-Hong; Xu, Jie; Wang, Yu-Ming; Xue, Chang-Hu; Kurihara, Hideyuki; Takahashi, Koretaro

    2016-04-01

    Nowadays, marine complex lipids, including starfish phospholipids (SFP) and cerebrosides (SFC) separated from Asterias amurensis as well as sea cucumber phospholipids (SCP) and cerebrosides (SCC) isolated from Cucumaria frondosa, have received much attention because of their potent biological activities. However, little information is known on the transport and uptake of these lipids in liposome forms in small intestinal cells. Therefore, this study was undertaken to investigate the effects of these complex lipid liposomes on transport and uptake in Caco-2 and M cell monolayer models. The results revealed that SFP and SCP contained 42% and 47.9% eicosapentaenoic acid (EPA), respectively. The average particle sizes of liposomes prepared in this study were from 169 to 189 nm. We found that the transport of the liposomes across the M cell monolayer model was much higher than the Caco-2 cell monolayer model. The liposomes consisting of SFP or SCP showed significantly higher transport and uptake than soy phospholipid (soy-PL) liposomes in both Caco-2 and M cell monolayer models. Our results also exhibited that treatment with 1 mM liposomes composed of SFP or SCP for 3 h tended to increase the EPA content in phospholipid fractions of both differentiated Caco-2 and M cells. Moreover, it was also found that the hybrid liposomes consisting of SFP/SFC/cholesterol (Chol) revealed higher transport and uptake across the M cell monolayer in comparison with other liposomes. Furthermore, treatment with SFP/SFC/Chol liposomes could notably decrease the trans-epithelial electrical resistance (TEER) values of Caco-2 and M cell monolayers. The present data also showed that the cell viability of differentiated Caco-2 and M cells was not affected after the treatment with marine complex lipids or soy-PL liposomes. Based on the data in this study, it was suggested that marine complex lipid liposomes exhibit prominent transport and uptake in small intestinal epithelial cell models.

  6. Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

    Science.gov (United States)

    Cousins, Fiona L; Murray, Alison; Esnal, Arantza; Gibson, Douglas A; Critchley, Hilary O D; Saunders, Philippa T K

    2014-01-01

    In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Mechanocellular models of epithelial morphogenesis.

    Science.gov (United States)

    Fletcher, Alexander G; Cooper, Fergus; Baker, Ruth E

    2017-05-19

    Embryonic epithelia achieve complex morphogenetic movements, including in-plane reshaping, bending and folding, through the coordinated action and rearrangement of individual cells. Technical advances in molecular and live-imaging studies of epithelial dynamics provide a very real opportunity to understand how cell-level processes facilitate these large-scale tissue rearrangements. However, the large datasets that we are now able to generate require careful interpretation. In combination with experimental approaches, computational modelling allows us to challenge and refine our current understanding of epithelial morphogenesis and to explore experimentally intractable questions. To this end, a variety of cell-based modelling approaches have been developed to describe cell-cell mechanical interactions, ranging from vertex and 'finite-element' models that approximate each cell geometrically by a polygon representing the cell's membrane, to immersed boundary and subcellular element models that allow for more arbitrary cell shapes. Here, we review how these models have been used to provide insights into epithelial morphogenesis and describe how such models could help future efforts to decipher the forces and mechanical and biochemical feedbacks that guide cell and tissue-level behaviour. In addition, we discuss current challenges associated with using computational models of morphogenetic processes in a quantitative and predictive way.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'. © 2017 The Author(s).

  9. Inflammatory Cytokine Tumor Necrosis Factor α Confers Precancerous Phenotype in an Organoid Model of Normal Human Ovarian Surface Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Joseph Kwong

    2009-06-01

    Full Text Available In this study, we established an in vitro organoid model of normal human ovarian surface epithelial (HOSE cells. The spheroids of these normal HOSE cells resembled epithelial inclusion cysts in human ovarian cortex, which are the cells of origin of ovarian epithelial tumor. Because there are strong correlations between chronic inflammation and the incidence of ovarian cancer, we used the organoid model to test whether protumor inflammatory cytokine tumor necrosis factor α would induce malignant phenotype in normal HOSE cells. Prolonged treatment of tumor necrosis factor α induced phenotypic changes of the HOSE spheroids, which exhibited the characteristics of precancerous lesions of ovarian epithelial tumors, including reinitiation of cell proliferation, structural disorganization, epithelial stratification, loss of epithelial polarity, degradation of basement membrane, cell invasion, and overexpression of ovarian cancer markers. The result of this study provides not only an evidence supporting the link between chronic inflammation and ovarian cancer formation but also a relevant and novel in vitro model for studying of early events of ovarian cancer.

  10. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model

    Science.gov (United States)

    Carey, Shawn P.; Starchenko, Alina; McGregor, Alexandra L.; Reinhart-King, Cynthia A.

    2013-01-01

    Solid tumors consist of genetically and phenotypically diverse subpopulations of cancer cells with unique capacities for growth, differentiation, and invasion. While the molecular and microenvironmental bases for heterogeneity are increasingly appreciated, the outcomes of such intratumor heterogeneity, particularly in the context of tumor invasion and metastasis, remain poorly understood. To study heterotypic cell-cell interactions and elucidate the biological consequences of intratumor heterogeneity, we developed a tissue-engineered multicellular spheroid (MCS) co-culture model that recapitulates the cellular diversity and fully three-dimensional cell-cell and cell-matrix interactions that characterize human carcinomas. We found that “invasion-competent” malignant cells induced the collective invasion of otherwise “invasion-incompetent” epithelial cells, and that these two cell types consistently exhibited distinct leader and follower roles during invasion. Analysis of extracellular matrix microarchitecture revealed that malignant cell invasion was accompanied by extensive extracellular matrix remodeling including matrix alignment and proteolytic track-making. Inhibition of cell contractility- and proteolysis-mediated matrix reorganization prevented leader-follower behavior and malignant cell-induced epithelial cell invasion. These results indicate that heterogeneous subpopulations within a tumor may possess specialized roles during tumor progression and suggest that complex interactions among the various subpopulations of cancer cells within a tumor may regulate critical aspects of tumor biology and affect clinical outcome. PMID:23328900

  11. Modeling Cystic Fibrosis Using Pluripotent Stem Cell-Derived Human Pancreatic Ductal Epithelial Cells.

    Science.gov (United States)

    Simsek, Senem; Zhou, Ting; Robinson, Christopher L; Tsai, Su-Yi; Crespo, Miguel; Amin, Sadaf; Lin, Xiangyi; Hon, Jane; Evans, Todd; Chen, Shuibing

    2016-05-01

    We established an efficient strategy to direct human pluripotent stem cells, including human embryonic stem cells (hESCs) and an induced pluripotent stem cell (iPSC) line derived from patients with cystic fibrosis, to differentiate into pancreatic ductal epithelial cells (PDECs). After purification, more than 98% of hESC-derived PDECs expressed functional cystic fibrosis transmembrane conductance regulator (CFTR) protein. In addition, iPSC lines were derived from a patient with CF carrying compound frameshift and mRNA splicing mutations and were differentiated to PDECs. PDECs derived from Weill Cornell cystic fibrosis (WCCF)-iPSCs showed defective expression of mature CFTR protein and impaired chloride ion channel activity, recapitulating functional defects of patients with CF at the cellular level. These studies provide a new methodology to derive pure PDECs expressing CFTR and establish a "disease in a dish" platform to identify drug candidates to rescue the pancreatic defects of patients with CF. An efficient strategy was established to direct human pluripotent stem cells, including human embryonic stem cells (hESCs) and an induced pluripotent stem cell line derived from patients with cystic fibrosis (CF-iPSCs), to differentiate into pancreatic ductal epithelial cells (PDECs). After purification, more than 98% of hESC-PDECs derived from CF-iPSCs showed defective expression of mature cystic fibrosis transmembrane conductance regulator (CFTR) protein and impaired chloride ion channel activity, recapitulating functional pancreatic defects of patients with CF at the cellular level. These studies provide a new methodology for deriving pure PDECs expressing CFTR, and they establish a "disease-in-a-dish" platform for identifying drug candidates to rescue the pancreatic defects of these patients. ©AlphaMed Press.

  12. Choroid plexus epithelial monolayers – a cell culture model from porcine brain

    Directory of Open Access Journals (Sweden)

    Reichel Valeska

    2006-12-01

    Full Text Available Abstract Background The goal of the present study was to develop an in vitro choroid plexus (CP epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF and vice versa. Methods Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR, were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1 and multidrug resistance protein 1 (Mrp1, Abcc1 was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments. Results Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER values in the range of 100 to 150 Ωcm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 ± 4.6 μl/cm2/h over 2–3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123. Conclusion It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic

  13. Choroid plexus epithelial monolayers – a cell culture model from porcine brain

    Science.gov (United States)

    Baehr, Carsten; Reichel, Valeska; Fricker, Gert

    2006-01-01

    Background The goal of the present study was to develop an in vitro choroid plexus (CP) epithelial cell culture model for studying transport of protein-mediated drug secretion from blood to cerebrospinal fluid (CSF) and vice versa. Methods Cells were isolated by mechanical and enzymatic treatment of freshly isolated porcine plexus tissue. Epithelial cell monolayers were grown and CSF secretion and transepithelial resistance were determined. The expression of f-actin as well as the choroid plexus marker protein transthyretin (TTR), were assessed. The expression of the export proteins p-glycoprotein (Pgp, Abcb1) and multidrug resistance protein 1 (Mrp1, Abcc1) was studied by RT-PCR, Western-blot and immunofluorescence techniques and their functional activity was assessed by transport and uptake experiments. Results Choroid plexus epithelial cells were isolated in high purity and grown to form confluent monolayers. Filter-grown monolayers displayed transendothelial resistance (TEER) values in the range of 100 to 150 Ωcm2. Morphologically, the cells showed the typical net work of f-actin and expressed TTR at a high rate. The cultured cells were able to secrete CSF at a rate of 48.2 ± 4.6 μl/cm2/h over 2–3 hours. The ABC-export protein Mrp1 was expressed in the basolateral (blood-facing) membranes of cell monolayers and intact tissue. P-glycoprotein showed only low expression within the apical (CSF directed) membrane but was located more in sub-apical cell compartments. This finding was paralleled by the lack of directed excretion of p-glycoprotein substrates, verapamil and rhodamine 123. Conclusion It was demonstrated that CP epithelium can be isolated and cultured, with cells growing into intact monolayers, fully differentiating and with properties resembling the tissue in vivo. Thus, the established primary porcine CP model, allowing investigation of complex transport processes, can be used as a reliable tool for analysis of xenobiotic transport across the blood

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

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    Marcos de Assis Moura

    2009-09-01

    Full Text Available 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.

  15. Cell Competition Drives the Formation of Metastatic Tumors in a Drosophila Model of Epithelial Tumor Formation

    DEFF Research Database (Denmark)

    Eichenlaub, Teresa; Cohen, Stephen M; Herranz, Héctor

    2016-01-01

    Cell competition is a homeostatic process in which proliferating cells compete for survival. Elimination of otherwise normal healthy cells through competition is important during development and has recently been shown to contribute to maintaining tissue health during organismal aging. The mechan......Cell competition is a homeostatic process in which proliferating cells compete for survival. Elimination of otherwise normal healthy cells through competition is important during development and has recently been shown to contribute to maintaining tissue health during organismal aging....... The mechanisms that allow for ongoing cell competition during adult life could, in principle, contribute to tumorigenesis. However, direct evidence supporting this hypothesis has been lacking. Here, we provide evidence that cell competition drives tumor formation in a Drosophila model of epithelial cancer. Cells...... of the Septin family protein Peanut. Cytokinesis failure due to downregulation of Peanut is required for tumorigenesis. This study provides evidence that the cellular mechanisms that drive cell competition during normal tissue growth can be co-opted to drive tumor formation and metastasis. Analogous mechanisms...

  16. Oral insulin stimulates intestinal epithelial cell turnover following massive small bowel resection in a rat and a cell culture model.

    Science.gov (United States)

    Ben Lulu, Shani; Coran, Arnold G; Shehadeh, Naim; Shamir, Raanan; Mogilner, Jorge G; Sukhotnik, Igor

    2012-02-01

    We have recently reported that oral insulin (OI) stimulates intestinal adaptation after bowel resection and that OI enhances enterocyte turnover in correlation with insulin receptor expression along the villus-crypt axis. The purpose of the present study was to evaluate the effect of OI on intestinal epithelial cell proliferation and apoptosis in a rat model of short bowel syndrome (SBS) and in a cell culture model. Caco-2 cells were incubated with increasing concentrations of insulin. Cell proliferation and apoptosis were determined by FACS cytometry. Cell viability was investigated using the Alamar Blue technique. Male rats were divided into three groups: Sham rats underwent bowel transection, SBS rats underwent a 75% bowel resection, and SBS-OI rats underwent bowel resection and were treated with OI given in drinking water (1 U/ml) from the third postoperative day. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined on day 15. Real time PCR was used to determine the level of bax and bcl-2 mRNA and western blotting was used to determine bax, bcl-2, p-ERK and AKT protein levels. Statistical analysis was performed using the one-way ANOVA test, with P cells with insulin resulted in a significant increase in cell proliferation (twofold increase after 24 h and 37% increase after 48 h) and cell viability (in a dose-dependent manner), but did not change cell apoptosis. In a rat model of SBS, treatment with OI resulted in a significant increase in all parameters of intestinal adaptation. Elevated cell proliferation rate in insulin treated rats was accompanied by elevated AKT and p-ERK protein levels. Decreased cell apoptosis in SBS-INS rats corresponded with a decreased bax/bcl-2 ratio. Oral insulin stimulates intestinal epithelial cell turnover after massive small bowel resection in a rat model of SBS and a cell culture model.

  17. Computational modeling of epithelial tissues.

    Science.gov (United States)

    Smallwood, Rod

    2009-01-01

    There is an extensive literature on the computational modeling of epithelial tissues at all levels from subcellular to whole tissue. This review concentrates on behavior at the individual cell to whole tissue level, and particularly on organizational aspects, and provides an indication of where information from other areas, such as the modeling of angiogenesis, is relevant. The skin, and the lining of all of the body cavities (lung, gut, cervix, bladder etc) are epithelial tissues, which in a topological sense are the boundary between inside and outside the body. They are thin sheets of cells (usually of the order of 0.5 mm thick) without extracellular matrix, have a relatively simple structure, and contain few types of cells. They have important barrier, secretory and transport functions, which are essential for the maintenance of life, so homeostasis and wound healing are important aspects of the behavior of epithelial tissues. Carcinomas originate in epithelial tissues.There are essentially two approaches to modeling tissues--to start at the level of the tissue (i.e., a length scale of the order of 1 mm) and develop generalized equations for behavior (a continuum approach); or to start at the level of the cell (i.e., a length scale of the order of 10 µm) and develop tissue behavior as an emergent property of cellular behavior (an individual-based approach). As will be seen, these are not mutually exclusive approaches, and they come in a variety of flavors.

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

    Science.gov (United States)

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

  19. Time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer.

    Science.gov (United States)

    Nakles, Rebecca E; Millman, Sarah L; Cabrera, M Carla; Johnson, Peter; Mueller, Susette; Hoppe, Philipp S; Schroeder, Timm; Furth, Priscilla A

    2013-02-08

    Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 μm x 700 μm fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

  20. Regulatory network modelling of iron acquisition by a fungal pathogen in contact with epithelial cells

    Directory of Open Access Journals (Sweden)

    Guthke Reinhard

    2010-11-01

    Full Text Available Abstract Background Reverse engineering of gene regulatory networks can be used to predict regulatory interactions of an organism faced with environmental changes, but can prove problematic, especially when focusing on complicated multi-factorial processes. Candida albicans is a major human fungal pathogen. During the infection process, this fungus is able to adapt to conditions of very low iron availability. Such adaptation is an important virulence attribute of virtually all pathogenic microbes. Understanding the regulation of iron acquisition genes will extend our knowledge of the complex regulatory changes during the infection process and might identify new potential drug targets. Thus, there is a need for efficient modelling approaches predicting key regulatory events of iron acquisition genes during the infection process. Results This study deals with the regulation of C. albicans iron uptake genes during adhesion to and invasion into human oral epithelial cells. A reverse engineering strategy is presented, which is able to infer regulatory networks on the basis of gene expression data, making use of relevant selection criteria such as sparseness and robustness. An exhaustive use of available knowledge from different data sources improved the network prediction. The predicted regulatory network proposes a number of new target genes for the transcriptional regulators Rim101, Hap3, Sef1 and Tup1. Furthermore, the molecular mode of action for Tup1 is clarified. Finally, regulatory interactions between the transcription factors themselves are proposed. This study presents a model describing how C. albicans may regulate iron acquisition during contact with and invasion of human oral epithelial cells. There is evidence that some of the proposed regulatory interactions might also occur during oral infection. Conclusions This study focuses on a typical problem in Systems Biology where an interesting biological phenomenon is studied using a small

  1. Autophagy controls an intrinsic host defense to bacteria by promoting epithelial cell survival: a murine model.

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    Sun-Young Chang

    Full Text Available Cell death is a critical host response to regulate the fate of bacterial infections, innate immune responses, and ultimately, disease outcome. Shigella spp. invade and colonize gut epithelium in human and nonhuman primates but adult mice are naturally resistant to intra-gastric Shigella infection. In this study, however, we found Shigella could invade the terminal ileum of the mouse small intestine by 1 hour after infection and be rapidly cleared within 24 h. These early phase events occurred shortly after oral infection resulting in epithelial shedding, degranulation of Paneth cells, and cell death in the intestine. During this process, autophagy proceeded without any signs of inflammation. In contrast, blocking autophagy in epithelial cells enhanced host cell death, leading to tissue destruction and to inflammation, suggesting that autophagic flow relieves cellular stress associated with host cell death and inflammation. Herein we propose a new concept of "epithelial barrier turnover" as a general intrinsic host defense mechanism that increases survival of host cells and inhibits inflammation against enteric bacterial infections, which is regulated by autophagy.

  2. Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia

    DEFF Research Database (Denmark)

    Petersen, Ole William; Gudjonsson, Thorarinn; Villadsen, René

    2003-01-01

    The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value of kn......% of breast cancers arise in TDLUs and more than 90% are also cytokeratin 19-positive, we suggest that this cell population contains a breast-cancer progenitor.......The majority of human breast carcinomas exhibit luminal characteristics and as such, are most probably derived from progenitor cells within the luminal epithelial compartment. This has been subdivided recently into at least three luminal subtypes based on gene expression patterns. The value...... of knowing the cellular origin of individual tumours is clear and should aid in designing effective therapies. To do this, however, we need strategies aimed at defining the nature of stem and progenitor cell populations in the normal breast. In this review, we will discuss our technical approach...

  3. Stem cells and corneal epithelial maintenance: insights from the mouse and other animal models.

    Science.gov (United States)

    Mort, Richard L; Douvaras, Panagiotis; Morley, Steven D; Dorà, Natalie; Hill, Robert E; Collinson, J Martin; West, John D

    2012-01-01

    Maintenance of the corneal epithelium is essential for vision and is a dynamic process incorporating constant cell production, movement and loss. Although cell-based therapies involving the transplantation of putative stem cells are well advanced for the treatment of human corneal defects, the scientific understanding of these interventions is poor. No definitive marker that discriminates stem cells that maintain the corneal epithelium from the surrounding tissue has been discovered and the identity of these elusive cells is, therefore, hotly debated. The key elements of corneal epithelial maintenance have long been recognised but it is still not known how this dynamic balance is co-ordinated during normal homeostasis to ensure the corneal epithelium is maintained at a uniform thickness. Most indirect experimental evidence supports the limbal epithelial stem cell (LESC) hypothesis, which proposes that the adult corneal epithelium is maintained by stem cells located in the limbus at the corneal periphery. However, this has been challenged recently by the corneal epithelial stem cell (CESC) hypothesis, which proposes that during normal homeostasis the mouse corneal epithelium is maintained by stem cells located throughout the basal corneal epithelium with LESCs only contributing during wound healing. In this chapter we review experimental studies, mostly based on animal work, that provide insights into how stem cells maintain the normal corneal epithelium and consider the merits of the alternative LESC and CESC hypotheses. Finally, we highlight some recent research on other stem cell systems and consider how this could influence future research directions for identifying the stem cells that maintain the corneal epithelium.

  4. A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes.

    Science.gov (United States)

    Inagawa, Yuki; Yamada, Kenji; Yugawa, Takashi; Ohno, Shin-ichi; Hiraoka, Nobuyoshi; Esaki, Minoru; Shibata, Tatsuhiro; Aoki, Kazunori; Saya, Hideyuki; Kiyono, Tohru

    2014-08-01

    Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Helicobacter pylori Alters Exogenous Antigen Absorption and Processing in a Digestive Tract Epithelial Cell Line Model

    OpenAIRE

    Matysiak-Budnik, Tamara; Terpend, Kathleen; Alain, Sophie; Sanson le Pors, Marie-José; Desjeux, Jehan-Francois; Mégraud, Francis; Heyman, Martine

    1998-01-01

    To study the influence of Helicobacter pylori on epithelial barrier function, bacteria, bacterial sonicates, or broth culture supernatants were incubated for 24 h with HT29-19A intestinal cells grown as monolayers. Subsequently, the monolayers were mounted in Ussing chambers, and electrical resistance (R), fluxes of 22Na (JNa) and 14C-mannitol (JMan) (markers of the paracellular pathway), and fluxes of horseradish peroxidase (HRP) in total (J3H-HRP), intact (JHRPi), and degraded forms were me...

  6. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells.

    Science.gov (United States)

    Zhu, Yaqi; Yang, Yan; Guo, Juanjuan; Dai, Ying; Ye, Lina; Qiu, Jianbin; Zeng, Zhihong; Wu, Xiaoting; Xing, Yanmei; Long, Xiang; Wu, Xufeng; Ye, Lin; Wang, Shubin; Li, Hui

    2017-02-28

    Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.

  7. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

    , deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

  8. Magnolol inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model.

    Science.gov (United States)

    Wei, Wang; Dejie, Liang; Xiaojing, Song; Tiancheng, Wang; Yongguo, Cao; Zhengtao, Yang; Naisheng, Zhang

    2015-02-01

    Mastitis comprises an inflammation of the mammary gland, which is almost always linked with bacterial infection. The treatment of mastitis concerns antimicrobial substances, but not very successful. On the other hand, anti-inflammatory therapy with Chinese traditional medicine becomes an effective way for treating mastitis. Magnolol is a polyphenolic binaphthalene compound extracted from the stem bark of Magnolia sp., which has been shown to exert a potential for anti-inflammatory activity. The purpose of this study was to investigate the protective effects of magnolol on inflammation in lipopolysaccharide (LPS)-induced mastitis mouse model in vivo and the mechanism of this protective effects in LPS-stimulated mouse mammary epithelial cells (MMECs) in vitro. The damage of tissues was determined by histopathology and myeloperoxidase (MPO) assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-kappa B (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and Toll-like receptor 4 (TLR4) were determined by Western blot. The results showed that magnolol significantly inhibit the LPS-induced TNF-α, IL-6, and IL-1β production both in vivo and vitro. Magnolol declined the phosphorylation of IκBα, p65, p38, ERK, and JNK in LPS-stimulated MMECs. Furthermore, magnolol inhibited the expression of TLR4 in LPS-stimulated MMECs. In vivo study, it was also observed that magnolol attenuated the damage of mastitis tissues in the mouse models. These findings demonstrated that magnolol attenuate LPS-stimulated inflammatory response by suppressing TLR4/NF-κB/mitogen-activated protein kinase (MAPK) signaling system. Thereby, magnolol may be a therapeutic agent against mastitis.

  9. Airway Epithelial Cells Are Crucial Targets of Glucocorticoids in a Mouse Model of Allergic Asthma.

    Science.gov (United States)

    Klaßen, Carina; Karabinskaya, Anna; Dejager, Lien; Vettorazzi, Sabine; Van Moorleghem, Justine; Lühder, Fred; Meijsing, Sebastiaan H; Tuckermann, Jan P; Bohnenberger, Hanibal; Libert, Claude; Reichardt, Holger M

    2017-07-01

    Although glucocorticoids (GCs) are a mainstay in the clinical management of asthma, the target cells that mediate their therapeutic effects are unknown. Contrary to our expectation, we found that GC receptor (GR) expression in immune cells was dispensable for successful therapy of allergic airway inflammation (AAI) with dexamethasone. Instead, GC treatment was compromised in mice expressing a defective GR in the nonhematopoietic compartment or selectively lacking the GR in airway epithelial cells. Further, we found that an intact GR dimerization interface was a prerequisite for the suppression of AAI and airway hyperresponsiveness by GCs. Our observation that the ability of dexamethasone to modulate gene expression in airway epithelial cells coincided with its potency to resolve AAI supports a crucial role for transcriptional regulation by the GR in this cell type. Taken together, we identified an unknown mode of GC action in the treatment of allergic asthma that might help to develop more specific therapies in the future. Copyright © 2017 by The American Association of Immunologists, Inc.

  10. Lack of liver X receptors leads to cell proliferation in a model of mouse dorsal prostate epithelial cell.

    Directory of Open Access Journals (Sweden)

    Julie Dufour

    Full Text Available Recent studies underline the implication of Liver X Receptors (LXRs in several prostate diseases such as benign prostatic hyperplasia (BPH and prostate cancer. In order to understand the molecular mechanisms involved, we derived epithelial cells from dorsal prostate (MPECs of wild type (WT or Lxrαβ-/- mice. In the WT MPECs, our results show that LXR activation reduces proliferation and correlates with the modification of the AKT-survival pathway. Moreover, LXRs regulate lipid homeostasis with the regulation of Abca1, Abcg1 and Idol, and, in a lesser extent, Srebp1, Fas and Acc. Conversely cells derived from Lxrαβ-/- mice show a higher basal phosphorylation and consequently activation of the survival/proliferation transduction pathways AKT and MAPK. Altogether, our data point out that the cell model we developed allows deciphering the molecular mechanisms inducing the cell cycle arrest. Besides, we show that activated LXRs regulate AKT and MAPK transduction pathways and demonstrate that LXRs could be good pharmacological targets in prostate disease such as cancer.

  11. Isolation and characterization of a primary proximal tubular epithelial cell model from human kidney by CD10/CD13 double labeling.

    Science.gov (United States)

    Van der Hauwaert, Cynthia; Savary, Grégoire; Gnemmi, Viviane; Glowacki, François; Pottier, Nicolas; Bouillez, Audrey; Maboudou, Patrice; Zini, Laurent; Leroy, Xavier; Cauffiez, Christelle; Perrais, Michaël; Aubert, Sébastien

    2013-01-01

    Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

  12. Isolation and characterization of a primary proximal tubular epithelial cell model from human kidney by CD10/CD13 double labeling.

    Directory of Open Access Journals (Sweden)

    Cynthia Van der Hauwaert

    Full Text Available Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.

  13. Major salivary gland hypertrophy model in immature rats: morphometric and histochemical epithelial cell characteristics

    Directory of Open Access Journals (Sweden)

    Vera V. Ivanova

    2017-01-01

    Full Text Available The purpose of the study is to estimate the functional state of epithelial cells of acini and ducts of major salivary glands with hypertrophy caused by repeated incisor amputations in immature rats.Materials and methods. The experiment was carried out on immature (20 days, white male rats, divided into 3 groups: intact, control and group of rats with repeated incisor amputations. Animals were taken out in 2d, 3d, 4th, 6th, 8th, 10th and 12th weeks after the first incisor amputation. Morphofunctional state of rat major salivary glands was assessed by histological (hematoxylin and eosin, histochemistrical (Alcian blue, PAS-reaction, Brachet method and morphometrical (acini area, intralobular ducts volume methods.Results. Repeated incisor amputations led to the increase of acini area and the decrease of intralobular duct volume in submandibular glands in 2nd–4th weeks of the experiment. Cytoplasm pyroninophilia of submandibular gland acinar cells was less pronounced and intensity of PAS-reaction was more pronounced than in intact animals in 3rd week of the experiment. Morphological and functional changes of parotid and sublingual gland epithelial cells were not observed after repeated amputations of incisors in immature rats.Conclusion. Repeated incisor amputations in immature male rats lead to submandibular gland acinar cell hypertrophy in the early stages of the experiment (2d–4th weeks with accumulation of glycoproteins and protein synthesis weakening in these cells. Hypertrophy of acinar cells are accompanied by retardation in the development of granular convoluted tubule cells which are the source of synthesis and secretion of the endocrine biologically active factors of submandibular glands.

  14. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    Science.gov (United States)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  15. Blue-light filtering alters angiogenic signaling in human retinal pigmented epithelial cells culture model.

    Science.gov (United States)

    Vila, Natalia; Siblini, Aya; Esposito, Evangelina; Bravo-Filho, Vasco; Zoroquiain, Pablo; Aldrees, Sultan; Logan, Patrick; Arias, Lluis; Burnier, Miguel N

    2017-11-02

    Light exposure and more specifically the spectrum of blue light contribute to the oxidative stress in Age-related macular degeneration (AMD). The purpose of the study was to establish whether blue light filtering could modify proangiogenic signaling produced by retinal pigmented epithelial (RPE) cells under different conditions simulating risk factors for AMD. Three experiments were carried out in order to expose ARPE-19 cells to white light for 48 h with and without blue light-blocking filters (BLF) in different conditions. In each experiment one group was exposed to light with no BLF protection, a second group was exposed to light with BLF protection, and a control group was not exposed to light. The ARPE-19 cells used in each experiment prior to light exposure were cultured for 24 h as follows: Experiment 1) Normoxia, Experiment 2) Hypoxia, and Experiment 3) Lutein supplemented media in normoxia. The media of all groups was harvested after light exposure for sandwich ELISA-based assays to quantify 10 pro-angiogenic cytokines. A significant decrease in angiogenin secretion levels and a significant increase in bFGF were observed following light exposure, compared to dark conditions, in both normoxia and hypoxia conditions. With the addition of a blue light-blocking filter in normoxia, a significant increase in angiogenin levels was observed. Although statistical significance was not achieved, blue light filters reduce light-induced secretion of bFGF and VEGF to near normal levels. This trend is also observed when ARPE-19 cells are grown under hypoxic conditions and when pre-treated with lutein prior to exposure to experimental conditions. Following light exposure, there is a decrease in angiogenin secretion by ARPE-19 cells, which was abrogated with a blue light - blocking filter. Our findings support the position that blue light filtering affects the secretion of angiogenic factors by retinal pigmented epithelial cells under normoxic, hypoxic, and lutein

  16. Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation

    National Research Council Canada - National Science Library

    Feng, Wenqiang; Guo, Juanjuan; Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    .... 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...

  17. Reprogramming of cell junction modules during stepwise epithelial to mesenchymal transition and accumulation of malignant features in vitro in a prostate cell model

    Energy Technology Data Exchange (ETDEWEB)

    Ke, Xi-song, E-mail: Xisong.Ke@gades.uib.no [The Gade Institute, University of Bergen, Bergen (Norway); Department of Microbiology, Haukeland, University Hospital, Bergen (Norway); Li, Wen-cheng [The Gade Institute, University of Bergen, Bergen (Norway); Urological Department, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hovland, Randi [Center of Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen (Norway); Department of Molecular Biology, University of Bergen, Bergen (Norway); Qu, Yi [The Gade Institute, University of Bergen, Bergen (Norway); Liu, Run-hui [Modern Research Center for Traditional Chinese Medicine, Second Military Medical University, Shanghai (China); McCormack, Emmet [Department of Medicine, Haukeland University Hospital, Bergen (Norway); Thorsen, Frits [Department of Biomedicine, University of Bergen, Bergen (Norway); Olsen, Jan Roger [The Gade Institute, University of Bergen, Bergen (Norway); Molven, Anders [The Gade Institute, University of Bergen, Bergen (Norway); Department of Pathology, Haukeland University Hospital, Bergen (Norway); Kogan-Sakin, Ira; Rotter, Varda [Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot (Israel); Akslen, Lars A. [The Gade Institute, University of Bergen, Bergen (Norway); Department of Pathology, Haukeland University Hospital, Bergen (Norway); Oyan, Anne Margrete [The Gade Institute, University of Bergen, Bergen (Norway); Department of Microbiology, Haukeland, University Hospital, Bergen (Norway); Kalland, Karl-Henning, E-mail: Kalland@gades.uib.no [The Gade Institute, University of Bergen, Bergen (Norway); Department of Microbiology, Haukeland, University Hospital, Bergen (Norway)

    2011-01-15

    Epithelial to mesenchymal transition (EMT) is pivotal in tumor metastasis. Our previous work reported an EMT model based on primary prostate epithelial cells (EP156T) which gave rise to cells with mesenchymal phenotype (EPT1) without malignant transformation. To promote prostate cell transformation, cells were maintained in saturation density cultures to select for cells overriding quiescence. Foci formed repeatedly following around 8 weeks in confluent EPT1 monolayers. Only later passage EPT1, but not EP156T cells of any passage, could form foci. Cells isolated from the foci were named EPT2 and formed robust colonies in soft agar, a malignant feature present neither in EP156T nor in EPT1 cells. EPT2 cells showed additional malignant traits in vitro, including higher ability to proliferate following confluence, higher resistance to apoptosis and lower dependence on exogenous growth factors than EP156T and EPT1 cells. Microarray profiling identified gene sets, many of which belong to cell junction modules, that changed expression from EP156T to EPT1 cells and continued to change from EPT1 to EPT2 cells. Our findings provide a novel stepwise cell culture model in which EMT emerges independently of transformation and is associated with subsequent accumulation of malignant features in prostate cells. Reprogramming of cell junction modules is involved in both steps.

  18. A bio-artificial renal epithelial cell system conveys survival advantage in a porcine model of septic shock.

    Science.gov (United States)

    Westover, Angela J; Buffington, Deborah A; Johnston, Kimberly A; Smith, Peter L; Pino, Christopher J; Humes, H David

    2017-03-01

    Renal cell therapy using the hollow fiber based renal assist device (RAD) improved survival time in an animal model of septic shock (SS) through the amelioration of cardiac and vascular dysfunction. Safety and ability of the RAD to improve clinical outcomes was demonstrated in a Phase II clinical trial, in which patients had high prevalence of sepsis. Even with these promising results, clinical delivery of cell therapy is hampered by manufacturing hurdles, including cell sourcing, large-scale device manufacture, storage and delivery. To address these limitations, the bioartificial renal epithelial cell system (BRECS) was developed. The BRECS contains human renal tubule epithelial cells derived from adult progenitor cells using enhanced propagation techniques. Cells were seeded onto trabeculated disks of niobium-coated carbon, held within cryopreservable, perfusable, injection-moulded polycarbonate housing. The study objective was to evaluate the BRECS in a porcine model of SS to establish conservation of efficacy after necessary cell sourcing and design modifications; a pre-clinical requirement to move back into clinical trials. SS was incited by peritoneal injection of E. coli simultaneous to insertion of BRECS (n=10) or control (n=15), into the ultrafiltrate biofeedback component of an extracorporeal circuit. Comparable to RAD, prolonged survival of the BRECS cohort was conveyed through stabilization of cardiac output and vascular leak. In conclusion, the demonstration of conserved efficacy with BRECS therapy in a porcine SS model represents a crucial step toward returning renal cell therapy to the clinical setting, initially targeting ICU patients with acute kidney injury requiring continuous renal replacement therapy. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  19. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  20. Spontaneous transformation of murine oviductal epithelial cells: A model system to investigate the onset of fallopian-derived tumors

    Directory of Open Access Journals (Sweden)

    MIchael P. Endsley

    2015-07-01

    Full Text Available High-grade serous carcinoma (HGSC is the most lethal ovarian cancer histotype. The fallopian tube secretory epithelial cells (FTSECs are a proposed progenitor cell type. Genetically altered FTSECs form tumors in mice; however, a spontaneous HGSC model has not been described. Apart from a subpopulation of genetically predisposed women, most women develop ovarian cancer spontaneously, which is associated with aging and lifetime ovulations. A murine oviductal cell line (MOELOW was developed and continuously passaged in culture to mimic cellular aging (MOEHIGH. The MOEHIGH cellular model exhibited a loss of acetylated tubulin consistent with an outgrowth of secretory epithelial cells in culture. MOEHIGH cells proliferated significantly faster than MOELOW, and the MOEHIGH cells produced more 2D foci and 3D soft agar colonies as compared to MOELOW. MOEHIGH were xenografted into athymic female nude mice both in the subcutaneous and the intraperiteonal compartments. Only the subcutaneous grafts formed tumors that were negative for cytokeratin, but positive for oviductal markers such as oviductal glycoprotein 1 and Pax8. These tumors were considered to be poorly differentiated carcinoma. The differential molecular profiles between MOEHIGH and MOELOW were determined using RNA-Seq and confirmed by protein expression to uncover pathways important in transformation, like the p53 pathway, the FOXM1 pathway, WNT signaling, and splicing. MOEHIGH had enhanced protein expression of c-myc, Cyclin E, p53 and FOXM1 with reduced expression of p21. MOEHIGH were also less sensitive to cisplatin and DMBA, which induce lesions typically repaired by base-excision repair. A model of spontaneous tumorogenesis was generated starting with normal oviductal cells. Their transition to cancer involved alterations in pathways associated with high-grade serous cancer in humans.

  1. Human Three-Dimensional Endometrial Epithelial Cell Model To Study Host Interactions with Vaginal Bacteria and Neisseria gonorrhoeae.

    Science.gov (United States)

    Łaniewski, Paweł; Gomez, Adriana; Hire, Geoffrey; So, Magdalene; Herbst-Kralovetz, Melissa M

    2017-03-01

    Colonization of the endometrium by pathogenic bacteria ascending from the lower female reproductive tract (FRT) is associated with many gynecologic and obstetric health complications. To study these host-microbe interactions in vitro, we developed a human three-dimensional (3-D) endometrial epithelial cell (EEC) model using the HEC-1A cell line and the rotating wall vessel (RWV) bioreactor technology. Our model, composed of 3-D EEC aggregates, recapitulates several functional/structural characteristics of human endometrial epithelial tissue, including cell differentiation, the presence of junctional complexes/desmosomes and microvilli, and the production of membrane-associated mucins and Toll-like receptors (TLRs). TLR function was evaluated by exposing the EEC aggregates to viral and bacterial products. Treatment with poly(I·C) and flagellin but not with synthetic lipoprotein (fibroblast-stimulating lipoprotein 1 [FSL-1]) or lipopolysaccharide (LPS) significantly induced proinflammatory mediators in a dose-dependent manner. To simulate ascending infection, we infected EEC aggregates with commensal and pathogenic bacteria: Lactobacillus crispatus, Gardnerella vaginalis, and Neisseria gonorrhoeae All vaginal microbiota and N. gonorrhoeae efficiently colonized the 3-D surface, localizing to crevices of the EEC model and interacting with multiple adjacent cells simultaneously. However, only infection with pathogenic N. gonorrhoeae and not infection with the other bacteria tested significantly induced proinflammatory mediators and significant ultrastructural changes to the host cells. The latter observation is consistent with clinical findings and illustrated the functional specificity of our system. Additionally, we highlighted the utility of the 3-D EEC model for the study of the pathogenesis of N. gonorrhoeae using a well-characterized ΔpilT mutant. Overall, this study demonstrates that the human 3-D EEC model is a robust tool for studying host

  2. Epithelial Ovarian Cancer Experimental Models

    Science.gov (United States)

    Lengyel, E; Burdette, JE; Kenny, HA; Matei, D; Pilrose, J; Haluska, P.; Nephew, KP; Hales, DB; Stack, MS

    2014-01-01

    Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase, and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge and many approaches used to study other solid tumors (lung, colon, and breast, for example) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment. This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients, and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis, and chemoresistance. As these new approaches more accurately recapitulate the complex tumor microenvironment, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge. PMID:23934194

  3. Identification of Wnt responsive genes using a murine mammary epithelial cell line model system

    Directory of Open Access Journals (Sweden)

    Pennica Diane

    2004-05-01

    Full Text Available Abstract Background The Wnt/Wg pathway plays an important role in the developmental program of many cells and tissues in a variety of organisms. In addition, many Wnts and components of their downstream signaling pathways, such as β-catenin and APC, have been implicated in tumorigenesis. Over the past years, several genes have been identified as Wnt responsive, including c-myc, siamois, and cyclin D1. Results In order to identify additional genes responsive to Wnt signaling that contribute to the transformed phenotype, we performed a cDNA subtractive hybridization screen between a mouse mammary epithelial cell line that overexpresses Wnt-1 (C57MG/Wnt-1 and the parental cell line (C57MG. The screen identified a total of 67 genes to be up-regulated in response to Wnt signaling. Of these 67 genes, the up-regulation of 62 was subsequently confirmed by Northern and dot blot analyses (and, for a subset, semi-quantitative PCR of RNA isolated from C57MG cells subjected to (1 an independent Wnt-1 retroviral infection, and (2 co-culture with Wnt-1 expressing cells. Among the confirmed Wnt-1 responsive genes, we further characterized a mouse homolog of the human transcription factor Basic Transcription Element Binding protein 2 (BTEB2, Wnt-1 Responsive Cdc42 homolog (Wrch-1, and Wnt-1 Induced Secreted Protein (WISP-1. Conclusion Several novel genes were identified in this screen, as well as others that have been shown previously to be regulated by Wnt signaling, such as connexin43. The results indicate that cDNA subtractive hybridization is a useful method for identifying genes involved in the process of Wnt-1-induced transformation.

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

    Directory of Open Access Journals (Sweden)

    Atsushi Yasukawa

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

  5. Protons sensitize epithelial cells to mesenchymal transition.

    Science.gov (United States)

    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.

  6. Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury.

    Science.gov (United States)

    Leaw, Bryan; Zhu, Dandan; Tan, Jean; Muljadi, Ruth; Saad, Mohamed I; Mockler, Joanne C; Wallace, Euan M; Lim, Rebecca; Tolcos, Mary

    2017-02-28

    Human amnion epithelial cells (hAECs) are clonogenic and have been proposed to reduce inflammatory-induced tissue injury. Perturbation of the immune response is implicated in the pathogenesis of perinatal brain injury; modulating this response could thus be a novel therapy for treating or preventing such injury. The immunomodulatory properties of hAECs have been shown in other animal models, but a detailed investigation of the effects on brain immune cells following injury has not been undertaken. Here, we investigate the effects of hAECs on microglia, the first immune responders to injury within the brain. We generated a mouse model combining neonatal inflammation and perinatal hyperoxia, both of which are risk factors associated with perinatal brain injury. On embryonic day 16 we administered lipopolysaccharide (LPS), or saline (control), intra-amniotically to C57Bl/6 J mouse pups. On postnatal day (P)0, LPS pups were placed in hyperoxia (65% oxygen) and control pups in normoxia for 14 days. Pups were given either hAECs or saline intravenously on P4. At P14, relative to controls, LPS and hyperoxia pups had reduced body weight, increased density of apoptotic cells (TUNEL) in the cortex, striatum and white matter, astrocytes (GFAP) in the white matter and activated microglia (CD68) in the cortex and striatum, but no change in total microglia density (Iba1). hAEC administration rescued the decreased body weight and reduced apoptosis and astrocyte areal coverage in the white matter, but increased the density of total and activated microglia. We then stimulated primary microglia (CD45lowCD11b+) with LPS for 24 h, followed by co-culture with hAEC conditioned medium for 48 h. hAEC conditioned medium increased microglial phagocytic activity, decreased microglia apoptosis and decreased M1 activation markers (CD86). Stimulating hAECs for 24 h with LPS did not alter release of cytokines known to modulate microglia activity. These data demonstrate that hAECs can

  7. An orientation-independent DIC microscope allows high resolution imaging of epithelial cell migration and wound healing in a cnidarian model.

    Science.gov (United States)

    Malamy, J E; Shribak, M

    2018-01-18

    Epithelial cell dynamics can be difficult to study in intact animals or tissues. Here we use the medusa form of the hydrozoan Clytia hemisphaerica, which is covered with a monolayer of epithelial cells, to test the efficacy of an orientation-independent differential interference contrast microscope for in vivo imaging of wound healing. Orientation-independent differential interference contrast provides an unprecedented resolution phase image of epithelial cells closing a wound in a live, nontransgenic animal model. In particular, the orientation-independent differential interference contrast microscope equipped with a 40x/0.75NA objective lens and using the illumination light with wavelength 546 nm demonstrated a resolution of 460 nm. The repair of individual cells, the adhesion of cells to close a gap, and the concomitant contraction of these cells during closure is clearly visualized. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

  8. Isolation and culture of biliary epithelial cells.

    OpenAIRE

    Joplin, R

    1994-01-01

    At one time it was thought that biliary epithelial cells simply formed the lining to the tubular conduits which constitute the biliary tract. Development of in vitro systems for culturing biliary epithelial cells has enabled functional studies which increasingly show that this is far from true, and that biliary epithelial cells do have important functional roles. Disruption of these functions may be involved in the generation of pathology. Most functional studies to date have utilised cells i...

  9. Enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model.

    Science.gov (United States)

    Naz, Neveda; Mills, Dominic C; Wren, Brendan W; Dorrell, Nick

    2013-10-22

    The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

  10. The effect of size on Ag nanosphere toxicity in macrophage cell models and lung epithelial cell lines is dependent on particle dissolution.

    Science.gov (United States)

    Hamilton, Raymond F; Buckingham, Sarah; Holian, Andrij

    2014-04-22

    Silver (Ag) nanomaterials are increasingly used in a variety of commercial applications. This study examined the effect of size (20 and 110 nm) and surface stabilization (citrate and PVP coatings) on toxicity, particle uptake and NLRP3 inflammasome activation in a variety of macrophage and epithelial cell lines. The results indicated that smaller Ag (20 nm), regardless of coating, were more toxic in both cell types and most active in the THP-1 macrophages. TEM imaging demonstrated that 20 nm Ag nanospheres dissolved more rapidly than 110 nm Ag nanospheres in acidic phagolysosomes consistent with Ag ion mediated toxicity. In addition, there were some significant differences in epithelial cell line in vitro exposure models. The order of the epithelial cell lines' sensitivity to Ag was LA4 > MLE12 > C10. The macrophage sensitivity to Ag toxicity was C57BL/6 AM > MARCO null AM, which indicated that the MARCO receptor was involved in uptake of the negatively charged Ag particles. These results support the idea that Ag nanosphere toxicity and NLRP3 inflammasome activation are determined by the rate of surface dissolution, which is based on relative surface area. This study highlights the importance of utilizing multiple models for in vitro studies to evaluate nanomaterials.

  11. The Effect of Size on Ag Nanosphere Toxicity in Macrophage Cell Models and Lung Epithelial Cell Lines Is Dependent on Particle Dissolution

    Directory of Open Access Journals (Sweden)

    Raymond F. Hamilton

    2014-04-01

    Full Text Available Silver (Ag nanomaterials are increasingly used in a variety of commercial applications. This study examined the effect of size (20 and 110 nm and surface stabilization (citrate and PVP coatings on toxicity, particle uptake and NLRP3 inflammasome activation in a variety of macrophage and epithelial cell lines. The results indicated that smaller Ag (20 nm, regardless of coating, were more toxic in both cell types and most active in the THP-1 macrophages. TEM imaging demonstrated that 20 nm Ag nanospheres dissolved more rapidly than 110 nm Ag nanospheres in acidic phagolysosomes consistent with Ag ion mediated toxicity. In addition, there were some significant differences in epithelial cell line in vitro exposure models. The order of the epithelial cell lines’ sensitivity to Ag was LA4 > MLE12 > C10. The macrophage sensitivity to Ag toxicity was C57BL/6 AM > MARCO null AM, which indicated that the MARCO receptor was involved in uptake of the negatively charged Ag particles. These results support the idea that Ag nanosphere toxicity and NLRP3 inflammasome activation are determined by the rate of surface dissolution, which is based on relative surface area. This study highlights the importance of utilizing multiple models for in vitro studies to evaluate nanomaterials.

  12. Progressive Depletion of Rough Endoplasmic Reticulum in Epithelial Cells of the Small Intestine in Monosodium Glutamate Mice Model of Obesity

    Directory of Open Access Journals (Sweden)

    Kazuhiko Nakadate

    2016-01-01

    Full Text Available Chronic obesity is a known risk factor for metabolic syndrome. However, little is known about pathological changes in the small intestine associated with chronic obesity. This study investigated cellular and subcellular level changes in the small intestine of obese mice. In this study, a mouse model of obesity was established by early postnatal administration of monosodium glutamate. Changes in body weight were monitored, and pathological changes in the small intestine were evaluated using hematoxylin-eosin and Nissl staining and light and electron microscopy. Consequently, obese mice were significantly heavier compared with controls from 9 weeks of age. Villi in the small intestine of obese mice were elongated and thinned. There was reduced hematoxylin staining in the epithelium of the small intestine of obese mice. Electron microscopy revealed a significant decrease in and shortening of rough endoplasmic reticulum in epithelial cells of the small intestine of obese mice compared with normal mice. The decrease in rough endoplasmic reticulum in the small intestine epithelial cells of obese mice indicates that obesity starting in childhood influences various functions of the small intestine, such as protein synthesis, and could impair both the defense mechanism against invasion of pathogenic microbes and nutritional absorption.

  13. Agent-based model of human alveoli predicts chemotactic signaling by epithelial cells during early Aspergillus fumigatus infection.

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    Johannes Pollmächer

    Full Text Available Aspergillus fumigatus is one of the most important human fungal pathogens, causing life-threatening diseases. Since humans inhale hundreds to thousands of fungal conidia every day, the lower respiratory tract is the primary site of infection. Current interaction networks of the innate immune response attribute fungal recognition and detection to alveolar macrophages, which are thought to be the first cells to get in contact with the fungus. At present, these networks are derived from in vitro or in situ assays, as the peculiar physiology of the human lung makes in vivo experiments, including imaging on the cell-level, hard to realize. We implemented a spatio-temporal agent-based model of a human alveolus in order to perform in silico experiments of a virtual infection scenario, for an alveolus infected with A. fumigatus under physiological conditions. The virtual analog captures the three-dimensional alveolar morphology consisting of the two major alveolar epithelial cell types and the pores of Kohn as well as the dynamic process of respiration. To the best of our knowledge this is the first agent-based model of a dynamic human alveolus in the presence of respiration. A key readout of our simulations is the first-passage-time of alveolar macrophages, which is the period of time that elapses until the first physical macrophage-conidium contact is established. We tested for random and chemotactic migration modes of alveolar macrophages and varied their corresponding parameter sets. The resulting first-passage-time distributions imply that randomly migrating macrophages fail to find the conidium before the start of germination, whereas guidance by chemotactic signals derived from the alveolar epithelial cell associated with the fungus enables a secure and successful discovery of the pathogen in time.

  14. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

    National Research Council Canada - National Science Library

    Choi, Chul Hee; Lee, Jun Sik; Lee, Yoo Chul; Park, Tae In; Lee, Je Chul

    2008-01-01

    ... of A. baumannii outer membrane protein A (AbOmpA) in interactions with epithelial cells. A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms...

  15. Medullary Thymic Epithelial Cells and Central Tolerance in Autoimmune Hepatitis Development: Novel Perspective from a New Mouse Model

    Directory of Open Access Journals (Sweden)

    Konstantina Alexandropoulos

    2015-01-01

    Full Text Available Autoimmune hepatitis (AIH is an immune-mediated disorder that affects the liver parenchyma. Diagnosis usually occurs at the later stages of the disease, complicating efforts towards understanding the causes of disease development. While animal models are useful for studying the etiology of autoimmune disorders, most of the existing animal models of AIH do not recapitulate the chronic course of the human condition. In addition, approaches to mimic AIH-associated liver inflammation have instead led to liver tolerance, consistent with the high tolerogenic capacity of the liver. Recently, we described a new mouse model that exhibited spontaneous and chronic liver inflammation that recapitulated the known histopathological and immunological parameters of AIH. The approach involved liver-extrinsic genetic engineering that interfered with the induction of T-cell tolerance in the thymus, the very process thought to inhibit AIH induction by liver-specific expression of exogenous antigens. The mutation led to depletion of specialized thymic epithelial cells that present self-antigens and eliminate autoreactive T-cells before they exit the thymus. Based on our findings, which are summarized below, we believe that this mouse model represents a relevant experimental tool towards elucidating the cellular and molecular aspects of AIH development and developing novel therapeutic strategies for treating this disease.

  16. An in vitro model of intra-epithelial expansion of transformed urothelial cells

    NARCIS (Netherlands)

    Rebel, J.M.J.; Boer, de W.I.; Thijssen, C.D.; Vermey, M.; Zwarthoff, E.C.; Kwast, van der T.H.

    1993-01-01

    Replacement of normal urothelium by pre-cancerous epithelium may explain the high recurrence rate of human bladder cancer. An in vitro model was designed in order to study the mechanisms of expansion of transformed urothelial cells at the expense of normal urothelium. For this purpose, mouse bladder

  17. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo, E-mail: zhuo.zhang@uky.edu

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit

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

    Science.gov (United States)

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

    2016-07-19

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

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

    Directory of Open Access Journals (Sweden)

    Jonathan M. Williams

    2013-11-01

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

  20. Senescent Fibroblasts Promote Neoplastic Transformation of Partially Transformed Ovarian Epithelial Cells in a Three-dimensional Model of Early Stage Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kate Lawrenson

    2010-04-01

    Full Text Available Most epithelial ovarian cancers are diagnosed postmenopausally, although the well-established epidemiological risk factors (parity, oral contraceptive use are premenopausal. We hypothesized that accumulation of senescent fibroblasts, together with concomitant loss of presenescent fibroblasts within the ovarian cortex, promotes initiation and early development of ovarian cancer from ovarian surface epithelial (OSE cells. To test this, we established immortalized OSE (IOSE cell lines that mimic early neoplastic transformation by overexpressing the CMYC oncogene (IOSECMYC and normal ovarian presenescent (PSN and senescent (SEN fibroblast cell lines. We then evaluated the ability of PSN and SEN fibroblasts to transform IOSE and IOSECMYC after coculture. SEN fibroblasts significantly enhanced neoplastic development of IOSECMYC cells; there was an up to 15-fold increase in migration of IOSECMYC cells cocultured with SEN fibroblasts compared with PSN fibroblasts. Conditioned medium from SEN fibroblasts promoted anchorage-independent growth of IOSECMYC cells. We studied fibroblast-epithelial cell interactions in heterotypic three-dimensional spheroid models. Dual immunohistochemical staining of spheroids for a proliferation marker (MIB-1 and cytokeratin-18 indicated that SEN fibroblasts induce approximately a five-fold increase in proliferation of IOSECMYC cells relative to cocultures with PSN fibroblasts. SEN, but not PSN fibroblasts, also induced nuclear atypia in epithelial cells in three-dimensional spheroids. These data suggest for the first time that the accumulation of senescent, or loss of presenescent fibroblasts, can promote neoplastic development of partially transformed OSE cells in vitro and illustrates the power of using three-dimensional heterotypic modeling to gain better insights into the etiology underlying the development of epithelial ovarian cancer.

  1. Epithelial Cells in Urine: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

  2. Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge.

    Science.gov (United States)

    Bannunah, Azzah M; Vllasaliu, Driton; Lord, Jennie; Stolnik, Snjezana

    2014-12-01

    This study investigated the effect of nanoparticle size (50 and 100 nm) and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number, and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below half maximal effective concentration (EC₅₀) revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol depletion (38%; via methyl-β-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in

  3. Increased COX-2 expression in epithelial and stromal cells of high mammographic density tissues and in a xenograft model of mammographic density.

    Science.gov (United States)

    Chew, G L; Huo, C W; Huang, D; Hill, P; Cawson, J; Frazer, H; Hopper, J L; Haviv, I; Henderson, M A; Britt, K; Thompson, E W

    2015-08-01

    Mammographic density (MD) adjusted for age and body mass index is one of the strongest known risk factors for breast cancer. Given the high attributable risk of MD for breast cancer, chemoprevention with a safe and available agent that reduces MD and breast cancer risk would be beneficial. Cox-2 has been implicated in MD-related breast cancer risk, and was increased in stromal cells in high MD tissues in one study. Our study assessed differential Cox-2 expression in epithelial and stromal cells in paired samples of high and low MD human breast tissue, and in a validated xenograft biochamber model of MD. We also examined the effects of endocrine treatment upon Cox-2 expression in high and low MD tissues in the MD xenograft model. Paired high and low MD human breast tissue samples were immunostained for Cox-2, then assessed for differential expression and staining intensity in epithelial and stromal cells. High and low MD human breast tissues were separately maintained in biochambers in mice treated with Tamoxifen, oestrogen or placebo implants, then assessed for percentage Cox-2 staining in epithelial and stromal cells. Percentage Cox-2 staining was greater for both epithelial (p = 0.01) and stromal cells (p tissues. In high MD biochamber tissues, percentage Cox-2 staining was greater in stromal cells of oestrogen-treated versus placebo-treated tissues (p = 0.05).

  4. Adult thymic epithelial cell (TEC) progenitors and TEC stem cells: Models and mechanisms for TEC development and maintenance.

    Science.gov (United States)

    Hamazaki, Yoko

    2015-11-01

    The thymus is the primary lymphoid organ for generating self-restricted and self-tolerant functional T cells. Its two distinct anatomical regions, the cortex and the medulla, are involved in positive and negative selection, respectively. Thymic epithelial cells (TECs) constitute the framework of this tissue and function as major stromal components. Extensive studies for more than a decade have revealed how TECs are generated during organogenesis; progenitors specific for medullary TECs (mTECs) and cortical TECs (cTECs) as well as bipotent progenitors for both lineages have been identified, and the signaling pathways required for the development and maturation of mTECs have been elucidated. However, little is known about the initial commitment of mTECs and cTECs during ontogeny, and how regeneration of both lineages is sustained in the postnatal/adult thymus. Recently, stem cell activities in TECs have been demonstrated, and TEC progenitors have been identified in the postnatal thymus. In this review, recent advances in studying the development and maintenance of TECs are summarized, and the possible mechanisms of thymic regeneration and involution are discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A Comprehensive, Multi-Scale Dynamical Model of ErbB Receptor Signal Transduction in Human Mammary Epithelial Cells

    Science.gov (United States)

    Helikar, Tomáš; Kochi, Naomi; Kowal, Bryan; Dimri, Manjari; Naramura, Mayumi; Raja, Srikumar M.; Band, Vimla; Band, Hamid; Rogers, Jim A.

    2013-01-01

    The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1) have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins and their post-translational modifications sites, and over 1,000 biochemical interactions. Using computer simulations of the model, we find it is able to reproduce a number of cellular phenomena. Furthermore, the model predicts that overexpression of Src results in increased endocytosis of EGFR in the absence/low amount of the epidermal growth factor (EGF). Our subsequent laboratory experiments also suggest increased internalization of EGFR upon Src overexpression under EGF-deprived conditions, further supporting this model-generated hypothesis. PMID:23637902

  6. A comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Tomáš Helikar

    Full Text Available The non-receptor tyrosine kinase Src and receptor tyrosine kinase epidermal growth factor receptor (EGFR/ErbB1 have been established as collaborators in cellular signaling and their combined dysregulation plays key roles in human cancers, including breast cancer. In part due to the complexity of the biochemical network associated with the regulation of these proteins as well as their cellular functions, the role of Src in EGFR regulation remains unclear. Herein we present a new comprehensive, multi-scale dynamical model of ErbB receptor signal transduction in human mammary epithelial cells. This model, constructed manually from published biochemical literature, consists of 245 nodes representing proteins and their post-translational modifications sites, and over 1,000 biochemical interactions. Using computer simulations of the model, we find it is able to reproduce a number of cellular phenomena. Furthermore, the model predicts that overexpression of Src results in increased endocytosis of EGFR in the absence/low amount of the epidermal growth factor (EGF. Our subsequent laboratory experiments also suggest increased internalization of EGFR upon Src overexpression under EGF-deprived conditions, further supporting this model-generated hypothesis.

  7. STORM: a general model to determine the number and adaptive changes of epithelial stem cells in teleost, murine and human intestinal tracts.

    Directory of Open Access Journals (Sweden)

    Zhengyuan Wang

    Full Text Available Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0-4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0-4.1 stem cells on each crypt section of murine small intestine and 1.8-3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions.

  8. Liver-Directed Human Amniotic Epithelial Cell Transplantation Improves Systemic Disease Phenotype in Hurler Syndrome Mouse Model.

    Science.gov (United States)

    Rodriguez, Natalie S; Yanuaria, Lisa; Parducho, Kevin Murphy R; Garcia, Irving M; Varghese, Bino A; Grubbs, Brendan H; Miki, Toshio

    2017-07-01

    Mucopolysaccharidosis type 1 (MPS1) is an inherited lysosomal storage disorder caused by a deficiency in the glycosaminoglycan (GAG)-degrading enzyme α-l-iduronidase (IDUA). In affected patients, the systemic accumulation of GAGs results in skeletal dysplasia, neurological degeneration, multiple organ dysfunction, and early death. Current therapies, including enzyme replacement and bone marrow transplant, improve life expectancy but the benefits to skeletal and neurological phenotypes are limited. In this study, we tested the therapeutic efficacy of liver-directed transplantation of a placental stem cell, which possesses multilineage differentiation potential, low immunogenicity, and high lysosomal enzyme activity. Unfractionated human amniotic epithelial cells (hAECs) were transplanted directly into the liver of immunodeficient Idua knockout mouse neonates. The hAECs engraftment was immunohistochemically confirmed with anti-human mitochondria staining. Enzyme activity assays indicated that hAECs transplantation restored IDUA function in the liver and significantly decreased urinary GAG excretion. Histochemical and micro-computed tomography analyses revealed reduced GAG deposition in the phalanges joints and composition/morphology improvement of cranial and facial bones. Neurological assessment in the hAEC treated mice showed significant improvement of sensorimotor coordination in the hAEC treated mice compared to untreated mice. Results confirm that partial liver cell replacement with placental stem cells can provide long-term (>20 weeks) and systemic restoration of enzyme function, and lead to significant phenotypic improvement in the MPS1 mouse model. This preclinical data indicate that liver-directed placental stem cell transplantation may improve skeletal and neurological phenotypes of MPS1 patients. Stem Cells Translational Medicine 2017;6:1583-1594. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of Alpha

  9. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

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

  10. Identification of epithelial cells in bronchoalveolar lavage.

    Science.gov (United States)

    Finotto, S; Rado, V; Dal Vecchio, A; Milani, G; Fabbri, L M; Maestrelli, P

    1993-01-01

    1. Damage to the bronchial epithelium occurs after the inhalation of toxic substances and allergens, and through virus infections and it may lead to increased desquamation of epithelial cells in bronchoalveolar lavage (BAL). 2. In this study we compared two methods of staining the epithelial cells of BAL, the conventional cytochemical May Grunwald-Giemsa stain (MGG) and an immunocytochemical technique using a monoclonal antibody anti-human cytokeratin (CK) detected with APAAP immuno-alkaline phosphatase. BAL was obtained from 13 subjects and the epithelial cells were cytocentrifuged either immediately after collection (fraction A) or after washing (fraction B). 3. Higher percentages of epithelial cells were identified in fraction A with CK (20.0 +/- 5.1%) than in fraction A with MGG (11.2 +/- 2.3%), which recognized only ciliated epithelial cells. In fact a proportion of CK-positive cells (34%) in fraction A were not ciliated. Underestimation of epithelial cells by MGG compared to CK was more pronounced in fraction B (8.0 +/- 2.9% and 22.9 +/- 3.0%, respectively) as there was a relative loss of ciliated CK+ cells after washings. 4. These results suggest that immunocytochemical staining with an anti-cytokeratin monoclonal antibody is more sensitive than using the MGG stain in detecting epithelial cells in BAL.

  11. Urinary Excretion of Tetrodotoxin Modeled in a Porcine Renal Proximal Tubule Epithelial Cell Line, LLC-PK1

    Directory of Open Access Journals (Sweden)

    Takuya Matsumoto

    2017-07-01

    Full Text Available This study examined the urinary excretion of tetrodotoxin (TTX modeled in a porcine renal proximal tubule epithelial cell line, LLC-PK1. Time course profiles of TTX excretion and reabsorption across the cell monolayers at 37 °C showed that the amount of TTX transported increased linearly for 60 min. However, at 4 °C, the amount of TTX transported was approximately 20% of the value at 37 °C. These results indicate that TTX transport is both a transcellular and carrier-mediated process. Using a transport inhibition assay in which cell monolayers were incubated with 50 µM TTX and 5 mM of a transport inhibitor at 37 °C for 30 min, urinary excretion was significantly reduced by probenecid, tetraethylammonium (TEA, l-carnitine, and cimetidine, slightly reduced by p-aminohippuric acid (PAH, and unaffected by 1-methyl-4-phenylpyridinium (MPP+, oxaliplatin, and cefalexin. Renal reabsorption was significantly reduced by PAH, but was unaffected by probenecid, TEA and l-carnitine. These findings indicate that TTX is primarily excreted by organic cation transporters (OCTs and organic cation/carnitine transporters (OCTNs, partially transported by organic anion transporters (OATs and multidrug resistance-associated proteins (MRPs, and negligibly transported by multidrug and toxic compound extrusion transporters (MATEs.

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

    Science.gov (United States)

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

    2016-05-14

    To analyze the effect of three-dimensional (3D)-arrangement on the expression of epithelial-to-mesenchymal transition markers in pancreatic adenocarcinoma (PDAC) cells. 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. 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. 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.

  13. Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer.

    Science.gov (United States)

    Timofeeva, Olga A; Palechor-Ceron, Nancy; Li, Guanglei; Yuan, Hang; Krawczyk, Ewa; Zhong, Xiaogang; Liu, Geng; Upadhyay, Geeta; Dakic, Aleksandra; Yu, Songtao; Fang, Shuang; Choudhury, Sujata; Zhang, Xueping; Ju, Andrew; Lee, Myeong-Seon; Dan, Han C; Ji, Youngmi; Hou, Yong; Zheng, Yun-Ling; Albanese, Chris; Rhim, Johng; Schlegel, Richard; Dritschilo, Anatoly; Liu, Xuefeng

    2017-04-04

    Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

  14. Indirubin Treatment of Lipopolysaccharide-Induced Mastitis in a Mouse Model and Activity in Mouse Mammary Epithelial Cells.

    Science.gov (United States)

    Lai, Jin-Lun; Liu, Yu-Hui; Peng, Yong-Chong; Ge, Pan; He, Chen-Fei; Liu, Chang; Chen, Ying-Yu; Guo, Ai-Zhen; Hu, Chang-Min

    2017-01-01

    Indirubin is a Chinese medicine extracted from indigo and known to be effective for treating chronic myelogenous leukemia, neoplasia, and inflammatory disease. This study evaluated the in vivo anti-inflammatory activity of indirubin in a lipopolysaccharide- (LPS-) induced mouse mastitis model. The indirubin mechanism and targets were evaluated in vitro in mouse mammary epithelial cells. In the mouse model, indirubin significantly attenuated the severity of inflammatory lesions, edema, inflammatory hyperemia, milk stasis and local tissue necrosis, and neutrophil infiltration. Indirubin significantly decreased myeloperoxidase activity and downregulated the production of tumor necrosis factor- α , interleukin-1 β (IL-1 β ), and IL-6 caused by LPS. In vitro, indirubin inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner. It also downregulated LPS-induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF- κ B) P65 protein and inhibitor of kappa B. In addition to its effect on the NF- κ B signaling pathway, indirubin suppressed the mitogen-activated protein kinase (MAPK) signaling by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), P38, and c-jun NH2-terminal kinase (JNK). Indirubin improved LPS-induced mouse mastitis by suppressing TLR4 and downstream NF- κ B and MAPK pathway inflammatory signals and might be a potential treatment of mastitis and other inflammatory diseases.

  15. Helicobacter pylori CagA and IL-1β Promote the Epithelial-to-Mesenchymal Transition in a Nontransformed Epithelial Cell Model

    Directory of Open Access Journals (Sweden)

    Haruki Arévalo-Romero

    2016-01-01

    Full Text Available Gastric cancer is the third cause of cancer death worldwide and infection by Helicobacter pylori (H. pylori is considered the most important risk factor, mainly by the activity of its virulence factor CagA. H. pylori/CagA-induced chronic inflammation triggers a series of gastric lesions of increased severity, starting with gastritis and ending with cancer. IL-1β has been associated with tumor development and invasiveness in different types of cancer, including gastric cancer. Currently, it is not clear if there is an association between CagA and IL-1β at a cellular level. In this study, we analyzed the effects of IL-1β and CagA on MCF-10A nontransformed cells. We found evidence that both CagA and IL-1β trigger the initiation of the epithelial-to-mesenchymal transition characterized by β-catenin nuclear translocation, increased expression of Snail1 and ZEB1, downregulation of CDH1, and morphological changes during MCF-10A acini formation. However, only CagA induced MMP9 activity and cell invasion. Our data support that IL-1β and CagA target the β-catenin pathway, with CagA leading to acquisition of a stage related to aggressive tumors.

  16. TRAM-Derived Decoy Peptides inhibits the inflammatory response in mouse mammary epithelial cells and a mastitis model in mice.

    Science.gov (United States)

    Hu, Xiaoyu; Tian, Yuan; Wang, Tiancheng; Zhang, Wenlong; Wang, Wei; Gao, Xuejiao; Qu, Shihui; Cao, Yongguo; Zhang, Naisheng

    2015-10-05

    It has been proved that TRAM-Derived Decoy peptides have anti-inflammatory properties. In this study, we synthesized a TRAM-Derived decoy peptide (TM6), belongs to TRAM TIR domain, of which sequence is "N"-RQIKIWFQNRRMKWK, KENFLRDTWCNFQFY-"C" and evaluated the effects of TM6 on lipopolysaccharide-induced mastitis in mice. In vivo, LPS-induced mice mastitis model was established by injection of LPS through the duct of mammary gland. TM6 was injected 1h before or after LPS treatment. In vitro, primary mouse mammary epithelial cells were used to investigate the effects of TM6 on LPS-induced inflammatory responses. The results showed that TM6 inhibited LPS-induced mammary gland histopathologic changes, MPO activity, and TNF-α, IL-1β and IL-6 production in mice. In vitro, TM6 significantly inhibited LPS-induced TNF-α and IL-6 production, as well as NF-κB and MAPKs activation. In conclusion, this study demonstrated that TM6 had protective effects on LPS-mastitis and may be a promising therapeutic reagent for mastitis treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Mitochondria-Rich Cells as Experimental Model in Studies of Epithelial Chloride Channels

    DEFF Research Database (Denmark)

    Willumsen, Niels J.; Amstrup, Jan; Møbjerg, Nadja

    2002-01-01

    in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl-]o is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion......The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl- channels, of which the function of only two types has been studied......-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl- pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via ß-adrenergic receptors, ion selectivity, genistein stimulation...

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

    Science.gov (United States)

    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 ...

  19. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  20. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

    Directory of Open Access Journals (Sweden)

    Alvaro Plaza Reyes

    2016-01-01

    Full Text Available Human embryonic stem cell (hESC-derived retinal pigment epithelial (RPE cells could replace lost tissue in geographic atrophy (GA but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

  1. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  2. Porcine small intestinal epithelial cell line (IPEC-J2) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics.

    Science.gov (United States)

    Liu, Fangning; Li, Guohua; Wen, Ke; Bui, Tammy; Cao, Dianjun; Zhang, Yanming; Yuan, Lijuan

    2010-04-01

    Previous studies of epithelial immune responses to rotavirus infection have been conducted in transformed cell lines. In this study, we evaluated a non-transformed porcine jejunum epithelial cell line (IPEC-J2) as an in-vitro model of rotavirus infection and probiotic treatment. Cell-culture-adapted porcine rotavirus (PRV) OSU strain, or human rotavirus (HRV) Wa strain, along with Lactobacillus acidophilus (LA) or Lactobacillus rhamnosus GG (LGG) were used to inoculate IPEC-J2 cells. LA or LGG treatment was applied pre- or post-rotavirus infection. We demonstrated that IPEC-J2 cells were productively infected by PRV. LA or LGG treatment of the cells did not reduce virus replication. PRV infection increased MUC3 mucin secretion. LGG treatment post-rotavirus infection reduced the mucin secretion response induced by PRV; LGG alone increased the production of membrane-associated MUC3 mucin. LA treatment prior to rotavirus infection significantly increased PRV replication and the IL-6 response to PRV infection, which is consistent with the adjuvant effect of LA. LGG treatment post-rotavirus infection downregulated the IL-6 response, confirming the anti-inflammatory effect of LGG. IPEC-J2 cells expressed toll-like receptor (TLR) 2, TLR3, and TLR9 constitutively. TLR2 expression was upregulated by LGG and peptidoglycan, corresponding to the decreased IL-6 response, indicating that the protective effect of LGG is associated with upregulation of TLR2 expression on intestinal epithelial cells. The IPEC-J2 cell model of PRV infection is a completely homologous system. It is a valuable model for studying the interactions among rotavirus-host-probiotics, and the mechanisms behind the immunomodulating effect of probiotic bacteria on innate immune responses.

  3. Cancer Stem Cells and Epithelial Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Sheetal Dyall

    2010-01-01

    Full Text Available The cancer stem cell hypothesis is becoming more widely accepted as a model for carcinogenesis. Tumours are heterogeneous both at the molecular and cellular level, containing a small population of cells that possess highly tumourigenic “stem-cell” properties. Cancer stem cells (CSCs, or tumour-initiating cells, have the ability to self-renew, generate xenografts reminiscent of the primary tumour that they were derived from, and are chemoresistant. The characterisation of the CSC population within a tumour that drives its growth could provide novel target therapeutics against these cells specifically, eradicating the cancer completely. There have been several reports describing the isolation of putative cancer stem cell populations in several cancers; however, no defined set of markers has been identified that conclusively characterises “stem-like” cancer cells. This paper highlights the current experimental approaches that have been used in the field and discusses their limitations, with specific emphasis on the identification and characterisation of the CSC population in epithelial ovarian cancer.

  4. Epithelial ovarian cancer: focus on genetics and animal models.

    Science.gov (United States)

    Shan, Weiwei; Liu, Jinsong

    2009-03-01

    Despite rapid advances in understanding ovarian cancer etiology, epithelial ovarian cancer remains the most lethal form of gynecologic cancers in the United States. The four morphologically-defined epithelial ovarian cancer subtypes-serous, endometrioid, mucinous, and clear cell carcinomas--are generally believed to originate from ovarian epithelial cells. Although it remains unclear how this single cell layer gives rise to morphologically distinct cancers, it has been suggested that early genetic events may direct the differentiation of ovarian epithelial cells. A number of genetic alterations are frequently encountered during ovarian tumorigenesis, including oncogenic activities of KRAS, BRAF and AKT, and silencing mutations of TP53, RB and PTEN. However, knowledge about how these genetic elements are coordinated during ovarian cancer initiation and progression is very limited. The establishment of cell-culture systems and rodent-based models has made big strides towards a better understanding of the genetic bases of human epithelial ovarian tumorigenesis. More importantly, the rise of genetically-engineered rodent and human models, particularly in the past five years, has provided key insight in the role of specific genes during ovarian tumorigenesis. In this review, we offer a comprehensive coverage of currently-available in vitro and in vivo models of human epithelial ovarian cancer, focusing on latest updates of genetically-modified rodent and human models and the valuable information conveyed by them.

  5. Human amniotic epithelial cells inhibit growth of epithelial ovarian cancer cells via TGF‑β1-mediated cell cycle arrest.

    Science.gov (United States)

    Bu, Shixia; Zhang, Qiuwan; Wang, Qian; Lai, Dongmei

    2017-11-01

    It is reported that human amniotic epithelial cells (hAECs) endow intrinsic antitumor effects on certain kinds of cancer. This research was designed to evaluate whether hAECs endowed potential anticancer properties on epithelial ovarian cancer (EOC) cells in vivo and in vitro, which has not been reported before. In this study, we established a xenografted BALB/c nude mouse model by subcutaneously co-injecting ovarian cancer cell line, SK-OV-3, and hAECs for 28 days. In ex vivo experiments, CCK‑8 cell viability assay, real-time PCR, cell counting assay, cell cycle analysis and immunohistochemistry (IHC) assay were used to detect the effects of hAEC‑secreted factors on the proliferation and cell cycle progression of EOC cells. A cytokine array was conducted to detect anticancer-related cytokines released from hAECs. Human recombinant TGF‑β1 and TGF‑β1 antibody were used to treat EOC cells and analyzed whether TGF‑β1 contributed to the cell cycle arrest. Results from in vivo and ex vivo experiments showed that hAEC-secreted factors and rhTGF‑β1 decreased proliferation of EOC cells and induced G0/G1 cell cycle arrest in cancer cells, which could be partially reversed by excess TGF‑β1 antibody. These data indicate that hAECs endow potential anticancer properties on epithelial ovarian cancer in vivo and in vitro which is partially mediated by hAEC‑secreted TGF‑β1-induced cell cycle arrest. This study suggests a potential application of hAEC‑based therapy against epithelial ovarian cancer.

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

    Directory of Open Access Journals (Sweden)

    Minhua DENG

    2017-02-01

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

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

    Science.gov (United States)

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

    2017-02-20

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

  8. Structure-activity relationship studies of 1,7-diheteroarylhepta-1,4,6-trien-3-ones with two different terminal rings in prostate epithelial cell models.

    Science.gov (United States)

    Wang, Rubing; Zhang, Xiaojie; Chen, Chengsheng; Chen, Guanglin; Sarabia, Cristian; Zhang, Qiang; Zheng, Shilong; Wang, Guangdi; Chen, Qiao-Hong

    2017-06-16

    To systematically investigate the structure-activity relationships of 1,7-diheteroarylhepta-1,4,6-trien-3-ones in three human prostate cancer cell models and one human prostate non-neoplastic epithelial cell model, thirty five 1,7-diarylhepta-1,4,6-trien-3-ones with different terminal heteroaromatic rings have been designed for evaluation of their anti-proliferative potency in vitro. These target compounds have been successfully synthesized through two sequential Horner-Wadsworth-Emmons reactions starting from the appropriate aldehydes and tetraethyl (2-oxopropane-1,3-diyl)bis(phosphonate). Their anti-proliferative potency against PC-3, DU-145 and LNCaP human prostate cancer cell lines can be significantly enhanced by the manipulation of the terminal heteroaromatic rings, further demonstrating the utility of 1,7-diarylhepta-1,4,6-trien-3-one as a potential scaffold for the development of anti-prostate cancer agents. The optimal analog 40 is 82-, 67-, and 39-fold more potent than curcumin toward the three prostate cancer cell lines, respectively. The experimental data also reveal that the trienones with two different terminal aromatic rings possess greater potency toward three prostate cancer cell lines, but also have greater capability of suppressing the proliferation of PWR-1E benign human prostate epithelial cells, as compared to the corresponding counterparts with two identical terminal rings and curcumin. The terminal aromatic rings also affect the cell apoptosis perturbation. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  9. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally,

  10. Cellular uptake but low permeation of human calcitonin-derived cell penetrating peptides and Tat(47-57) through well-differentiated epithelial models

    DEFF Research Database (Denmark)

    Tréhin, Rachel; Krauss, Ulrike; Beck-Sickinger, Annette G

    2004-01-01

    To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers.......To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers....

  11. Elucidation of epithelial-mesenchymal transition-related pathways in a triple-negative breast cancer cell line model by multi-omics interactome analysis

    DEFF Research Database (Denmark)

    Pauling, Josch K; Christensen, Anne G; Batra, Richa

    2014-01-01

    gene expression, protein expression or post-translational modifications. To overcome single omics analysis, we developed a set of computational methods that allow a combined analysis of data collections from multiple omics fields utilizing hybrid interactome networks. We apply these methods to data...... obtained from a triple-negative breast cancer cell line model, combining data sets of gene and protein expression as well as protein phosphorylation. We focus on alterations associated with the phenotypical differences arising from epithelial-mesenchymal transition in two breast cancer cell lines...

  12. Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model.

    Directory of Open Access Journals (Sweden)

    Elhaseen Elamin

    Full Text Available BACKGROUND: Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. METHODS AND FINDINGS: Caco-2 cells were grown in a basement membrane matrix (Matrigel™ to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10-40 mM or acetaldehyde (25-200 µM for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. CONCLUSIONS: These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in

  13. Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy.

    Science.gov (United States)

    Petrus-Reurer, Sandra; Bartuma, Hammurabi; Aronsson, Monica; Westman, Sofie; Lanner, Fredrik; André, Helder; Kvanta, Anders

    2017-02-01

    Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA). Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry. Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate. In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.

  14. Airway epithelial cell tolerance to Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Verghese Margrith W

    2005-04-01

    Full Text Available Abstract Background The respiratory tract epithelium is a critical environmental interface that regulates inflammation. In chronic infectious airway diseases, pathogens may permanently colonize normally sterile luminal environments. Host-pathogen interactions determine the intensity of inflammation and thus, rates of tissue injury. Although many cells become refractory to stimulation by pathogen products, it is unknown whether the airway epithelium becomes either tolerant or hypersensitive in the setting of chronic infection. Our goals were to characterize the response of well-differentiated primary human tracheobronchial epithelial cells to Pseudomonas aeruginosa, to understand whether repeated exposure induced tolerance and, if so, to explore the mechanism(s. Methods The apical surface of well-differentiated primary human tracheobronchial epithelial cell cultures was repetitively challenged with Pseudomonas aeruginosa culture filtrates or the bacterial media control. Toxicity, cytokine production, signal transduction events and specific effects of dominant negative forms of signaling molecules were examined. Additional experiments included using IL-1β and TNFα as challenge agents, and performing comparative studies with a novel airway epithelial cell line. Results An initial challenge of the apical surface of polarized human airway epithelial cells with Pseudomonas aeruginosa culture filtrates induced phosphorylation of IRAK1, JNK, p38, and ERK, caused degradation of IκBα, generation of NF-κB and AP-1 transcription factor activity, and resulted in IL-8 secretion, consistent with activation of the Toll-like receptor signal transduction pathway. These responses were strongly attenuated following a second Pseudomonas aeruginosa, or IL-1β, but not TNFα, challenge. Tolerance was associated with decreased IRAK1 protein content and kinase activity and dominant negative IRAK1 inhibited Pseudomonas aeruginosa -stimulated NF-κB transcriptional

  15. Contrasting models for the roles of Aire in the differentiation program of epithelial cells in the thymic medulla.

    Science.gov (United States)

    Matsumoto, Mitsuru

    2011-01-01

    The current prevailing view regarding the role of Aire in self-tolerance is that it is involved in the transcriptional control of many tissue-restricted self-antigen genes in thymic epithelial cells in the medulla (mTECs); however, accumulating evidence also suggests that Aire has other roles, e.g. in mTEC differentiation, and furthermore that Aire can either promote or inhibit the mTEC differentiation program, i.e. Aire does not play a neutral role in mTEC differentiation. This review discusses when and how Aire plays an important role in controlling the organization of mTECs required for the expression of self-antigen genes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. AT1R blocker losartan attenuates intestinal epithelial cell apoptosis in a mouse model of Crohn's disease.

    Science.gov (United States)

    Liu, Tian-Jing; Shi, Yong-Yan; Wang, En-Bo; Zhu, Tong; Zhao, Qun

    2016-02-01

    Angiotensin II, which is the main effector of the renin‑angiotensin system, has an important role in intestinal inflammation via the angiotensin II type 1 receptor (AT1R). The present study aimed to investigate the protective effects of the AT1R blocker losartan on 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. Losartan was administered to male adult C57BL/6 J mice 2 weeks prior to the induction of colitis, and images of the whole colon were captured to record changes, scored according to a microscopic scoring system, and reverse transcription-quantitative polymerase chain reaction were performed in order to investigate colonic inflammation. In addition, intestinal epithelial barrier permeability was evaluated, and intestinal epithelial cell (IEC) apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and apoptosis-related protein expression levels were detected by western blotting. Losartan was able to attenuate TNBS-induced body weight loss and colonic damage. Furthermore, T helper 1-mediated proinflammatory cytokines were suppressed by losartan, and gut permeability was largely preserved. TUNEL staining revealed reduced IEC apoptosis in the losartan-treated mice. Losartan also increased the B-cell lymphoma 2 (Bcl2)/Bcl-2-associated X protein (Bax) ratio and suppressed caspase-3 induction. These results suggested that the AT1R blocker losartan may attenuate TNBS-induced colitis by inhibiting the apoptosis of IECs. The effects of losartan were partially mediated through increasing the Bcl-2/Bax ratio and subsequently suppressing the induction of the proapoptotic mediator caspase-3.

  17. Osmosignaling and volume regulation in intestinal epithelial cells.

    Science.gov (United States)

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

    2007-01-01

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

  18. Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

    NARCIS (Netherlands)

    Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

    2012-01-01

    The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-02-01

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

  20. Porcine epidermal stem cells as a biomedical model for wound healing and normal/malignant epithelial cell propagation

    Czech Academy of Sciences Publication Activity Database

    Motlík, Jan; Klíma, Jiří; Dvořánková, B.; Smetana, K. Jr.

    2007-01-01

    Roč. 67, - (2007), s. 105-111 ISSN 0093-691X Grant - others:GA ČR(CZ) GA304/04/0171 Institutional research plan: CEZ:AV0Z50450515 Keywords : pig * stem cell * epidermis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.911, year: 2007

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

    Science.gov (United States)

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

    2015-10-13

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

  2. Melanosome Motility in Fish Retinal Pigment Epithelial (RPE) Cells.

    Science.gov (United States)

    King-Smith, Christina

    2016-01-01

    Several model systems have been developed to investigate mechanism and regulation of intracellular organelle motility. The fish retinal pigment epithelial (RPE) cell represents a novel yet simple system for the study of organelle motility. Primary cultures of dissociated RPE cells are easily prepared and amenable to motility studies. In vivo, melanin-containing pigment granules (melanosomes) within fish RPE migrate distances up to 100 μm in response to light flux. When dissociated from the epithelial layer and cultured in vitro, RPE cells attach to the substrate with the apical projections extending radially from the central cell body. Melanosomes can be chemically triggered to aggregate or disperse throughout the projections, and are easily observed using phase contrast microscopy. Melanosome migration in RPE apical projections is dependent on actin filaments, and thus renders this model system useful for investigations of actin-dependent organelle motility.

  3. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Directory of Open Access Journals (Sweden)

    Ivana Viktorinová

    2017-11-01

    Full Text Available Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  4. Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

    Science.gov (United States)

    Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

    2017-11-01

    Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

  5. Symbiotic Plant Peptides Eliminate Candida albicans Both In Vitro and in an Epithelial Infection Model and Inhibit the Proliferation of Immortalized Human Cells

    Directory of Open Access Journals (Sweden)

    Lilla Ördögh

    2014-01-01

    Full Text Available The increasing number of multidrug-resistant microbes now emerging necessitates the identification of novel antimicrobial agents. Plants produce a great variety of antimicrobial peptides including hundreds of small, nodule-specific cysteine-rich NCR peptides that, in the legume Medicago truncatula, govern the differentiation of endosymbiotic nitrogen fixing bacteria and, in vitro, can display potent antibacterial activities. In this study, the potential candidacidal activity of 19 NCR peptides was investigated. Cationic NCR peptides having an isoelectric point above 9 were efficient in killing Candida albicans, one of the most common fungal pathogens of humans. None of the tested NCR peptides were toxic for immortalized human epithelial cells at concentrations that effectively killed the fungus; however, at higher concentrations, some of them inhibited the division of the cells. Furthermore, the cationic peptides successfully inhibited C. albicans induced human epithelial cell death in an in vitro coculture model. These results highlight the therapeutic potential of cationic NCR peptides in the treatment of candidiasis.

  6. Cells of Origin of Epithelial Ovarian Cancers

    Science.gov (United States)

    2015-09-01

    lethal malignancy of the female reproductive system, largely due to the fact that most EOCs are diagnosed only after the cancer has metastasized into the...Epithelial ovarian cancer (EOC) is the most lethal malignancy of the female reproductive system, largely due to the fact that most EOCs are diagnosed only...experience in ovary research (ovarian physiology , oogonial stem cells) to work on this project. We also ! 5! obtained approval of our animal

  7. Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

    Science.gov (United States)

    Hyun, Dong Won; Kim, Yun Hee; Koh, Ah Young; Lee, Hyun Ju; Wee, Won Ryang; Jeon, Saewha; Kim, Mee Kum

    2017-03-01

    The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

    Science.gov (United States)

    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

  9. Renal epithelial cells can release ATP by vesicular fusion

    Directory of Open Access Journals (Sweden)

    Randi G Bjaelde

    2013-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Cornelia Blume

    Full Text Available 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.

  11. Comparison of autologous versus allogeneic epithelial-like stem cell treatment in an in vivo equine skin wound model.

    Science.gov (United States)

    Broeckx, Sarah Y; Borena, Bizunesh M; Van Hecke, Lore; Chiers, Koen; Maes, Sofie; Guest, Deborah J; Meyer, Evelyne; Duchateau, Luc; Martens, Ann; Spaas, Jan H

    2015-10-01

    Several studies report beneficial effects of autologous and allogeneic stem cells on wound healing. However, no comparison between autologous versus allogeneic epithelial-like stem cells (EpSCs) has been made so far. For this reason, we first hypothesize that both EpSC types enhance wound healing in comparison to vehicle treatment and untreated controls. Second, on the basis of other studies, we hypothesized that there would be no difference between autologous and allogeneic EpSCs. Twelve full-thickness skin wounds were created in six horses. Each horse was subjected to (i) autologous EpSCs, (ii) allogeneic EpSCs, (iii) vehicle treatment or (iv) untreated control. Wound evaluation was performed at day 3, 7 and 14 through wound exudates and at week 1, 2 and 5 through biopsies. Wound circumference and surface were significantly smaller in autologous EpSC-treated wounds. A significantly lower amount of total granulation tissue (overall) and higher vascularization (week 1) was observed after both EpSC treatments. Significantly more major histocompatibility complex II-positive and CD20-positive cells were noticed in EpSC-treated wounds at week 2. In autologous and allogeneic groups, the number of EpSCs in center biopsies was low after 1 week (11.7% and 6.1%), decreased to 7.6% and 1.7%, respectively (week 2), and became undetectable at week 5. These results confirm the first hypothesis and partially support the second hypothesis. Besides macroscopic improvements, both autologous and allogeneic EpSCs had similar effects on granulation tissue formation, vascularization and early cellular immune response. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Padilla-Nash, Hesed M; McNeil, Nicole E; Yi, Ming; Nguyen, Quang-Tri; Hu, Yue; Wangsa, Danny; Mack, David L; Hummon, Amanda B; Case, Chanelle; Cardin, Eric; Stephens, Robert; Difilippantonio, Michael J; Ried, Thomas

    2013-08-01

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

  13. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob

    2003-01-01

    Studies have suggested that in ulcerative colitis (UC), intercellular adhesion molecule-1 (ICAM-1) is involved in migration of leukocytes toward the colonic epithelium. A suitable in vitro model of chronic colonic inflammation does not exist, and the role of the epithelium is based on monolayers...... of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium....

  14. Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing.

    Directory of Open Access Journals (Sweden)

    Chi-Chin Sun

    Full Text Available 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.

  15. Computational investigation of epithelial cell dynamic phenotype in vitro

    Directory of Open Access Journals (Sweden)

    Debnath Jayanta

    2009-05-01

    Full Text Available Abstract Background When grown in three-dimensional (3D cultures, epithelial cells typically form cystic organoids that recapitulate cardinal features of in vivo epithelial structures. Characterizing essential cell actions and their roles, which constitute the system's dynamic phenotype, is critical to gaining deeper insight into the cystogenesis phenomena. Methods Starting with an earlier in silico epithelial analogue (ISEA1 that validated for several Madin-Darby canine kidney (MDCK epithelial cell culture attributes, we built a revised analogue (ISEA2 to increase overlap between analogue and cell culture traits. Both analogues used agent-based, discrete event methods. A set of axioms determined ISEA behaviors; together, they specified the analogue's operating principles. A new experimentation framework enabled tracking relative axiom use and roles during simulated cystogenesis along with establishment of the consequences of their disruption. Results ISEA2 consistently produced convex cystic structures in a simulated embedded culture. Axiom use measures provided detailed descriptions of the analogue's dynamic phenotype. Dysregulating key cell death and division axioms led to disorganized structures. Adhering to either axiom less than 80% of the time caused ISEA1 to form easily identified morphological changes. ISEA2 was more robust to identical dysregulation. Both dysregulated analogues exhibited characteristics that resembled those associated with an in vitro model of early glandular epithelial cancer. Conclusion We documented the causal chains of events, and their relative roles, responsible for simulated cystogenesis. The results stand as an early hypothesis–a theory–of how individual MDCK cell actions give rise to consistently roundish, cystic organoids.

  16. Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

    Science.gov (United States)

    Lazarevic, Ivana; Engelhardt, Britta

    2016-01-29

    The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the

  17. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

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

  18. MUCOUS CELLS IN THE EPITHELIAL LINING OF DENTIGEROUS CYST

    OpenAIRE

    Pratiksha; Sangeeta; Sumanta; Prashanti; Sathyajitraje; Rajkumar

    2014-01-01

    The epithelial lining of both the developmental and inflammatory cysts of odontogenic origin are primarily composed of squamous epithelium. Various forms of metaplasia and degenerations are observed in these epithelial linings e.g. mucous cells, ciliated cells, para and/or orthokeratinization and formation of hyaline bodies. The present study was designed to investigate the incidences of mucous cells in the epithelial lining of dentigerous cyst. Mucous cells were observed ...

  19. The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function.

    Science.gov (United States)

    Kreft, Mateja Erdani; Jerman, Urška Dragin; Lasič, Eva; Hevir-Kene, Neli; Rižner, Tea Lanišnik; Peternel, Luka; Kristan, Katja

    2015-03-10

    In this study we have investigated the effects of different cell culture conditions on the Calu-3 epithelial cell model. Calu-3 cells were cultured in media A-MEM at the air-liquid (A-L) or liquid-liquid (L-L) interface for one or three wks (weeks). Different cryomethods were tested and the cell line was characterized using histochemistry, immunofluorescence, transmission and scanning electron microscopy, transepithelial resistance (TEER) measurements, permeability studies, and gene profiling of 84 drug transporters. Cell culture was successful in A-MEM with only 2.5% FBS. Cell proliferation and viability depended on the cryopreservation method. All Calu-3 models expressed CK7, occludin, and E-cadherin. The A-L interface resulted in a more biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles than at the L-L interface, where the epithelium was cuboidal, but exhibited higher TEER values and lower dextran permeabilities. Longer time in culture significantly decreased dextran permeability and increased the expression of specific drug transporters. Drug transporter expression was also notably influenced by the culture interface, where the A-L interface yielded a higher expression of drug transporter genes than the L-L interface. Since cell culture interface and time in culture affect Calu-3 cell differentiation, barrier integrity, permeability properties, and drug transporter expression, culture conditions need to be considered and standardized when using the Calu-3 cell line as an in vitro model for aerosol drug delivery and screening of bronchial drug candidates. Copyright © 2015 Elsevier B.V. All rights reserved.

  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.

    Science.gov (United States)

    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.

  1. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  2. The cell line NCl-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier.

    Science.gov (United States)

    Salomon, Johanna J; Muchitsch, Viktoria E; Gausterer, Julia C; Schwagerus, Elena; Huwer, Hanno; Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

    2014-03-03

    The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors

  3. Jagged1 upregulation in prostate epithelial cells promotes formation of reactive stroma in the Pten null mouse model for prostate cancer.

    Science.gov (United States)

    Su, Q; Zhang, B; Zhang, L; Dang, T; Rowley, D; Ittmann, M; Xin, L

    2017-02-02

    The role of Notch signaling in prostate cancer has not been defined definitively. Several large scale tissue microarray studies have revealed that the expression of some Notch signaling components including the Jagged1 ligand are upregulated in advanced human prostate cancer specimens. Jagged1 expressed by tumor cells may activate Notch signaling in both adjacent tumor cells and cells in tumor microenvironment. However, it remains undetermined whether increased Jagged1 expression reflects a cause for or a consequence of tumor progression in vivo. To address this question, we generated a novel R26-LSL-JAG1 mouse model that enables spatiotemporal Jagged1 expression. Prostate specific upregulation of Jagged1 neither interferes with prostate epithelial homeostasis nor significantly accelerates tumor initiation or progression in the prostate-specific Pten deletion mouse model for prostate cancer. However, Jagged1 upregulation results in increased inflammatory foci in tumors and incidence of intracystic adenocarcinoma. In addition, Jagged1 overexpression upregulates Tgfβ signaling in prostate stromal cells and promotes progression of a reactive stromal microenvironment in the Pten null prostate cancer model. Collectively, Jagged1 overexpression does not significantly accelerate prostate cancer initiation and progression in the context of loss-of-function of Pten, but alters tumor histopathology and microenvironment. Our study also highlights an understudied role of Notch signaling in regulating prostatic stromal homeostasis.

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

    Science.gov (United States)

    Wilson, Tyler J; Bergstralh, Dan T

    2017-06-01

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

  5. Small molecule mediated proliferation of primary retinal pigment epithelial cells.

    Science.gov (United States)

    Swoboda, Jonathan G; Elliott, Jimmy; Deshmukh, Vishal; de Lichtervelde, Lorenzo; Shen, Weijun; Tremblay, Matthew S; Peters, Eric C; Cho, Charles Y; Lu, Bin; Girman, Sergej; Wang, Shaomei; Schultz, Peter G

    2013-07-19

    Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of RPE cells results in retinal disorders such as age-related macular degeneration. Cell transplant strategies have potential therapeutic value for such disorders; however, risks associated with an inadequate supply of donor cells limit their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for transplantation. Here, we report that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.

  6. Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2

    DEFF Research Database (Denmark)

    Boyd, Mette; Coskun, Mehmet; Lilje, Berit

    2014-01-01

    The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated...... genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52...... promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers....

  7. Effects of epiplakin-knockdown in cultured corneal epithelial cells

    OpenAIRE

    Kokado, Masahide; Okada, Yuka; Miyamoto, Takeshi; Yamanaka, Osamu; Saika, Shizuya

    2016-01-01

    Background To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. Methods Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and prolifer...

  8. Novel application of artificial dermis plus autologous vital epithelial cells: improved wound epithelialization.

    Science.gov (United States)

    Lee, Li-Tzu; Kwan, Po-Cheung; Wong, Yong-Kie

    2010-02-01

    The purpose of this study was to evaluate artificial dermis with the simultaneous addition of autologous epithelial cells for oral lesion defect reconstruction. Surgical wounds reconstructed with artificial dermis plus scraped epithelial cells were evaluated in 5 patients with oral benign lesions or squamous cell carcinoma. Clinical follow-up indices included scar formation and tissue surface texture observation. The neomucosal layers were analyzed histologically to establish the degree of epithelialization. Clinical observation showed that the oral mucosal texture was smoother in artificial dermis with added epithelial cells at 4 weeks postoperation compared with artificial dermis alone. The wound contraction and scar formation processes were slow. Viable epithelial cells with flat rete ridges remained in the artificial dermis, and a neoepithelial layer was present in the histological findings. We showed that healthy granulation tissue and neoepithelial formation in artificial dermis with epithelial cells was beneficial for the repair of oral defects. Scraping oral epithelial cells and applying them to artificial dermis assisted in the early preparation of composite grafts and minimized requirement for donor sites. This technique may improve the treatment of patients with oral benign tumors and early-stage squamous cell carcinoma. Copyright 2010 Elsevier. Published by Elsevier B.V. All rights reserved.

  9. Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma.

    Science.gov (United States)

    Hua, Yuanyuan; Choi, Pui-Wah; Trachtenberg, Alexander J; Ng, Allen C; Kuo, Winston P; Ng, Shu-Kay; Dinulescu, Daniela M; Matzuk, Martin M; Berkowitz, Ross S; Ng, Shu-Wing

    2016-10-04

    Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

  10. Effects of Supernatants from Escherichia coli Nissle 1917 and Faecalibacterium prausnitzii on Intestinal Epithelial Cells and a Rat Model of 5-Fluorouracil-Induced Mucositis.

    Science.gov (United States)

    Wang, Hanru; Jatmiko, Yoga D; Bastian, Susan E P; Mashtoub, Suzanne; Howarth, Gordon S

    2017-01-01

    Faecalibacterium prausnitzii (Fp) and Escherichia coli Nissle 1917 (EcN) are probiotics, which have been reported to ameliorate certain gastrointestinal disorders. We evaluated the effects of supernatants (SN) derived from Fp and EcN on 5-fluorouracil (5-FU)-treated intestinal cells and in a rat model of mucositis. In vitro: IEC-6, Caco-2, and T-84 cells were analyzed for viability and monolayer permeability. In vivo: Female dark agouti rats were gavaged with Fp or EcN SN and injected intraperitoneally with saline (control) or 5-FU to induce mucositis. Rats were euthanized and intestinal tissues collected for myeloperoxidase assay and histological analyses. In vitro: Caco-2 cell viability was further reduced when treated with Fp SN + 5-FU compared to 5-FU controls. In both Caco-2 and T-84 cells, Fp SN partially prevented the decrease in transepithelial electrical resistance (TER) caused by 5-FU administration. In vivo: 5-FU-injected rats administered Fp SN or EcN SN partly prevented body weight loss and normalized water intake compared to 5-FU controls. These results suggest a growth inhibitory mechanism of Fp SN action on transformed epithelial cells that could be mediated by effects on tight junctions. Factors derived from Fp SN and EcN SN could have a role in reducing the severity of intestinal mucositis.

  11. Modulation of epithelial cell polarity by bacterial pathogens.

    Science.gov (United States)

    Tapia, Rocio; Kralicek, Sarah E; Hecht, Gail A

    2017-10-01

    Epithelial cells constitute a physical barrier that aids in protecting the host from microbial pathogens. Polarized epithelial cells contain distinct apical and basolateral membrane domains separated by intercellular junctions, including tight junctions (TJs), which contribute to the maintenance of apical-basal polarity. Polarity complexes also contribute to the establishment of TJ formation. Several pathogens perturb epithelial TJ barrier function and structure in addition to causing a loss of apical-basal polarity. Here, we review the impact of pathogenic bacteria on the disruption of cell-cell junctions and epithelial polarity. © 2017 New York Academy of Sciences.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-14

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

  13. Nuclear microscopy of rat colon epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, M., E-mail: phyrenmq@nus.edu.sg [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Rajendran, Reshmi [Lab of Molecular Imaging, Singapore Bioimaging Consotium, 11 Biopolis Way, 02-02 Helios, Singapore 138667 (Singapore); Ng, Mary [Department of Pharmacology, National University of Singapore (Singapore); Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank [Centre for Ion Beam Applications (CIBA), Department of Physics, National University of Singapore, Singapore 117542 (Singapore); Jenner, Andrew Michael [Illawara Health and Medical Research Institute (IHMRI), University of Wollongong, NSW 2522 (Australia)

    2011-10-15

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

  14. Impact of Bacillus thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) - a new in vitro model for safety assessment of recombinant food compounds.

    Science.gov (United States)

    Bondzio, Angelika; Stumpff, Friederike; Schön, Jennifer; Martens, Holger; Einspanier, Ralf

    2008-06-01

    The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.

  15. The Contribution of the Airway Epithelial Cell to Host Defense

    OpenAIRE

    Frauke Stanke

    2015-01-01

    In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how t...

  16. The all-trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.

    Science.gov (United States)

    Kim, So R; Jang, Young P; Jockusch, Steffen; Fishkin, Nathan E; Turro, Nicholas J; Sparrow, Janet R

    2007-12-04

    The bis-retinoid pigments that accumulate in retinal pigment epithelial cells as lipofuscin are associated with inherited and age-related retinal disease. In addition to A2E and related cis isomers, we previously showed that condensation of two molecules of all-trans-retinal leads to the formation of a protonated Schiff base conjugate, all-trans-retinal dimer-phosphatidylethanolamine. Here we report the characterization of the related pigments, all-trans-retinal dimer-ethanolamine and unconjugated all-trans-retinal dimer, in human and mouse retinal pigment epithelium. In eyecups of Abcr(-/-) mice, a model of recessive Stargardt macular degeneration, all-trans-retinal dimer-phosphatidylethanolamine was increased relative to wild type and was more abundant than A2E. Total pigment of the all-trans-retinal dimer series (sum of all-trans-retinal dimer-phosphatidylethanolamine, all-trans-retinal dimer-ethanolamine, and all-trans-retinal dimer) increased with age in Abcr(-/-) mice and was modulated by amino acid variants in Rpe65. In in vitro assays, enzyme-mediated hydrolysis of all-trans-retinal dimer-phosphatidylethanolamine generated all-trans-retinal dimer-ethanolamine, and protonation/deprotonation of the Schiff base nitrogen of all-trans-retinal dimer-ethanolamine was pH-dependent. Unconjugated all-trans-retinal dimer was a more efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more reactive with singlet oxygen than was A2E. By analyzing chromatographic properties and UV-visible spectra together with mass spectrometry, mono- and bis-oxygenated all-trans-retinal dimer photoproducts were detected in Abcr(-/-) mice. The latter findings are significant to an understanding of the adverse effects of retinal pigment epithelial cell lipofuscin.

  17. Intestinal transport: studies with isolated epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kimmich, G.A.

    1979-12-01

    Isolated intestinal epithelial cells have been extremely useful for characterizing the nature of intestinal absorption processes and for providing insight into the energetics of Na/sup +/-dependent transport systems. This report describes a number of experimental approaches which have been used for investigating the specific epithelial transport systems involved in sugar absorption, but provides information which ultimately should prove useful for characterizing a number of different intestinal transport events. Similar experiments should also prove useful for exploring the effect of environmental agents on the function of intestinal tissue. In the case of sugars, net absorption is accomplished via a mucosal, Na/sup +/-dependent concentrative transport system acting in sequence with a passive serosal system which does not require Na/sup +/. The serosal system limits the full gradient-forming capability of the muscosal system. Agents such as phloretin or cytochalasin B which inhibit serosal transport allow the cells to establish sugar gradients as high as 70 fold in contrast to 10 to 15 fold gradients observed for control cells. Sevety-fold sugar gradients cannot be explained in terms of the energy available in the electrochemical potential for Na/sup +/ if the Na/sub 2/: sugar coupling stoichiometry is 1:1 as commonly assumed. New information indicates that the true Na/sup +/:sugar stoichiometry is in fact 2:1. Flow of two Na/sup +/ ions per sugar molecule down the transmembrane electrochemical potential for Na/sup +/ provides more than sufficient energy to account for observed 70 fold sugar gradients. If flow of sugar by other routes could be completely inhibited, theoretical sugar gradients as high as 400 could be achieved assuming that the cells maintain a membrane potential of -36 mV as measured for intact tissue.

  18. Uranium induces apoptosis in lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Periyakaruppan, Adaikkappan; Sarkar, Shubhashish; Sadanandan, Bindu; Thomas, Renard; Wilson, Bobby L. [Texas Southern University, Environmental Toxicology Program, Department of Chemistry, Houston, TX (United States); Ravichandran, Prabakaran; Sharma, Chidananda S.; Ramesh, Vani; Hall, Joseph C.; Ramesh, Govindarajan T. [Norfolk State University, Molecular Toxicology Laboratory, Department of Biology, Center for Biotechnology and Biomedical Sciences, Norfolk, VA (United States)

    2009-06-15

    Uranium is a naturally occurring radioactive material present everywhere in the environment. It is toxic because of its chemical or radioactive properties. Uranium enters environment mainly from mines and industry and cause threat to human health by accumulating in lungs as a result of inhalation. In our previous study, we have shown the effectiveness of antioxidant system response to the oxidative stress induced by uranyl acetate (UA) in rat lung epithelial (LE) cells. As part of our continuing studies; here, we investigated the mechanism underlying when LE cells are exposed to different concentration of UA. Oxidative stress may lead to apoptotic signaling pathways. LE cells treated with 0.25, 0.5 and 1 mM of UA results in dose and time-dependent increase in activity of both caspases-3 and -8. Increase in the concentration of cytochrome-c oxidase in cytosol was seen in LE cells treated with 1 mM UA as a result of mitochondria membrane permeability. The cytochrome-c leakage may trigger the apoptotic pathway. TUNEL assay performed in LE cells treated with 1 mM of UA showed significant incorporation of dNTPs in the nucleus after 24 h. In the presence of the caspase inhibitors, we observed the significant decrease in the activity of caspases-8 and -3 in 0.5 and 1 mM UA-treated LE cells. (orig.)

  19. Interaction between submicron COD crystals and renal epithelial cells.

    Science.gov (United States)

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

    2012-01-01

    This study aims to investigate the adhesion characteristics between submicron calcium oxalate dihydrate (COD) with a size of 150 ± 50 nm and African green monkey kidney epithelial cells (Vero cells) before and after damage, and to discuss the mechanism of kidney stone formation. Vero cells were oxidatively injured by hydrogen peroxide to establish a model of injured cells. Scanning electron microscopy was used to observe Vero-COD adhesion. Inductively coupled plasma emission spectrometry was used to quantitatively measure the amount of adhered COD microcrystals. Nanoparticle size analyzer and laser scanning confocal microscopy were performed to measure the change in the zeta potential on the Vero cell surface and the change in osteopontin expression during the adhesion process, respectively. The level of cell injury was evaluated by measuring the changes in malonaldehyde content, and cell viability during the adhesion process. The adhesion capacity of Vero cells in the injury group to COD microcrystals was obviously stronger than that of Vero cells in the control group. After adhesion to COD, cell viability dropped, both malonaldehyde content and cell surface zeta potential increased, and the fluorescence intensity of osteopontin decreased because the osteopontin molecules were successfully covered by COD. Submicron COD further damaged the cells during the adhesion process, especially for Vero cells in the control group, leading to an elevated amount of attached microcrystals. Submicron COD can further damage injured Vero cells during the adhesion process. The amount of attached microcrystals is proportional to the degree of cell damage. The increased amount of microcrystals that adhered to the injured epithelial cells plays an important role in the formation of early-stage kidney stones.

  20. 8-prenylnaringenin and tamoxifen inhibit the shedding of irradiated epithelial cells and increase the latency period of radiation-induced oral mucositis. Cell culture and murine model

    Energy Technology Data Exchange (ETDEWEB)

    Ryck, Tine de; Impe, Annouchka van; Bracke, Marc E. [Ghent University, Laboratory of Experimental Cancer Research, Department Radiation Oncology and Experimental Cancer Research, Ghent (Belgium); Vanhoecke, Barbara W. [Ghent University, Laboratory of Experimental Cancer Research, Department Radiation Oncology and Experimental Cancer Research, Ghent (Belgium); Ghent University, Laboratory of Microbial Ecology and Technology (LabMET), Ghent (Belgium); Heyerick, Arne [Ghent University, Laboratory of Pharmacognosy and Phytochemistry, Ghent (Belgium); Vakaet, Luc; Neve, Wilfried de [Ghent University Hospital, Department of Radiation Oncology, Ghent (Belgium); Mueller, Doreen [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); Schmidt, Margret [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); German Cancer Consortium (DKTK) partner site Dresden and German Cancer Center (DKFZ), Heidelberg (Germany); Doerr, Wolfgang [Medical Faculty and University Hospital Carl Gustav Carus, Technische Universitaet Dresden, Department of Radiotherapy and Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Dresden (Germany); Medical University, Department of Radiation Oncology, CCC, and CD-Laboratory RadOnc, Vienna (Austria)

    2015-05-01

    The major component in the pathogenesis of oral radiation-induced mucositis is progressive epithelial hypoplasia and eventual ulceration. Irradiation inhibits cell proliferation, while cell loss at the surface continues. We conceived to slow down this desquamation by increasing intercellular adhesion, regulated by the E-cadherin/catenin complex. We investigated if 8-prenylnaringenin (8-PN) or tamoxifen (TAM) decrease the shedding of irradiated human buccal epithelial cells in vitro and thus delay the ulcerative phase of radiation-induced mucositis in vivo. In vitro, aggregates of buccal epithelial cells were irradiated and cultured in suspension for 11 days. 8-PN or TAM were investigated regarding their effect on cell shedding. In vivo, the lower tongue surface of mice was irradiated with graded single doses of 25 kV X-rays. The incidence, latency, and duration of the resulting mucosal ulcerations were analyzed after topical treatment with 8-PN, TAM or solvent. 8-PN or TAM prevented the volume reduction of the irradiated cell aggregates during the incubation period. This was the result of a higher residual cell number in the treated versus the untreated irradiated aggregates. In vivo, topical treatment with 8-PN or TAM significantly increased the latency of mucositis from 10.9 to 12.1 and 12.4 days respectively, while the ulcer incidence was unchanged. 8-PN and TAM prevent volume reduction of irradiated cell aggregates in suspension culture. In the tongues of mice, these compounds increase the latency period. This suggests a role for these compounds for the amelioration of radiation-induced mucositis in the treatment of head and neck tumors. (orig.) [German] Die wesentliche Komponente in der Pathogenese der radiogenen Mukositis ist eine progressive epitheliale Hypoplasie und letztendlich Ulzeration. Die Bestrahlung hemmt die Zellproliferation, waehrend der Zellverlust an der Oberflaeche fortbesteht. Wir versuchten, diese Desquamation durch eine Stimulation der

  1. Nutrients released by gastric epithelial cells enhance Helicobacter pylori growth

    NARCIS (Netherlands)

    van Amsterdam, Karin; van der Ende, Arie

    2004-01-01

    Background. Helicobacter pylori survives and proliferates in the human gastric mucosa. In this niche, H. pylori adheres to the gastric epithelial cells near the tight junctions. In vitro, H. pylori proliferated well in tissue-culture medium near gastric epithelial cells. However, in the absence of

  2. In vitro effect of smokeless tobacco on gingival epithelial cells

    Directory of Open Access Journals (Sweden)

    Arzu Beklen

    2017-05-01

    Epithelial cells are the first line of defense against pathogens in the oral cavity. The results suggest that smokeless tobacco not only inhibits the growth of epithelial cells but also induce the generation of inflammatory cytokines which leads to smokeless tobacco-exacerbated disease.

  3. The application of silver nano-particles on developing potential treatment for chronic rhinosinusitis: Antibacterial action and cytotoxicity effect on human nasal epithelial cell model.

    Science.gov (United States)

    Chen, Shyuan-Yow; Huang, Min-Tsan; Pender, Sylvia L F; Ruslin, Muhammad; Chou, Hsin-Hua; Ou, Keng-Liang

    2017-11-01

    Chronic rhinosinusitis (CRS) has raised attentions both in many countries due to its high relapsing rate and the resistance of certain treatments especially antibiotics application on both acute and chronic bacterial rhinosinusitis. The aim of this research is stressing on developing an effective alternative treatment for treating CRS and reducing the use of antibiotics to avoid further resistance forming. The antibacterial functions of silver nano-particles (AgNPs) are well known according to previous reports and studies. However, for developing a suitable treatment for further clinical application, a variety of AgNPs cell cytotoxicity experiments and AgNPs antibacterial properties experiments were examined in vitro in this study. For imitating the clinical condition of CRS, the human nasal epithelial cell line (RPMI2650) has chosen as experimental model. Moreover, Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Escherichia coli (E. coli) were selected for antibacterial function experiments. The analytical results demonstrated that 5ppm of AgNPs not only maintains >80% of cell activity to RPMI2650, but also possesses >80% of antibacterial function to S. aureus and 100% of antibacterial function to E. coli. Therefore, 5ppm of AgNPs might be considered as a promising antibacterial agent for treating CRS. Copyright © 2017. Published by Elsevier B.V.

  4. Thymic epithelial cell-specific deletion of Jmjd6 reduces Aire protein expression and exacerbates disease development in a mouse model of autoimmune diabetes.

    Science.gov (United States)

    Yanagihara, Toyoshi; Tomino, Takahiro; Uruno, Takehito; Fukui, Yoshinori

    2017-07-15

    Thymic epithelial cells (TECs) establish spatially distinct microenvironments in which developing T cells are selected to mature or die. A unique property of medullary TECs is their expression of thousands of tissue-restricted self-antigens that is largely under the control of the transcriptional regulator Aire. We previously showed that Jmjd6, a lysyl hydroxylase for splicing regulatory proteins, is important for Aire protein expression and that transplantation of Jmjd6-deficient thymic stroma into athymic nude mice resulted in multiorgan autoimmunity. Here we report that TEC-specific deletion of Jmjd6 exacerbates development of autoimmune diabetes in a mouse model, which express both ovalbumin (OVA) under the control of the rat insulin gene promoter and OT-I T cell receptor specific for OVA peptide bound to major histocompatibility complex class I K b molecules. We found that Aire protein expression in mTECs was reduced in the absence of Jmjd6, with retention of intron 2 in Aire transcripts. Our results thus demonstrate the importance of Jmjd6 in establishment of immunological tolerance in a more physiological setting. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. ING-1(heMAb, a Monoclonal Antibody to Epithelial Cell Adhesion Molecule, Inhibits Tumor Metastases in a Murine Cancer Model

    Directory of Open Access Journals (Sweden)

    Harry H. Ruan

    2003-11-01

    Full Text Available ING-1(heMAb, a human-engineered monoclonal antibody (MAb that specifically targets the epithelial cell adhesion molecule (Ep-CAM, kills adenocarcinoma cells in vitro and inhibits tumor growth in vivo. In the current study, we evaluated the efficacy of ING-1(heMAb in a murine model of cancer metastases. Mice received intravenous dosing of 1 mg/kg ING-1(heMAb, twice a week, starting on day 2 or day 5. A negative control group received 1 mg/kg human immunoglobulin G with the same dose frequency starting on day 2. A positive control group received weekly 100 mg/kg 54lurouracil/leucovorin starting on day 2. ING-1(heMAb/day 2 treatment significantly reduced both the number of visible tumor nodules in body cavities (P < .01 and the number of metastases on lung surfaces (P < .005. The treatment also resulted in a 91% reduction of micrometastases in lung tissues (P <.0001. Delaying ING-1(heMAb treatment until day 5 caused 54% reduction in micrometastases (P <.005. Our results indicate that a number of parameters, including treatment starting day, dose level, and dose frequency, are critical in achieving the optimal efficacy of ING-1(heMAb. We conclude that ING-1(heMAb effectively reduced tumor metastases in a murine cancer model. Immunotherapy with ING-1(heMAb may be beneficial in treating human metastatic diseases.

  6. Preventive Effect of TU-100 on a Type-2 Model of Colitis in Mice: Possible Involvement of Enhancing Adrenomedullin in Intestinal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Atsushi Kaneko

    2013-01-01

    Full Text Available Purpose. Crohn's disease (CD and ulcerative colitis (UC, the two major forms of inflammatory bowel disease (IBD, have histopathologically and immunologically different characteristics. We previously reported that a traditional Japanese medicine, daikenchuto (TU-100, ameliorated a trinitrobenzenesulfonic acid- (TNBS- induced type-1 model colitis exhibiting histopathological features of CD through adrenomedullin (ADM enhancement. Our current aims were to examine whether TU-100 ameliorates a type-2 model colitis that histologically resembles UC and identify the active ingredients. Methods. TU-100 was administered orally to mice with oxazolone- (OXN- induced type-2 model colitis. The morbidity was evaluated by body weight loss and the macroscopic score of colonic lesions. ADM was quantified using an EIA kit. Results. TU-100 prevented weight loss and colon ulceration. ADM production by intestinal epithelial cells was increased by TU-100 addition. Screening to identify active ingredients showed that [6]-shogaol and hydroxy α-sanshool enhanced ADM production. Conclusions. TU-100 exerted a protective effect in OXN-induced type-2 model colitis, indicating that TU-100 may be a beneficial agent for treatment of UC.

  7. Isolation of fibroblasts and epithelial cells in bronchoalveolar lavage (BAL).

    Science.gov (United States)

    Pollock, Kathryn; Albares, Luke; Wendt, Chris; Hubel, Allison

    2013-04-01

    The long-term outcome of lung transplants is poor with 60%-70% of patients developing chronic rejection. Chronic rejection is manifested histologically by obliterative bronchiolitis with bronchiolitis obliterans syndrome (BOS), the clinical surrogate. Recent studies suggest that fibroblasts and epithelial cells present in bronchoalveolar lavage (BAL) may be a clinically relevant biomarker for BOS. The goal of this investigation was to develop a fast, repeatable method to individually isolate these low-frequency cell types. Fibroblasts and epithelial cells were isolated from BAL using attachment methods and the phenotype of the cells confirmed using immunostaining for vimentin (fibroblasts) and epithelial cell adhesion molecule (EpCAM, epithelial cells). Both fibroblasts and epithelial cells were isolated in every sample of BAL processed with the frequency of fibroblasts ranging from 0.03% to 0.48% and epithelial cells ranging from 0.05% to 1.5% of the total sample. Additional studies were performed using cytospins of cells after macrophages were depleted; cells exhibiting characteristics of both fibroblasts and epithelial cells were observed. The frequency of the cells of interest suggests that conventional methods of immunomagnetic isolation will not be effective in isolating these subpopulations. Finally, some of the low-frequency cells isolated via cytospin exhibit characteristics of epithelial to mesenchymal transition (which was not observed in plating incubations), indicating that the epithelial to mesenchymal cell transition fibroblasts may be nonadherent. In future studies, this technique and dataset may be of use to statistically correlate low-frequency cell type abundance to the onset and development of BOS.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells.

    Science.gov (United States)

    Krautkrämer, Ellen; Lehmann, Maik J; Bollinger, Vanessa; Zeier, Martin

    2012-11-30

    Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC). In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection. Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV) and Puumala (PUUV) virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells. Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.

  10. Polar release of pathogenic Old World hantaviruses from renal tubular epithelial cells

    Directory of Open Access Journals (Sweden)

    Krautkrämer Ellen

    2012-11-01

    Full Text Available Abstract Background Epithelio- and endotheliotropic viruses often exert polarized entry and release that may be responsible for viral spread and dissemination. Hantaviruses, mostly rodent-borne members of the Bunyaviridae family infect epithelial and endothelial cells of different organs leading to organ dysfunction or even failure. Endothelial and renal epithelial cells belong to the target cells of Old World hantavirus. Therefore, we examined the release of hantaviruses in several renal epithelial cell culture models. We used Vero cells that are commonly used in hantavirus studies and primary human renal epithelial cells (HREpC. In addition, we analyzed MDCKII cells, an epithelial cell line of a dog kidney, which represents a widely accepted in vitro model of polarized monolayers for their permissiveness for hantavirus infection. Results Vero C1008 and primary HREpCs were grown on porous-support filter inserts for polarization. Monolayers were infected with hantavirus Hantaan (HTNV and Puumala (PUUV virus. Supernatants from the apical and basolateral chamber of infected cells were analyzed for the presence of infectious particles by re-infection of Vero cells. Viral antigen and infectious particles of HTNV and PUUV were exclusively detected in supernatants collected from the apical chamber of infected Vero C1008 cells and HREpCs. MDCKII cells were permissive for hantavirus infection and polarized MDCKII cells released infectious hantaviral particles from the apical surface corresponding to the results of Vero and primary human epithelial cells. Conclusions Pathogenic Old World hantaviruses are released from the apical surface of different polarized renal epithelial cells. We characterized MDCKII cells as a suitable polarized cell culture model for hantavirus infection studies.

  11. Epithelial disruptions, but not immune cell invasion, induced secretory dysfunction following innate immune activation in a novel model of acute salivary gland injury.

    Science.gov (United States)

    Shaalan, Abeer; Carpenter, Guy; Proctor, Gordon

    2017-11-21

    Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and salivary gland injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production. C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre-treating mice with RB6-8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva-driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na-K- CL-Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), were characterized using RT-qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO-1) and basement membrane (BM) protein; laminin were assessed by immunohistochemistry. Innate immune challenge prompted dysfunction in the exocrine salivary glands (SGs). Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)-positive, acute inflammatory cells. In the present study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Paula A Vasquez

    2016-08-01

    Full Text Available 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.

  14. A dynamic cellular vertex model of growing epithelial tissues

    Science.gov (United States)

    Lin, Shao-Zhen; Li, Bo; Feng, Xi-Qiao

    2017-04-01

    Intercellular interactions play a significant role in a wide range of biological functions and processes at both the cellular and tissue scales, for example, embryogenesis, organogenesis, and cancer invasion. In this paper, a dynamic cellular vertex model is presented to study the morphomechanics of a growing epithelial monolayer. The regulating role of stresses in soft tissue growth is revealed. It is found that the cells originating from the same parent cell in the monolayer can orchestrate into clustering patterns as the tissue grows. Collective cell migration exhibits a feature of spatial correlation across multiple cells. Dynamic intercellular interactions can engender a variety of distinct tissue behaviors in a social context. Uniform cell proliferation may render high and heterogeneous residual compressive stresses, while stress-regulated proliferation can effectively release the stresses, reducing the stress heterogeneity in the tissue. The results highlight the critical role of mechanical factors in the growth and morphogenesis of epithelial tissues and help understand the development and invasion of epithelial tumors.

  15. Human amniotic epithelial cells combined with silk fibroin scaffold in the repair of spinal cord injury

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    Ting-gang Wang

    2016-01-01

    Full Text Available Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithelial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the transplant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial cells combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

  16. Larazotide acetate promotes tight junction assembly in epithelial cells.

    Science.gov (United States)

    Gopalakrishnan, Shobha; Tripathi, Amit; Tamiz, Amir P; Alkan, Sefik S; Pandey, Niranjan B

    2012-05-01

    Tight junctions (TJ) control paracellular permeability and apical-basolateral polarity of epithelial cells. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. TJ formation is dependent on E-cadherin-mediated cell-cell adhesion and actin rearrangement, and is regulated by the Rho family GTPase and aPKC signaling pathways. Larazotide acetate, an 8-mer peptide and TJ modulator, inhibits TJ disassembly and dysfunction caused by endogenous and exogenous stimuli in intestinal epithelial cells. Here, we examined the effect of larazotide acetate on de novo TJ assembly using 2 different model systems. In MDCK cells, larazotide acetate promoted TJ assembly in a calcium switch assay. Larazotide acetate also promoted actin rearrangement, and junctional distribution of zonula occludens-1 (ZO-1), occludin, claudins, and E-cadherin. Larazotide acetate promoted TJ maturation and decreased paracellular permeability in "leaky" Caco-2 cells. Taken together, our data indicate that larazotide acetate enhances TJ assembly and barrier function by promoting actin rearrangement and redistribution of TJ and AJ proteins. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Ho Chih-Ming

    2012-02-01

    Full Text Available Abstract Background At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA. Methods Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium. Results The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44high, CD24low, and AC133+. These cells also demonstrated high BMP-2, BMP4, TGF-β, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α2β1, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α2β1, and CD146 surface marker expression than the epithelial type cells. Conclusion The established culture system provides an in vitro model for the selection of drugs that target cancer

  18. Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma.

    Science.gov (United States)

    Ho, Chih-Ming; Chang, Shwu-Fen; Hsiao, Chih-Chiang; Chien, Tsai-Yen; Shih, Daniel Tzu-Bi

    2012-02-14

    At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA). Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium. The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44(high), CD24(low), and AC133(+). These cells also demonstrated high BMP-2, BMP4, TGF-β, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α2β1, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α2β1, and CD146 surface marker expression than the epithelial type cells. The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian

  19. Epithelial cells as alternative human biomatrices for comet assay

    OpenAIRE

    Emilio eRojas; Yolanda eLorenzo; Kristiane eHuag-Berg; Bjørn eNicolaissen; Mahara eValverde

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many org...

  20. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment......ophthalmology, age-related macular degeneration, transplantation, retinal pigment epithelial cells, treatment...

  1. Transplantation of retinal pigment epithelial cells - a possible future treatment for age-related macular degeneration

    DEFF Research Database (Denmark)

    Wiencke, Anne Katrine

    2001-01-01

    ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment......ophthalmology, age-related macular degeneration, retinal pigment epithelial cells, transplantation, treatment...

  2. Quantitative assessment of cytosolic Salmonella in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Leigh A Knodler

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

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

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    Araki Hiromasa

    2007-04-01

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

  4. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

    NARCIS (Netherlands)

    P. Aparicio-Domingo (Patricia); M. Romera-Hernandez (Monica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

    2015-01-01

    textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

  5. Modulation of Abnormal Metabolic Brain Networks by Experimental Therapies in a Nonhuman Primate Model of Parkinson Disease: An Application to Human Retinal Pigment Epithelial Cell Implantation.

    Science.gov (United States)

    Peng, Shichun; Ma, Yilong; Flores, Joseph; Cornfeldt, Michael; Mitrovic, Branka; Eidelberg, David; Doudet, Doris J

    2016-10-01

    Abnormal covariance pattern of regional metabolism associated with Parkinson disease (PD) is modulated by dopaminergic pharmacotherapy. Using high-resolution (18)F-FDG PET and network analysis, we previously derived and validated a parkinsonism-related metabolic pattern (PRP) in nonhuman primate models of PD. It is currently not known whether this network is modulated by experimental therapeutics. In this study, we examined changes in network activity by striatal implantation of human levodopa-producing retinal pigment epithelial (hRPE) cells in parkinsonian macaques and evaluated the reproducibility of network activity in a small test-retest study. (18)F-FDG PET scans were acquired in 8 healthy macaques and 8 macaques with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced bilateral nigrostriatal dopaminergic lesions after unilateral putaminal implantation of hRPE cells or sham surgery. PRP activity was measured prospectively in all animals and in a subset of test-retest animals using a network quantification approach. Network activity and regional metabolic values were compared on a hemispheric basis between animal groups and treatment conditions. All individual macaques showed clinical improvement after hRPE cell implantation compared with the sham surgery. PRP activity was elevated in the untreated MPTP hemispheres relative to those of the normal controls (P therapy and other symptomatic interventions. With further validation in large samples, (18)F-FDG PET imaging with network analysis may provide a viable biomarker for assessing treatment response in animal models of PD after experimental therapies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  6. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  7. Epithelial cells as alternative human biomatrices for comet assay

    Directory of Open Access Journals (Sweden)

    Emilio eRojas

    2014-11-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  9. Sodium selectivity of Reissner's membrane epithelial cells

    Directory of Open Access Journals (Sweden)

    Kim Kyunghee X

    2011-02-01

    Full Text Available Abstract Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC, which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196, RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3. By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala

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

    Directory of Open Access Journals (Sweden)

    Gan L

    2016-11-01

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

  11. Anti-inflammatory activity of a novel family of aryl ureas compounds in an endotoxin-induced airway epithelial cell injury model.

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    Nuria E Cabrera-Benitez

    Full Text Available Despite our increased understanding of the mechanisms involved in acute lung injury (ALI and the acute respiratory distress syndrome (ARDS, there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS-induced ALI/ARDS.After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103 was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4 and nuclear factor kappa B inhibitor alpha (IκBα was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings.Using a novel screening methodology, we identified a compound - CKT0103 - with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and sepsis-induced ALI/ARDS.

  12. Expression of proliferating cell nuclear antigen in cultured middle ear epithelial cells of the guinea pig.

    Science.gov (United States)

    Takeno, S; Hamamura, N; Hirakawa, K; Yajin, K

    1996-01-01

    Primary cultures of middle ear epithelium from the guinea pig were successfully established on type I collagen coated dishes. To characterize cellular outgrowth, antibodies to the proliferating cell nuclear antigen were used as a marker for spreading cells in the S phase of the cell cycle. A number of migrating epithelial cells positively stained for proliferating cell nuclear antigen after 7 and 14 days in culture. Confocal laser scanning microscopy was used to evaluate the localization pattern of this antigen, and the fluorescence intensity was quantified in different areas of the migrating epithelial sheet after various times in culture. Two distinct areas proved to be major sites of proliferating cell nuclear antigen expression. One was at the edge of the tissue explants from which multilayered epithelial cells had begun to migrate. The other was along the margin of the outgrowth, where the cells often had elongated shapes and were aligned in rows. The cells in both areas were identified as nonciliated cells; ciliated cells in the outgrowth showed little staining. We hypothesized that the outgrowth cells in this experiment might be identical to the migrating cells usually observed in renewing epithelia after injury. This model may provide a simple and reproducible method of evaluating the regenerative ability of the middle ear epithelium.

  13. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

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    Chang Rae Rho

    Full Text Available Granulocyte-macrophage colony-stimulating factor (GM-CSF is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs. We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF. An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml. MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  14. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Science.gov (United States)

    Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

    2015-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  15. Invasion of epithelial cells by Trichinella spiralis: in vitro observations

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    Romarís F.

    2001-06-01

    Full Text Available It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine, however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. The recent demonstration that invasion also occurs in vitro when infective larvae of T. spiralis are inoculated onto cultures of epithelial cells provides a model that allows the direct observation of the process by which the parasite recognizes, invades and migrates within the epithelium. The finding that penetration of the cell membrane or Induction of plasma membrane wounds by larvae do not always result in invasion argue in favor of some kind of host-parasite communication in successful invasion. In this sense, the in vitro model of invasion provides a readily manipulated and controlled system to investigate both parasite, and host cell requirements for invasion.

  16. Intracellular localization of the extracellular matrix enzyme lysyl oxidase in polarized epithelial cells

    Science.gov (United States)

    Jansen, Matthias K.; Csiszar, Katalin

    2007-01-01

    Considerable evidence supports novel functions for lysyl oxidase (LOX) beyond its traditional role in initiating crosslinkages in collagen and elastin within the extracellular matrix. These novel roles are particularly relevant during the transition of malignant epithelial cells towards a migratory and invasive phenotype. However, knowledge on cellular and matrix functions of LOX has been generated almost exclusively in mesenchymal cell types. But it is becoming increasingly evident that these cell types are not adequate to address these novel and highly significant roles for LOX in epithelial tissues. In this initial report, we demonstrate that active LOX is expressed by polarized MDCK II kidney and MCF-10A breast epithelial cells. Furthermore, we show evidence for the presence of mature LOX in the cytoplasm and establish these cell lines as models for epithelial LOX studies. PMID:17074474

  17. Regulated Mucin Secretion from Airway Epithelial Cells

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    Kenneth Bruce Adler

    2013-09-01

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

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

    OpenAIRE

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

    2015-01-01

    This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb3248 Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epi...

  19. Proximal location of mouse prostate epithelial stem cells

    OpenAIRE

    Tsujimura, Akira; Koikawa, Yasuhiro; Salm, Sarah; Takao, Tetsuya; Coetzee, Sandra; Moscatelli, David; Shapiro, Ellen; Lepor, Herbert; Sun, Tung-Tien; Wilson, E. Lynette

    2002-01-01

    Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propos...

  20. SWCNTs induced autophagic cell death in human bronchial epithelial cells.

    Science.gov (United States)

    Park, Eun-Jung; Zahari, Nur Elida M; Lee, Eun-Woo; Song, Jaewhan; Lee, Jae-Hyeok; Cho, Myung-Haing; Kim, Jae-Ho

    2014-04-01

    Carbon nanotubes are being actively introduced in electronics, computer science, aerospace, and other industries. Thus, the urgent need for toxicological studies on CNTs is mounting. In this study, we investigated the alterations in cellular response with morphological changes induced by single-walled carbon nanotubes (SWCNTs) in BEAS-2B cells, a human bronchial epithelial cell line. At 24h after exposure, SWCNTs rapidly decreased ATP production and cell viability as well a slight increase in the number of cells in the subG1 and G1 phases. In addition, SWCNTs increased the expression of superoxide dismutase (SOD)-1, but not SOD-2, and the number of cells generating ROS. The concentration of Cu and Zn ions also increased in a dose-dependent manner in cells exposed to SWCNTs. SWCNTs significantly enhanced the release of nitric oxide, interleukin (IL)-6, and IL-8 and up-regulated the expression of chemokine- and cytokine-related genes. Furthermore, the levels of autophagy-related genes, especially the DRAM1 gene, and the autophagosome formation-related proteins, were clearly up-regulated together with an increase of autophagosome-like vacuoles. Based on these results, we suggest that SWCNTs induce autophagic cell death through mitochondrial dysfunction and cytosolic damage in human bronchial epithelial cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Epithelial cells line all cavities and surfaces throughout the body and play a substantial role in maintaining tissue homeostasis. Asthma and other atopic diseases are increasing worldwide and allergic disorders are hypothesized to be a consequence of a combination of dysregulation of the epithel......Epithelial cells line all cavities and surfaces throughout the body and play a substantial role in maintaining tissue homeostasis. Asthma and other atopic diseases are increasing worldwide and allergic disorders are hypothesized to be a consequence of a combination of dysregulation...... environment of both potential allergens and microflora. Limitations of these models prompt a need to develop new human cell-based in vitro models. A variety of co-culture systems for modelling the respiratory epithelium exist and are available to the scientific community. The models have become increasingly......-culture models to become powerful tools in the discovery of key molecules dictating immunity and/or tolerance, and for understanding the complex interplay that takes place between mucosa, airway epithelium and resident or infiltrating immune cells. This review focuses on current knowledge and the advantages...

  2. Aldose reductase inhibition prevents metaplasia of airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Umesh C S Yadav

    Full Text Available BACKGROUND: Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR regulates the mucus cell metaplasia in vitro and in vivo. METHODOLOGY/FINDINGS: Metaplasia in primary human small airway epithelial cells (SAEC was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE. CONCLUSIONS: The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors

  3. Aldose reductase inhibition prevents metaplasia of airway epithelial cells.

    Science.gov (United States)

    Yadav, Umesh C S; Aguilera-Aguirre, Leopoldo; Ramana, Kota V; Boldogh, Istvan; Srivastava, Satish K

    2010-12-28

    Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo. Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE. The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors such as fidarestat could be developed as therapeutic agents to

  4. The Paracrine Effect of Transplanted Human Amniotic Epithelial Cells on Ovarian Function Improvement in a Mouse Model of Chemotherapy-Induced Primary Ovarian Insufficiency

    Directory of Open Access Journals (Sweden)

    Xiaofen Yao

    2016-01-01

    Full Text Available Human amnion epithelial cells (hAECs transplantation via tail vein has been reported to rescue ovarian function in mice with chemotherapy-induced primary ovarian insufficiency (POI. To test whether intraperitoneally transplanted hAECs could induce therapeutic effect and to characterize the paracrine effect of transplanted hAECs, we utilized a chemotherapy induced mice model of POI and investigated the ability of hAECs and conditioned medium collected from cultured hAECs (hAECs-CM to restore ovarian function. We found that transplantation of hAECs or hAECs-CM either 24 hours or 7 days after chemotherapy could increase follicle numbers and partly restore fertility. By PCR analysis of recipient mice ovaries, the presence of SRY gene was only detected in mice transplanted with male hAECs 24 hours following chemotherapy. Further, the gene expression level of VEGFR1 and VEGFR2 in the ovaries decreased, although VEGFA increased 2 weeks after chemotherapy. After treatment with hAECs or hAEC-CM, the expression of both VEGFR1 and VEGFR2 increased, consistent with the immunohistochemical analysis. In addition, both hAECs and hAECs-CM treatment enhanced angiogenesis in the ovaries. The results suggested that hAECs-CM, like hAECs, could partly restore ovarian function, and the therapeutic function of intraperitoneally transplanted hAECs was mainly induced by paracrine-mediated ovarian protection and angiogenesis.

  5. Kalman filter parameter estimation for a nonlinear diffusion model of epithelial cell migration using stochastic collocation and the Karhunen-Loeve expansion.

    Science.gov (United States)

    Barber, Jared; Tanase, Roxana; Yotov, Ivan

    2016-06-01

    Several Kalman filter algorithms are presented for data assimilation and parameter estimation for a nonlinear diffusion model of epithelial cell migration. These include the ensemble Kalman filter with Monte Carlo sampling and a stochastic collocation (SC) Kalman filter with structured sampling. Further, two types of noise are considered -uncorrelated noise resulting in one stochastic dimension for each element of the spatial grid and correlated noise parameterized by the Karhunen-Loeve (KL) expansion resulting in one stochastic dimension for each KL term. The efficiency and accuracy of the four methods are investigated for two cases with synthetic data with and without noise, as well as data from a laboratory experiment. While it is observed that all algorithms perform reasonably well in matching the target solution and estimating the diffusion coefficient and the growth rate, it is illustrated that the algorithms that employ SC and KL expansion are computationally more efficient, as they require fewer ensemble members for comparable accuracy. In the case of SC methods, this is due to improved approximation in stochastic space compared to Monte Carlo sampling. In the case of KL methods, the parameterization of the noise results in a stochastic space of smaller dimension. The most efficient method is the one combining SC and KL expansion. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Sequential Salinomycin Treatment Results in Resistance Formation through Clonal Selection of Epithelial-Like Tumor Cells

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    Florian Kopp

    2014-12-01

    Full Text Available Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC–specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.

  7. The Contribution of the Airway Epithelial Cell to Host Defense.

    Science.gov (United States)

    Stanke, Frauke

    2015-01-01

    In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

  8. The Contribution of the Airway Epithelial Cell to Host Defense

    Directory of Open Access Journals (Sweden)

    Frauke Stanke

    2015-01-01

    Full Text Available In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

  9. GM-CSF produced by nonhematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa.

    Science.gov (United States)

    Egea, Laia; McAllister, Christopher S; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F

    2013-02-15

    GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, as well as dendritic cell differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn's disease in humans and colitis in murine models has mainly been considered to reflect its activity on myeloid cells. We used GM-CSF-deficient (GM-CSF(-/-)) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS), at doses that resulted in little epithelial damage and mucosal ulceration in wild type mice, caused marked colon ulceration and delayed ulcer healing in GM-CSF(-/-) mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF(-/-) mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF(-/-) mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Nonhematopoietic cells, and not myeloid cells, produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury, as revealed by bone marrow chimera and dendritic cell-depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell-produced GM-CSF has a novel nonredundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium.

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

    Directory of Open Access Journals (Sweden)

    Jeanette Wagener

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

  11. A hybrid computational model to explore the topological characteristics of epithelial tissues.

    Science.gov (United States)

    González-Valverde, Ismael; García-Aznar, José Manuel

    2017-11-01

    Epithelial tissues show a particular topology where cells resemble a polygon-like shape, but some biological processes can alter this tissue topology. During cell proliferation, mitotic cell dilation deforms the tissue and modifies the tissue topology. Additionally, cells are reorganized in the epithelial layer and these rearrangements also alter the polygon distribution. We present here a computer-based hybrid framework focused on the simulation of epithelial layer dynamics that combines discrete and continuum numerical models. In this framework, we consider topological and mechanical aspects of the epithelial tissue. Individual cells in the tissue are simulated by an off-lattice agent-based model, which keeps the information of each cell. In addition, we model the cell-cell interaction forces and the cell cycle. Otherwise, we simulate the passive mechanical behaviour of the cell monolayer using a material that approximates the mechanical properties of the cell. This continuum approach is solved by the finite element method, which uses a dynamic mesh generated by the triangulation of cell polygons. Forces generated by cell-cell interaction in the agent-based model are also applied on the finite element mesh. Cell movement in the agent-based model is driven by the displacements obtained from the deformed finite element mesh of the continuum mechanical approach. We successfully compare the results of our simulations with some experiments about the topology of proliferating epithelial tissues in Drosophila. Our framework is able to model the emergent behaviour of the cell monolayer that is due to local cell-cell interactions, which have a direct influence on the dynamics of the epithelial tissue. Copyright © 2017 John Wiley & Sons, Ltd.

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

    Energy Technology Data Exchange (ETDEWEB)

    Malizia, Andrea P.; Lacey, Noreen [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland); Walls, Dermot [School of Biotechnology, Dublin City University. Dublin, 9. Ireland (Ireland); Egan, Jim J. [Advanced Lung Disease and Lung Transplant Program, Mater Misericordiae University Hospital. 44, Eccles Street. Dublin, 7. Ireland (Ireland); Doran, Peter P., E-mail: peter.doran@ucd.ie [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland)

    2009-07-01

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

  13. Airway epithelial cells produce neurotrophins and promote the survival of eosinophils during allergic airway inflammation.

    Science.gov (United States)

    Hahn, Christian; Islamian, Ariyan Pirayesh; Renz, Harald; Nockher, Wolfgang Andreas

    2006-04-01

    Eosinophil-epithelial cell interactions make a major contribution to asthmatic airway inflammation. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and other members of the neurotrophin family, originally defined as a class of neuronal growth factors, are now recognized to support the survival and activation of immune cells. Neurotrophin levels are increased in bronchoalveolar lavage fluid during allergic asthma. We sought to investigate the role of neurotrophins as inflammatory mediators in eosinophil-epithelial cell interactions during the allergic immune response. Neurotrophin expression in the lung was investigated by means of immunohistochemistry and ELISA in a mouse model of chronic experimental asthma. Coculture experiments were performed with airway epithelial cells and bronchoalveolar lavage fluid eosinophils. Neurotrophin levels increased continuously during chronic allergic airway inflammation, and airway epithelial cells were the major source of NGF and BDNF within the inflamed lung. Epithelial neurotrophin production was upregulated by IL-1beta, TNF-alpha, and T(H)2 cytokines. Lung eosinophils expressed the BDNF and NGF receptors tropomyosin-related kinase (Trk) A and TrkB, and coculture with airway epithelial cells resulted in enhanced epithelial neurotrophin production, as well as in prolonged survival of eosinophils. Eosinophil survival was completely abolished in the presence of the neurotrophin receptor Trk antagonist K252a. During allergic inflammation, airway epithelial cells express increased amounts of NGF and BDNF that promote the survival of tissue eosinophils. Controlling epithelial neurotrophin production might be an important therapeutic target to prevent allergic airway eosinophilia. Attenuating the release of inflammatory mediators from the activated airway epithelium will become an important strategy to disrupt the pathogenesis of chronic allergic asthma.

  14. Native and synthetic scaffolds for limbal epithelial stem cell transplantation.

    Science.gov (United States)

    Nguyen, Kim N; Bobba, Samantha; Richardson, Alexander; Park, Mijeong; Watson, Stephanie L; Wakefield, Denis; Di Girolamo, Nick

    2018-01-01

    Limbal stem cell deficiency (LSCD) is a complex blinding disease of the cornea, which cannot be treated with conventional corneal transplants. Instead, a stem cell (SC) graft is required to replenish the limbal epithelial stem cell (LESC) reservoir, which is ultimately responsible for regenerating the corneal epithelium. Current therapies utilize limbal tissue biopsies that harbor LESCs as well as tissue culture expanded cells. Typically, this tissue is placed on a scaffold that supports the formation of corneal epithelial cell sheets, which are then transferred to diseased eyes. A wide range of biological and synthetic materials have been identified as carrier substrates for LESC, some of which have been used in the clinic, including amniotic membrane, fibrin, and silicon hydrogel contact lenses, each with their own advantages and limitations. This review will provide a brief background of LSCD, focusing on bio-scaffolds that have been utilized in limbal stem cell transplantation (LSCT) and materials that are being developed as potentially novel therapeutics for patients with this disease. The outcome of patients with corneal blindness that receive stem cell grafts to restore eye health and correct vision varies considerably and may be due to the different biological and synthetic scaffolds used to deliver these cells to the ocular surface. This review will highlight the positive attributes and limitations of the myriad of carriers developed for clinical use as well as those that are being trialled in pre-clinical models. The overall focus is on developing a standardized therapy for patients, however due to the multiple causes of corneal blindness, a personal regenerative medicine approach may be the best option. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  15. Three-dimensional cultures of mouse mammary epithelial cells.

    Science.gov (United States)

    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 laboratory

  16. The transport pathways of polymer nanoparticles in MDCK epithelial cells.

    Science.gov (United States)

    He, Bing; Jia, Zengrong; Du, Wenwen; Yu, Chao; Fan, Yuchen; Dai, Wenbing; Yuan, Lan; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Zhang, Qiang

    2013-06-01

    Epithelial cell membranes as the typical biological barrier constitute the prime obstacle for the transport of therapeutic agents including nanomedicines. The previous studies on the interaction between nanomedicines and cells are mostly emphasized on cellular uptake and intracellular trafficking, but seldom on epithelial cells, although more and more oral nanomedicines are available now. In an attempt to clarify the transport pathways of nanomedicines in epithelial cells, the different molecular mechanisms among endocytosis, exocytosis and transcytosis processes were carefully studied and compared here using a kind of polymer nanoparticles (PNs) and MDCK epithelial cells as models. As the result, their similarity and difference were demonstrated. The similarities among all the three processes included the mediation of lipid rafts, the involvement of some protein kinases such as protein tyrosine kinase (PTK), protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), and the existence of multiple pathways. However, the difference among these processes was very significant, including different pathways, and especially the disparate effects of lipid rafts and protein kinases for different processes. The endocytosis involved both lipid raft and clathrin mechanisms but no macropinocytosis, via the invagination of membrane but no pore formation, the exocytosis contained ER/Golgi and Golgi/PM pathways, and transcytosis included AEE/CE/BSE and Golgi/BSE pathways. The roles of lipid rafts on endocytosis were positive but that on exocytosis and transcytosis was negative. The impacts of PTK and PKC on endocytosis were positive, while the influences of PTK, PKC and P13K on AEE/CE/BSE, as well as PTK and P13K on Golgi/BSE transcytosis pathways were negative. Moreover, the discrepancy between inward and outward transport of PNs elucidated an interesting fact that the endocytosis was rather easy and outward transport including exocytosis and transcytosis was rather

  17. Proteoglycan synthesis and Golgi organization in polarized epithelial cells.

    Science.gov (United States)

    Dick, Gunnar; Akslen-Hoel, Linn K; Grøndahl, Frøy; Kjos, Ingrid; Prydz, Kristian

    2012-12-01

    A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.

  18. PAX8 expression in ovarian surface epithelial cells.

    Science.gov (United States)

    Adler, Emily; Mhawech-Fauceglia, Paulette; Gayther, Simon A; Lawrenson, Kate

    2015-07-01

    High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells, a considerable body of evidence suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in fallopian tube secretory epithelial cells, but there are conflicting reports about PAX8 expression in OSECs. The purposes of this study were to comprehensively characterize PAX8 expression in a large series of OSECs and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 messenger RNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44% to 71% of OSECs. Calretinin and E-cadherin were frequently coexpressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P = .028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P = .003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs, and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs, PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures

    Science.gov (United States)

    Cerruti, Benedetta; Puliafito, Alberto; Shewan, Annette M.; Yu, Wei; Combes, Alexander N.; Little, Melissa H.; Chianale, Federica; Primo, Luca; Serini, Guido; Mostov, Keith E.; Celani, Antonio

    2013-01-01

    The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell–cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell–cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis. PMID:24145168

  20. Cell volume regulation in epithelial physiology and cancer

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Hoffmann, Else Kay; Novak, Ivana

    2013-01-01

    regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes...... such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered...

  1. Formation of reactive oxygen species in rat epithelial cells upon ...

    Indian Academy of Sciences (India)

    Unknown

    2.1 Cell culture. RLE-6TN epithelial cells (ATCC Rockville, MD, USA) have been immortalized by transfection of rat alveolar type II cells with SV40 DNA (Driscoll et al 1995). Cells were maintained in Dulbecco's Modified .... candidate because it is bound to desferrioxamine with a stability constant near 1031. The hint that ...

  2. Metformin inhibits the proliferation of benign prostatic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Zongwei Wang

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

  3. Dynamic cellular finite-element method for modelling large-scale cell migration and proliferation under the control of mechanical and biochemical cues: a study of re-epithelialization.

    Science.gov (United States)

    Zhao, Jieling; Cao, Youfang; DiPietro, Luisa A; Liang, Jie

    2017-04-01

    Computational modelling of cells can reveal insight into the mechanisms of the important processes of tissue development. However, current cell models have limitations and are challenged to model detailed changes in cellular shapes and physical mechanics when thousands of migrating and interacting cells need to be modelled. Here we describe a novel dynamic cellular finite-element model (DyCelFEM), which accounts for changes in cellular shapes and mechanics. It also models the full range of cell motion, from movements of individual cells to collective cell migrations. The transmission of mechanical forces regulated by intercellular adhesions and their ruptures are also accounted for. Intra-cellular protein signalling networks controlling cell behaviours are embedded in individual cells. We employ DyCelFEM to examine specific effects of biochemical and mechanical cues in regulating cell migration and proliferation, and in controlling tissue patterning using a simplified re-epithelialization model of wound tissue. Our results suggest that biochemical cues are better at guiding cell migration with improved directionality and persistence, while mechanical cues are better at coordinating collective cell migration. Overall, DyCelFEM can be used to study developmental processes when a large population of migrating cells under mechanical and biochemical controls experience complex changes in cell shapes and mechanics. © 2017 The Author(s).

  4. Environmentally prevalent polycyclic aromatic hydrocarbons can elicit co-carcinogenic properties in an in vitro murine lung epithelial cell model.

    Science.gov (United States)

    Bauer, Alison K; Velmurugan, Kalpana; Plöttner, Sabine; Siegrist, Katelyn J; Romo, Deedee; Welge, Peter; Brüning, Thomas; Xiong, Ka-Na; Käfferlein, Heiko U

    2017-11-23

    Low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAH) are the most abundant PAHs environmentally, occupationally, and are in cigarette smoke; however, little is known about their carcinogenic potential. We hypothesized that LMW PAHs act as co-carcinogens in the presence of a known carcinogen (benzo[a]pyrene (B[a]P)) in a mouse non-tumorigenic type II cell line (C10 cells). Gap junctions are commonly suppressed and inflammation induced during tumor promotion, while DNA-adduct formation is observed during the initiation stage of cancer. We used these endpoints together as markers of carcinogenicity in these lung adenocarcinoma progenitor cells. LMW PAHs (1-methylanthracene and fluoranthene, 1-10 µM total in a 1:1 ratio) were used based on previous studies as well as B[a]P (0-3 µM) as the classic carcinogen; non-cytotoxic doses were used. B[a]P-induced inhibition of gap junctional intercellular communication (GJIC) was observed at low doses and further reduced in the presence of the LMW PAH mixture (P carcinogenic potential. Future studies will further address the mechanisms of co-carcinogenesis driving these responses.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    2015-07-15

    The pyloric antral hormone gastrin plays a role in remodeling of the gastric epithelium, but the specific targets of gastrin that mediate these effects are poorly understood. Glandular epithelial cells of the gastric corpus express matrix metalloproteinase (MMP)-1, which is a potential determinant of tissue remodeling; some of these cells express the CCK-2 receptor at which gastrin acts. We have now examined the hypothesis that gastrin stimulates expression of MMP-1 in the stomach. We determined MMP-1 transcript abundance in gastric mucosal biopsies from Helicobacter pylori negative human subjects with normal gastric mucosal histology, who had a range of serum gastrin concentrations due in part to treatment with proton pump inhibitors (PPI). The effects of gastrin were studied on gastric epithelial AGS-GR cells using Western blot and migration assays. In human subjects with increased serum gastrin due to PPI usage, MMP-1 transcript abundance was increased 2-fold; there was also increased MMP-7 transcript abundance but not MMP-3. In Western blots, gastrin increased proMMP-1 abundance, as well that of a minor band corresponding to active MMP-1, in the media of AGS-GR cells, and the response was mediated by protein kinase C and p42/44 MAP kinase. There was also increased MMP-1 enzyme activity. Gastrin-stimulated AGS-GR cell migration in both scratch wound and Boyden chamber assays was inhibited by MMP-1 immunoneutralization. We conclude that MMP-1 expression is a target of gastrin implicated in mucosal remodeling. Copyright © 2015 the American Physiological Society.

  7. Engineering human renal epithelial cells for transplantation in regenerative medicine.

    Science.gov (United States)

    Manzoli, Vita; Colter, David C; Dhanaraj, Sridevi; Fornoni, Alessia; Ricordi, Camillo; Pileggi, Antonello; Tomei, Alice A

    2017-10-01

    Cellular transplantation may treat several human diseases by replacing damaged cells and/or providing a local source of trophic factors promoting regeneration. We utilized human renal epithelial cells (hRECs) isolated from cadaveric donors as a cell model. For efficacious implementation of hRECs for treatment of kidney diseases, we evaluated a novel encapsulation strategy for immunoisolation of hRECs and lentiviral transduction of the Green Fluorescent Protein (GFP) as model gene for genetic engineering of hRECs to secrete desired trophic factors. In specific, we determined whether encapsulation through conformal coating and/or GFP transduction of hRECs allowed preservation of cell viability and of their trophic factor secretion. To that end, we optimized cultures of hRECs and showed that aggregation in three-dimensional spheroids significantly preserved cell viability, proliferation, and trophic factor secretion. We also showed that both wild type and GFP-engineered hRECs could be efficiently encapsulated within conformal hydrogel coatings through our fluid dynamic platform and that this resulted in further improvement of cell viability and trophic factors secretion. Our findings may lay the groundwork for future therapeutics based on transplantation of genetically engineered human primary cells for treatment of diseases affecting kidneys and potentially other tissues. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  8. Characterisation of cell adhesion in airway epithelial cell types using electric cell-substrate impedance sensing

    NARCIS (Netherlands)

    Heijink, I H; Brandenburg, S M; Noordhoek, J A; Postma, D S; Slebos, D-J; van Oosterhout, A J M

    Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance

  9. In Vitro Compatibility of Contact Lenses With Corneal Epithelial Cells.

    Science.gov (United States)

    Vijay, Ajay K; Fadli, Zohra; Lakkis, Carol; Coles-Brennan, Chantal; Willcox, Mark D P

    2017-07-18

    To determine the interaction of contact lenses of different materials with corneal epithelial cells grown in tissue culture. Two different corneal epithelial cell lines were grown to confluence in culture media. Two hydrogel contact lenses with and without polyvinylpyrrolidone (PVP) {1-DAY ACUVUE MOIST (1-Day ACUVUE [hydrogel lenses]) and a silicone hydrogel contact lens, AIR OPTIX NIGHT & DAY} were removed from their blister packs, washed in phosphate-buffered saline, and applied to the cells. After exposure for 24 hr at 37°C, lenses were removed, and the corneal cells and supernatants processed. Supernatants from the cell assays were used to quantify the amount of 17 different cytokines that were produced using a multiplex bead assay. Cells were stained to assess amount of cell death (apoptosis or necrosis) or stained to determine the level of mitochondrial activity. Stimulants of necrotic death (latex) or apoptotic death (sorbitol) were used as positive controls. Cells produced cytokines during normal growth. Exposure of cells to the hydrogel lenses resulted in only minimal changes to normal production of cytokines, but latex or sorbitol produced the most change. Exposure of the cells to all three lenses caused 4% to 23% reduction in mitochondrial activity, whereas exposure to the positive controls caused 71% to 98% reduction in mitochondrial activity. Exposure of the corneal epithelial cells to contact lenses produced minimal morphological changes, whereas exposure to latex or sorbitol produced significant changes to the human corneal epithelial cell line. Exposure of corneal epithelial cells to contact lenses had minimal impact on their physiology. There was no difference in epithelial cell responses to hydrogel with or without PVP compared with the silicone hydrogel contact lens.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-10-15

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

  11. Probiotics promote endocytic allergen degradation in gut epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Song, Chun-Hua [Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou (China); Liu, Zhi-Qiang [Department of Gastroenterology, The Second Hospital, Zhengzhou University, Zhengzhou (China); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada); Huang, Shelly [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada); Zheng, Peng-Yuan, E-mail: medp7123@126.com [Department of Gastroenterology, The Second Hospital, Zhengzhou University, Zhengzhou (China); Yang, Ping-Chang, E-mail: yangp@mcmaster.ca [Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON (Canada)

    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.

  12. Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

    Science.gov (United States)

    Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

    2017-04-01

    The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

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

    Science.gov (United States)

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

    2015-11-01

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

  14. Lingual Epithelial Stem Cells and Organoid Culture of Them

    Directory of Open Access Journals (Sweden)

    Hiroko Hisha

    2016-01-01

    Full Text Available 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.

  15. Effects of epiplakin-knockdown in cultured corneal epithelial cells.

    Science.gov (United States)

    Kokado, Masahide; Okada, Yuka; Miyamoto, Takeshi; Yamanaka, Osamu; Saika, Shizuya

    2016-05-20

    To investigate effects of knockdown of epiplakin gene expression on the homeostasis of cultured corneal epithelial cell line. We previously reported acceleration of corneal epithelial wound healing in an epiplakin-null mouse. Gene expression of epiplakin was knockdowned by employing siRNA transfection in SV40-immortalized human corneal epithelial cell line. Protein expression of E-cadherin, keratin 6 and vimentin was examined by western blotting. Cell migration and proliferation were examined by using scratch assay and Alamar blue assay, respectively. Scratch assay and Alamar blue assay showed migration and proliferation of the cells was accelerated by epiplakin knockdown. siRNA-knockdown of epiplakin suppressed protein expression of E-cadherin, keratin 6 and vimentin. Decreased expression of E-cadherin, keratin 6 and vimentin might be included in the mechanisms of cell migration acceleration in the absence of epiplakin. The mechanism of cell proliferation stimulation by epiplakin knockdown is to be investigated.

  16. Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

    Directory of Open Access Journals (Sweden)

    Guillaume Golovkine

    2016-01-01

    Full Text Available To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

  17. Ion transport in epithelial spheroids derived from human airway cells

    DEFF Research Database (Denmark)

    Pedersen, P S; Frederiksen, O; Holstein-Rathlou, N H

    1999-01-01

    -CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial...... electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+ absorption followed by water. Transformation to a Cl- secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine......-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model...

  18. [Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims].

    Science.gov (United States)

    Cristovam, Priscila Cardoso; Glória, Maria Aparecida da; Melo, Gustavo Barreto; Gomes, José Alvaro Pereira

    2008-01-01

    To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.

  19. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... However, troglitazone had protective effects of EPCR on injured cells. Key words: Endothelial protein C receptor, renal tubular epithelial cell, troglitazone, tumor necrosis factor-α, interleukin-1β; high glucose. ..... induce apoptosis of vascular smooth muscle cells through an extracellular signal- regulated ...

  20. Toxic mechanisms of copper oxide nanoparticles in epithelial kidney cells

    DEFF Research Database (Denmark)

    Thit, Amalie; Selck, Henriette; Bjerregaard, Henning F.

    2015-01-01

    CuO NPs have previously been reported as toxic to a range of cell cultures including kidney epithelial cells from the frog, Xenopus laevis (A6). Here we examine the molecular mechanisms affecting toxicity of Cu in different forms and particle sizes. A6 cells were exposed to ionic Cu (Cu2+) or CuO...

  1. Mesenchymal stem cell therapy stimulates endogenous host progenitor cells to improve colonic epithelial regeneration.

    Directory of Open Access Journals (Sweden)

    Alexandra Sémont

    Full Text Available Patients who undergo pelvic radiotherapy may develop severe and chronic complications resulting from gastrointestinal alterations. The lack of curative treatment highlights the importance of novel and effective therapeutic strategies. We thus tested the therapeutic benefit of mesenchymal stem cells (MSC treatment and proposed molecular mechanisms of action. MSC efficacy was tested in an experimental model of radiation-induced severe colonic ulceration histologically similar to that observed in patients. In this model, MSC from bone marrow were administered intravenously, immediately or three weeks (established lesions after irradiation. MSC therapy reduces radiation-induced colonic ulceration and increases animal survival. MSC treatment induces therapeutic efficacy whatever the time of cell infusion. Infused-MSC engraft in the colon but also increase endogenous MSC mobilization in blood that have lasting benefits over time. In vitro analysis demonstrates that the MSC effect is mediated by paracrine mechanisms through the non-canonical WNT (Wingless integration site pathway. In irradiated rat colons, MSC treatment increases the expression of the non-canonical WNT4 ligand by epithelial cells. The epithelial regenerative process is improved after MSC injection by stimulation of colonic epithelial cells positive for SOX9 (SRY-box containing gene 9 progenitor/stem cell markers. This study demonstrates that MSC treatment induces stimulation of endogenous host progenitor cells to improve the regenerative process and constitutes an initial approach to arguing in favor of the use of MSC to limit/reduce colorectal damage induced by radiation.

  2. [The entry of Epstein-Barr virus into B lymphocytes and epithelial cells during infection].

    Science.gov (United States)

    Zuo, Lie-Lian; Zhu, Mei-Juan; Du, Shu-Juan; Lu, Jian-Hong; Li, Gui-Yuan

    2014-07-01

    Epstein-Barr virus (EBV) is a human herpesvirus associated with important human diseases, including infectious mononucleosis syndrome, malignant lymphoma, and nasopharyngeal carcinoma. The mechanism of EBV entry into host cells remains a subject of intensive research. After decades of study, researchers have identified several key proteins and different patterns of EBV intrusion into host cells. The viral surface glycoproteins, gp350/220, gp42, gB, gH, and gL, are involved in interactions with the CR2 receptor on the surface of B lymphocytes during viral entry. However, the majority of epithelial cells lack CR2 receptor expression, which makes viral invasion much more complex than in B lymphocytes. Three different models have been proposed to explain how EBV enters epithelial cells: (1) "transfer of infection", mediated by B lymphocytes or Langerhans cells; (2) EBV utilizes its own proteins during the process of fusion with the cell membrane; and (3) progeny virions arising from EBV-infected epithelial cells cross lateral membranes into adjacent epithelial cells. This review will discuss the relevant mechanism of viral entry into B lymphocytes and epithelial cells during EBV infection.

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

    Science.gov (United States)

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

    2016-10-15

    The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalized airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Obesity reversibly depletes the basal cell population and enhances mammary epithelial cell estrogen receptor alpha expression and progenitor activity.

    Science.gov (United States)

    Chamberlin, Tamara; D'Amato, Joseph V; Arendt, Lisa M

    2017-11-29

    Obesity is correlated with an increased risk for developing postmenopausal breast cancer. Since obesity rates continue to rise worldwide, it is important to understand how the obese microenvironment influences normal mammary tissue to increase breast cancer risk. We hypothesized that obesity increases the proportion of luminal progenitor cells, which are thought to be the cells of origin for the most common types of breast cancer, potentially leading to an increased risk for breast cancer. To study the obese microenvironment within the mammary gland, we used a high-fat diet mouse model of obesity and human breast tissue from reduction mammoplasty surgery. We identified changes in breast epithelial cell populations using flow cytometry for cell surface markers, in vitro functional assays and expression of markers on breast tissue sections. In both obese female mice and women, mammary epithelial cell populations demonstrated significant decreases in basal/myoepithelial cells, using either flow cytometry or cell-type-specific markers (SMA and p63). Estrogen receptor alpha (ERα) expression was significantly increased in luminal cells in obese mammary tissue, compared with control mice or breast tissue from lean women. Functional assays demonstrated significantly enhanced mammary epithelial progenitor activity in obese mammary epithelial cells and elevated numbers of ERα-positive epithelial cells that were co-labeled with markers of proliferation. Weight loss in a group of obese mice reversed increases in progenitor activity and ERα expression observed in obese mammary tissue. Obesity enhances ERα-positive epithelial cells, reduces the number of basal/myoepithelial cells, and increases stem/progenitor activity within normal mammary tissue in both women and female mice. These changes in epithelial cell populations induced by obesity are reversible with weight loss. Our findings support further studies to examine how obesity-induced changes in stem/progenitor cells

  5. Alveolar type II epithelial cell dysfunction in rat experimental hepatopulmonary syndrome (HPS.

    Directory of Open Access Journals (Sweden)

    Wenli Yang

    Full Text Available The hepatopulmonary syndrome (HPS develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2 play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL. We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064 induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.

  6. Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Shimpei Gotoh

    2014-09-01

    Full Text Available No methods for isolating induced alveolar epithelial progenitor cells (AEPCs from human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs, we identified carboxypeptidase M (CPM as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Increased epithelial-free areas in thymuses with altered EphB-mediated thymocyte-thymic epithelial cell interactions.

    Science.gov (United States)

    García-Ceca, Javier; Montero-Herradón, Sara; Alfaro, David; Zapata, Agustín G

    2017-10-01

    Epithelial-free areas, present in both thymic cortex and medulla, have been studied in WT and EphB-deficient mice that have important alterations in the development of thymic epithelium due to the lack of proper thymocyte-thymic epithelial cell interactions. In both WT and mutant thymuses, the number and size of epithelial-free areas are significantly larger in the medulla than in the cortex. The two parameters show a reverse correlation: low numbers of these areas course with large epithelial-free areas and vice versa. However, their structure and cell content are similar in mutant and WT thymuses. Cortical epithelial-free areas just contain DP thymocytes, while the medullary ones consist of SP cells, blood vessels, mesenchyme-derived ER-TR7+ cells and components of the extracellular matrix (i.e., collagen IV, fibronectin, laminin). Other components, such as desmin, αSMA, PDGFRβ and Ng2, frequently associated with blood vessel walls, also appear. Vimentin, although present in medullary epithelial-free areas, does not co-express with epithelial cells. Other markers related to epithelial-mesenchymal transitions, such as Snail, Slug or FSP1, are not expressed. These results suggest that alterations in the cell interactions between distinct thymic cell components that induce both increased proportions of apoptotic thymic epithelial cells and altered behavior of the mesenchyme associated with the medullary vasculature could explain the appearance of these areas and their differences in the cortex and medulla.

  9. Neurogenic effects of β-amyloid in the choroid plexus epithelial cells in Alzheimer's disease.

    Science.gov (United States)

    Bolos, Marta; Spuch, Carlos; Ordoñez-Gutierrez, Lara; Wandosell, Francisco; Ferrer, Isidro; Carro, Eva

    2013-08-01

    β-amyloid (Aβ) can promote neurogenesis, both in vitro and in vivo, by inducing neural progenitor cells to differentiate into neurons. The choroid plexus in Alzheimer's disease (AD) is burdened with amyloid deposits and hosts neuronal progenitor cells. However, neurogenesis in this brain tissue is not firmly established. To investigate this issue further, we examined the effect of Aβ on the neuronal differentiation of choroid plexus epithelial cells in several experimental models of AD. Here we show that Aβ regulates neurogenesis in vitro in cultured choroid plexus epithelial cells as well as in vivo in the choroid plexus of APP/Ps1 mice. Treatment with oligomeric Aβ increased proliferation and differentiation of neuronal progenitor cells in cultured choroid plexus epithelial cells, but decreased survival of newly born neurons. These Aβ-induced neurogenic effects were also observed in choroid plexus of APP/PS1 mice, and detected also in autopsy tissue from AD patients. Analysis of signaling pathways revealed that pre-treating the choroid plexus epithelial cells with specific inhibitors of TyrK or MAPK diminished Aβ-induced neuronal proliferation. Taken together, our results support a role of Aβ in proliferation and differentiation in the choroid plexus epithelial cells in Alzheimer's disease.

  10. Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis.

    Science.gov (United States)

    Zhang, Yue; Li, Fengyin; Song, Yongli; Sheng, Xiaole; Ren, Fazheng; Xiong, Kai; Chen, Lei; Zhang, Hongquan; Liu, Dequan; Lengner, Christopher J; Xue, Lixiang; Yu, Zhengquan

    2016-10-01

    Mammary epithelium is comprised of an inner layer of luminal epithelial cells and an outer layer of contractile myoepithelial cells with mesenchymal properties. These two compartments interact throughout mammary morphogenesis to form branching ducts during puberty and terminate in secretory alveoli during lactation. It is not known how the myoepithelial cell lineage is specified, nor how signals in myoepithelial cells contribute to lactogenesis. Here, we show that Numb and Numbl are enriched in mammary myoepithelial cells, with their expression peaking during pregnancy. We use conditional Numb- and Numbl-knockout mouse models to demonstrate that loss of Numb/Numbl compromised the myoepithelial layer and expanded the luminal layer, led epithelial cells to undergo epithelial-to-mesenchymal transition, and resulted in lactation failure as a result of abnormal alveolar formation during pregnancy. Numb and Numbl function via repression of the Notch signaling pathway and of the p53-p21 axis during mammary gland development. These findings highlight the importance of Numb and Numbl in the control of myoepithelial cell fate determination, epithelial identity, and lactogenesis.-Zhang Y., Li, F., Song, Y., Sheng, X., Ren, F., Xiong, K., Chen, L., Zhang, H., Liu, D., Lengner, C. J., Xue, L., Yu, Z. Numb and Numbl act to determine mammary myoepithelial cell fate, maintain epithelial identity, and support lactogenesis. © FASEB.

  11. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells.

    Science.gov (United States)

    Huff, Ryan D; Hsu, Alan C-Y; Nichol, Kristy S; Jones, Bernadette; Knight, Darryl A; Wark, Peter A B; Hansbro, Philip M; Hirota, Jeremy A

    2017-01-01

    The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines.

  12. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ryan D Huff

    Full Text Available The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production.Allergen and cigarette smoke mouse models were performed using house dust mite (HDM and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies.HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4 inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells.Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines.

  13. Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

    Science.gov (United States)

    Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

    2017-01-01

    Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

  14. Differential nephron HO-1 expression following glomerular epithelial cell injury.

    Science.gov (United States)

    Datta, Prasun K; Reddy, Sreenivas; Sharma, Mukut; Lianos, Elias A

    2006-01-01

    In proteinuria of glomerular origin there is upregulation of heme-oxygenase (HO), the rate-limiting enzyme of heme degradation, in the nephron in a segment-specific manner. To better characterize this phenomenon, we employed a model of proteinuria resulting from disruption of the glomerular capillary permeability barrier to protein by administration of the glomerular epithelial cell toxin puromycin aminonucleoside (PAN) to rats. In this model, we assessed nephron distribution of the expression of the inducible HO isoform, HO-1, and the role of free radicals in modulating HO-1 expression. Rats were injected with either vehicle (dimethyl sulfoxide) or PAN or the spin trap free radical stabilizer alpha-phenyl-N-tert butyl nitrone (PBN), or with both PAN and PBN. Ten days following the PAN injection, urine protein, creatinine, nitric oxide (NO) and malonyldialdehyde (MDA) were measured. Kidney sections and protein lysates were assessed for changes in HO-1 expression by immunohistochemistry and Western blot analysis. In control animals (DMSO or PBN alone) there was no proteinuria and very weak or absent HO-1 staining in nephron segments. PAN treatment induced proteinuria and increased urine MDA excretion. In these animals, there was a robust HO-1 expression mainly in tubules and in glomerular parietal but not visceral epithelial cells. Unilateral ureteral obstruction to interrupt glomerular filtration in animals treated with PAN abrogated tubular HO-1 expression in the kidney ipsilateral to the obstruction. Administration of PBN to PAN-treated animals reduced proteinuria and MDA excretion while it markedly augmented tubular HO-1 expression. This augmentation was prominent in tubular cells of the inner cortex/outer medulla. These observations indicate that upregulation of nephron HO-1 following disruption of the glomerular permeability barrier occurs at sites downstream of this barrier and is mediated by a filtered HO-1 inducer(s). Scavenging of free radicals potentiates

  15. Aberrant Smad3 phosphoisoforms in cyst-lining epithelial cells in the cpk mouse, a model of autosomal recessive polycystic kidney disease.

    Science.gov (United States)

    Hama, Taketsugu; Nakanishi, Koichi; Sato, Masashi; Mukaiyama, Hironobu; Togawa, Hiroko; Shima, Yuko; Miyajima, Masayasu; Nozu, Kandai; Nagao, Shizuko; Takahashi, Hisahide; Sako, Mayumi; Iijima, Kazumoto; Yoshikawa, Norishige; Suzuki, Hiroyuki

    2017-12-01

    Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved; however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L), COOH-terminal regions (pSmad3C), and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in the TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, cyclin-dependent kinase (CDK) 4, and c-Myc were evaluated by Western blotting. Cophosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-Smooth muscle actin, which indicates mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4, and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was cophosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/cpk double-mutant mice revealed amelioration of cpk abnormalities. These findings suggest that upregulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD and may be a target for disease-specific intervention. Copyright © 2017 the American Physiological Society.

  16. Foxc2 influences alveolar epithelial cell differentiation during lung development.

    Science.gov (United States)

    Tsuji, Mayoko; Morishima, Masae; Shimizu, Kazuhiko; Morikawa, Shunichi; Heglind, Mikael; Enerbäck, Sven; Ezaki, Taichi; Tamaoki, Jun

    2017-08-01

    FOXC2, a forkhead transcriptional factor, is a candidate gene for congenital heart diseases and lymphedema-distichiasis syndrome and yellow nail syndrome; however, there are no reports on Foxc2 and the development of the lung. We have identified lung abnormalities in Foxc2-knockout embryos during investigation of cardiac development. The aim of this study was to clarify the morphological characteristics during lung development using ICR-Foxc2 knockout lungs. Mutant fetuses at embryonic days 10.5-18.5 were obtained from mating of Foxc2+/- mice and then analyzed. Notably, Foxc2-knockout lungs appeared parenchymatous and much smaller than those of the wild-type littermates. In the Foxc2 knockout lungs, the capillary beds remained distant from the alveolar epithelium until the late stages, the number of type2 alveolar cells per alveolar progenitor cell was lower and the type1 alveolar cells were thicker in Foxc2 knockout mice. In contrast, Foxc2 expression was only detected in the mesenchyme of the lung buds at E10.5, and it disappeared at E11.5 in Foxc2-LacZ knockin mice. Furthermore, the expression of Lef1 was significantly inhibited in E11.5 lungs. All of these results suggest that the abnormalities in Foxc2 knockout mice may involve maldifferentiation of alveolar epithelial cells and capillary vessel endothelial-alveolar epithelial approach as well as lymph vessel malformation. This is the first report about relationship between Foxc2 and lung development. This animal model might provide an important clue for elucidating the mechanism of lung development and the cause of respiratory diseases. © 2017 Japanese Society of Developmental Biologists.

  17. Drug permeation across intestinal epithelial cells using porous silicon nanoparticles.

    Science.gov (United States)

    Bimbo, Luis M; Mäkilä, Ermei; Laaksonen, Timo; Lehto, Vesa-Pekka; Salonen, Jarno; Hirvonen, Jouni; Santos, Hélder A

    2011-04-01

    Mesoporous silicon particles hold great potential in improving the solubility of otherwise poorly soluble drugs. To effectively translate this feature into the clinic, especially via oral or parenteral administration, a thorough understanding of the interactions of the micro- and nanosized material with the physiological environment during the delivery process is required. In the present study, the behaviour of thermally oxidized porous silicon particles of different sizes interacting with Caco-2 cells (both non-differentiated and polarized monolayers) was investigated in order to establish their fate in a model of intestinal epithelial cell barrier. Particle interactions and TNF-α were measured in RAW 264.7 macrophages, while cell viabilities, reactive oxygen species and nitric oxide levels, together with transmission electron microscope images of the polarized monolayers, were assessed with both the Caco-2 cells and RAW 264.7 macrophages. The results showed a concentration and size dependent influence on cell viability and ROS-, NO- and TNF-α levels. There was no evidence of the porous nanoparticles crossing the Caco-2 cell monolayers, yet increased permeation of the loaded poorly soluble drug, griseofulvin, was shown. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. No junctional communication between epithelial cells in hydra

    DEFF Research Database (Denmark)

    de Laat, S W; Tertoolen, L G; Grimmelikhuijzen, C J

    1980-01-01

    Diffusion gradients of morphogens have been inferred as a basis for the control of morphogenesis in hydra, and morphogenetic substances have been found which, on the basis of their molecular weight (MW), should be able to pass gap junctions. There have been several reports of the presence of gap...... junctions between epithelial cells of hydra. However, until now, there has been no report published on whether these junctions enable the epithelial cells to exchange molecules of small molecular weight, as has been described in other organisms. Therefore we decided to investigate the communicative...... properties of the junctional membranes by electrophysiological methods and by intracellular-dye iontophoresis. We report here that no electrotonic coupling is detectable between epithelial cells of Hydra attenuata in: (1) intact animals, (2) head-regenerating animals, (3) cell re-aggregates, and (4) hydra...

  19. High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells.

    Science.gov (United States)

    Chen, Yu-Ching; Statt, Sarah; Wu, Reen; Chang, Hao-Teng; Liao, Jiunn-Wang; Wang, Chien-Neng; Shyu, Woei-Cherng; Lee, Chen-Chen

    2016-01-07

    Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway.

  20. Curcumin and anthocyanin inhibit pepsin-mediated cell damage and carcinogenic changes in airway epithelial cells.

    Science.gov (United States)

    Samuels, Tina L; Pearson, Amy C S; Wells, Clive W; Stoner, Gary D; Johnston, Nikki

    2013-10-01

    Laryngopharyngeal reflux (LPR) is associated with inflammatory and neoplastic airway diseases. Gastric pepsin internalized by airway epithelial cells during reflux contributes to oxidative stress, inflammation, and carcinogenesis. Several plant extracts and compounds inhibit digestive enzymes and inflammatory or neoplastic changes to the esophagus in models of gastroesophageal reflux. This study examined the potential of chemoprotective phytochemicals to inhibit peptic activity and mitigate pepsin-mediated damage of airway epithelial cells. Cultured human laryngeal and hypopharyngeal epithelial cells were pretreated with curcumin (10 micromol/L), ecabet sodium (125 microg/mL), and anthocyanin-enriched black-raspberry extract (100 microg/mL) 30 minutes before treatment with pepsin (0.1 mg/mL; 1 hour; pH 7). Controls were treated with media pH 7 or pepsin pH 7 without phytochemicals. Cell damage and proliferative changes were assessed by electron microscopy, cell count, thymidine analog incorporation, and real-time polymerase chain reaction array. Pepsin inhibition was determined by in vitro kinetic assay. Micromolar concentrations of curcumin, ecabet sodium, and black-raspberry extract inhibited peptic activity and pepsin-induced mitochondrial damage and hyperproliferation. Curcumin abrogated pepsin-mediated depression of tumor suppressor gene expression and altered the subcellular localization of pepsin following endocytosis. Several phytochemicals inhibit the pepsin-mediated cell damage underlying inflammatory or neoplastic manifestations of LPR. Dietary supplementation or adjunctive therapy with phytochemicals may represent novel preventive or therapeutic strategies for LPR-attributed disease.

  1. Vulnerability of Normal Human Mammary Epithelial Cells to Oncogenic Transformation

    Science.gov (United States)

    2012-04-01

    escape senescence and acquire genomic changes. Nature 2001;409:633–7. 10. Olsen CL, Gardie B, Yaswen P, Stampfer MR. Raf-1- induced growth arrest in...p16INK4a. Cell 88:593–602. 10. Olsen CL, Gardie B, Yaswen P, Stampfer MR (2002) Raf-1-induced growth arrest in human mammary epithelial cells is p16...Cycle 3, 244–246. Olsen CL, Gardie B, Yaswen P, Stampfer MR (2002) Raf-1-induced growth arrest in human mammary epithelial cells is p16-independent and

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

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

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

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

    Science.gov (United States)

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

    2014-07-04

    Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

    Science.gov (United States)

    Jeppsson, Sabrina; Srinivasan, Shanthi; Chandrasekharan, Bindu

    2017-02-01

    We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However, the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSS) were used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY -/- ) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 µM) on epithelial proliferation and apoptosis in vitro. DSS-WT mice exhibited enhanced intestinal inflammation, polyp size, and polyp number (7.5 ± 0.8) compared with DSS-NPY -/- mice (4 ± 0.5, P inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and downregulation of miR-375. Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

  5. Replication of influenza A virus in swine umbilical cord epithelial stem-like cells.

    Science.gov (United States)

    Khatri, Mahesh; Chattha, Kuldeep S

    2015-01-01

    In this study, we describe the isolation and characterization of epithelial stem-like cells from the swine umbilical cord and their susceptibility to influenza virus infection. Swine umbilical cord epithelial stem cells (SUCECs) expressed stem cell and pluripotency associated markers such as SSEA-1, SSEA-4, TRA 1-60 and TRA 1-81 and Oct4. Morphologically, cells displayed polygonal morphology and were found to express epithelial markers; pancytokeratin, cytokeratin-18 and occludin; mesenchymal cell markers CD44, CD90 and haematopoietic cell marker CD45 were not detected on these cells. The cells had extensive proliferation and self- renewal properties. The cells also possessed immunomodulatory activity and inhibited the proliferation of T cells. Also, higher levels of anti-inflammatory cytokine IL-10 were detected in SUCEC-T cell co-cultures. The cells were multipotent and differentiated into lung epithelial cells when cultured in epithelial differentiation media. We also examined if SUCECs are susceptible to infection with influenza virus. SUCECs expressed sialic acid receptors, used by influenza virus for binding to cells. The 2009 pandemic influenza virus and swine influenza virus replicated in these cells. SUCECs due to their differentiation and immunoregulatory properties will be useful as cellular therapy in a pig model for human diseases. Additionally, our data indicate that influenza virus can infect SUCECs and may transmit influenza virus from mother to fetus through umbilical cord and transplantation of influenza virus-infected stem cells may transmit infection to recipients. Therefore, we propose that umbilical cord cells, in addition to other agents, should also be tested for influenza virus before cryopreservation for future use as a cell therapy for disease conditions.

  6. Effect of curcumin on aging retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Zhu W

    2015-09-01

    Full Text Available Wei Zhu,1,* Yan Wu,2,* Yi-Fang Meng,1 Jin-Yu Wang,1 Ming Xu,1 Jian-Jun Tao,1 Jiong Lu1 1Department of Ophthalmology, Changshu No 2 People’s Hospital, Changshu, 2Department of Ophthalmology, The First People’s Hospital of Kunshan Affiliated with Jiangsu University, Suzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Age-related macular degeneration (AMD is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD. Keywords: curcumin, retinal pigment epithelium, apoptosis, age-related macular degeneration

  7. The origin of epithelial neoplasms after allogeneic stem cell transplantation.

    NARCIS (Netherlands)

    Smith, M.J.; Cleef, P.H. van; Schattenberg, A.V.M.B.; Krieken, J.H.J.M. van

    2006-01-01

    We analyzed five women, who have developed epithelial neoplasms after sex-mismatched stem cell transplants. Using in situ hybridization for sex chromosome-specific DNA probes and immunohistochemistry we identified the origin of the tumor cells. We conclude that none of the non-hematologic

  8. Effect of Helicobacter pylori on gastric epithelial cells

    Science.gov (United States)

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

    2014-01-01

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

  9. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    The endothelial protein C receptor (EPCR) plays an important role within the protein C pathway in regulating coagulation and inflammation. It was reported that EPCR was expressed in large vessels, placenta, heart, liver and lung endothelial cell. However, there are a few studies concerned about renal epithelial cells.

  10. The role of epithelial cell adhesion molecule N-glycosylation on apoptosis in breast cancer cells.

    Science.gov (United States)

    Zhang, Dandan; Liu, Xue; Gao, Jiujiao; Sun, Yan; Liu, Tingjiao; Yan, Qiu; Yang, Xuesong

    2017-03-01

    Glycosylation of cell surface proteins plays an important role in the regulation of apoptosis. It has been demonstrated that knockdown of epithelial cell adhesion molecule promoted apoptosis, inhibited cell proliferation, and caused cell-cycle arrest. In this study, we investigated whether and how N-glycosylation of epithelial cell adhesion molecule influenced the apoptosis in breast cancer cells. We applied the N-glycosylation mutation epithelial cell adhesion molecule plasmid to express deglycosylation of epithelial cell adhesion molecule and then to study its function. Our results showed that deglycosylation of epithelial cell adhesion molecule promoted apoptosis and inhibited cell proliferation. Deglycosylation of epithelial cell adhesion molecule enhanced the cytotoxic effect of 5-fluorouracil, promoting apoptosis by downregulating the expression of the anti-apoptotic protein Bcl-2 and upregulating the expression of the pro-apoptotic proteins Bax and Caspase 3 via the extracellular-signal-regulated kinase 1/2 and c-Jun N-terminal kinase mitogen-activated protein kinase signaling pathways in MCF-7 and MDA-MB-231 cells. These findings are important for a better understanding of epithelial cell adhesion molecule apoptosis regulation and suggest epithelial cell adhesion molecule as a potential target for the treatment of breast cancer.

  11. Epstein-Barr virus infection of polarized epithelial cells via the basolateral surface by memory B cell-mediated transfer infection.

    Science.gov (United States)

    Shannon-Lowe, Claire; Rowe, Martin

    2011-05-01

    Epstein Barr virus (EBV) exhibits a distinct tropism for both B cells and epithelial cells. The virus persists as a latent infection of memory B cells in healthy individuals, but a role for infection of normal epithelial is also likely. Infection of B cells is initiated by the interaction of the major EBV glycoprotein gp350 with CD21 on the B cell surface. Fusion is triggered by the interaction of the EBV glycoprotein, gp42 with HLA class II, and is thereafter mediated by the core fusion complex, gH/gL/gp42. In contrast, direct infection of CD21-negative epithelial cells is inefficient, but efficient infection can be achieved by a process called transfer infection. In this study, we characterise the molecular interactions involved in the three stages of transfer infection of epithelial cells: (i) CD21-mediated co-capping of EBV and integrins on B cells, and activation of the adhesion molecules, (ii) conjugate formation between EBV-loaded B cells and epithelial cells via the capped adhesion molecules, and (iii) interaction of EBV glycoproteins with epithelial cells, with subsequent fusion and uptake of virions. Infection of epithelial cells required the EBV gH and gL glycoproteins, but not gp42. Using an in vitro model of normal polarized epithelia, we demonstrated that polarization of the EBV receptor(s) and adhesion molecules restricted transfer infection to the basolateral surface. Furthermore, the adhesions between EBV-loaded B cells and the basolateral surface of epithelial cells included CD11b on the B cell interacting with heparan sulphate moieties of CD44v3 and LEEP-CAM on epithelial cells. Consequently, transfer infection was efficiently mediated via CD11b-positive memory B cells but not by CD11b-negative naïve B cells. Together, these findings have important implications for understanding the mechanisms of EBV infection of normal and pre-malignant epithelial cells in vivo.

  12. Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

    Science.gov (United States)

    Anabazhagan, Arivarasu N; Chatterjee, Ishita; Priyamvada, Shubha; Kumar, Anoop; Tyagi, Sangeeta; Saksena, Seema; Alrefai, Waddah A; Dudeja, Pradeep K; Gill, Ravinder K

    2017-03-16

    The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin

  13. Stem cell factor expression after renal ischemia promotes tubular epithelial survival.

    Directory of Open Access Journals (Sweden)

    Geurt Stokman

    Full Text Available BACKGROUND: Renal ischemia leads to apoptosis of tubular epithelial cells and results in decreased renal function. Tissue repair involves re-epithelialization of the tubular basement membrane. Survival of the tubular epithelium following ischemia is therefore important in the successful regeneration of renal tissue. The cytokine stem cell factor (SCF has been shown to protect the tubular epithelium against apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In a mouse model for renal ischemia/reperfusion injury, we studied how expression of c-KIT on tubular epithelium and its ligand SCF protect cells against apoptosis. Administration of SCF specific antisense oligonucleotides significantly decreased specific staining of SCF following ischemia. Reduced SCF expression resulted in impaired renal function, increased tubular damage and increased tubular epithelial apoptosis, independent of inflammation. In an in vitro hypoxia model, stimulation of tubular epithelial cells with SCF activated survival signaling and decreased apoptosis. CONCLUSIONS/SIGNIFICANCE: Our data indicate an important role for c-KIT and SCF in mediating tubular epithelial cell survival via an autocrine pathway.

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

    OpenAIRE

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

    2017-01-01

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated ?-Galactosid...

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

    Science.gov (United States)

    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

  16. Factors influencing the exfoliation of cervicovaginal epithelial cells.

    Science.gov (United States)

    Basu, J; Mikhail, M S; Palan, P R; Payraudeau, P H; Romney, S L

    1992-12-01

    Our objective was to investigate the association of smoking and exfoliation of cervicovaginal epithelial cells while controlling for other factors that may potentially influence cell exfoliation (e.g., presence of cervical intraepithelial neoplasia or koilocytes, the use of oral contraceptives, age, and the phase of the menstrual cycle). Cervicovaginal lavage specimens and epidemiologic questionnaires were obtained with informed consent from 190 women. The cervicovaginal lavage samples were processed to separate other contaminants. The number of squamous epithelial cells counted was expressed as cells per milliliter of lavage. Multiple linear regression analysis revealed that the number of exfoliated epithelial cells was significantly higher in smokers (p < 0.01) and also in women with cervical intraepithelial neoplasia (p < 0.05). The other studied variables had no detectable effect. The findings suggest that smoking or the presence of cervical intraepithelial neoplasia may induce an acceleration in the exfoliation of cervicovaginal epithelial cells. This may alter cell maturation and may be a factor in the oncogenic process.

  17. Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

    Science.gov (United States)

    Kon, Shunsuke; Ishibashi, Kojiro; Katoh, Hiroto; Kitamoto, Sho; Shirai, Takanobu; Tanaka, Shinya; Kajita, Mihoko; Ishikawa, Susumu; Yamauchi, Hajime; Yako, Yuta; Kamasaki, Tomoko; Matsumoto, Tomohiro; Watanabe, Hirotaka; Egami, Riku; Sasaki, Ayana; Nishikawa, Atsuko; Kameda, Ikumi; Maruyama, Takeshi; Narumi, Rika; Morita, Tomoko; Sasaki, Yoshiteru; Enoki, Ryosuke; Honma, Sato; Imamura, Hiromi; Oshima, Masanobu; Soga, Tomoyoshi; Miyazaki, Jun-Ichi; Duchen, Michael R; Nam, Jin-Min; Onodera, Yasuhito; Yoshioka, Shingo; Kikuta, Junichi; Ishii, Masaru; Imajo, Masamichi; Nishida, Eisuke; Fujioka, Yoichiro; Ohba, Yusuke; Sato, Toshiro; Fujita, Yasuyuki

    2017-05-01

    Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

  18. Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells

    NARCIS (Netherlands)

    Kistemaker, Loes E. M.; Hiemstra, Pieter S.; Bos, I. Sophie T.; Bouwman, Susanne; van den Berge, Maarten; Hylkema, Machteld N.; Meurs, Herman; Kerstjens, Huib A. M.; Gosens, Reinoud

    BACKGROUND: It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct

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

    Science.gov (United States)

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

    2017-11-01

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

  20. Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface.

    Science.gov (United States)

    O'Boyle, Nicky; Sutherland, Erin; Berry, Catherine C; Davies, Robert L

    2017-01-01

    The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω × cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω × cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments.

  1. Inhibitory effects of ameloblastin on epithelial cell proliferation.

    Science.gov (United States)

    Saito, Noriko; Ariyoshi, Wataru; Okinaga, Toshinori; Kamegawa, Mariko; Matsukizono, Miho; Akebiyama, Yasuo; Kitamura, Chiaki; Nishihara, Tatsuji

    2014-08-01

    Ameloblastin is an enamel matrix protein expressed in several tissues. Many potential mechanisms have been identified by which ameloblastin functions as an extracellular matrix protein. However, the biological effects of ameloblastin on gingival epithelial cells remain unclear. In the present study, we established a novel system to purify recombinant human ameloblastin and clarified its biological functions in epithelial cells in vitro. Recombinant human ameloblastin was isolated from COS-7 cells overexpressing HaloTag-fused human ameloblastin by the HaloTag system and then purified further by reverse-phase high-performance liquid chromatography. SCC-25 cells, derived from human oral squamous cell carcinoma, were treated with recombinant ameloblastin and then cell survival was assessed by a WST-1 assay. Cell cycle analysis was performed by flow cytometry. The novel purification system allowed effective recovery of the recombinant ameloblastin proteins at a high purity. Recombinant ameloblastin protein was found to suppress the proliferation of SCC-25 cells. Flow cytometric analysis showed that ameloblastin treatment induced cell cycle arrest G1 phase. We developed a procedure for production of highly purified recombinant human ameloblastin. Biological analyses suggest that ameloblastin induces cell cycle arrest in epithelial cells and regulates the progression of periodontitis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Human Bronchial Epithelial Cell Response to Heavy Particle Exposure

    Science.gov (United States)

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

    2012-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Nagai A

    2003-09-01

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

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

    Science.gov (United States)

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

    2017-05-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices. Failure of most biomaterials originates from the inability to predict and control the influence of their surface properties on biological phenomena, particularly protein adsorption, and cellular behaviour, which subsequently results in unfavourable host response. Here, we introduce a surface-proteomic screening approach using a label-free mass spectrometry technique to decipher the adsorption profile of extracellular matrix (ECM) proteins on different biomaterials, and correlate it with cellular behaviour. We demonstrated that the way a biomaterial selectively interacts with specific ECM proteins of a given tissue seems to determine the interactions between the cells of that tissue and biomaterials. Accordingly, this approach can

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  7. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J., E-mail: tokare@niehs.nih.gov

    2015-07-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

  8. CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Miura, Yuka; Hagiwara, Natsumi [Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, 2-1 Gakuen, Sanda 669-1337 Japan (Japan); Radisky, Derek C. [Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 32225 (United States); Hirai, Yohei, E-mail: y-hirai@kwansei.ac.jp [Department of Bioscience, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, 2-1 Gakuen, Sanda 669-1337 Japan (Japan)

    2014-09-10

    Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells. Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.

  9. Apicobasal Polarity Controls Lymphocyte Adhesion to Hepatic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Natalia Reglero-Real

    2014-09-01

    Full Text Available Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1 adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α. We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.

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

    Directory of Open Access Journals (Sweden)

    Isabella Eckerle

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

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

    Science.gov (United States)

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

    2014-01-01

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

  12. Myoferlin depletion in breast cancer cells promotes mesenchymal to epithelial shape change and stalls invasion.

    Directory of Open Access Journals (Sweden)

    Ruth Li

    Full Text Available Myoferlin (MYOF is a mammalian ferlin protein with homology to ancestral Fer-1, a nematode protein that regulates spermatic membrane fusion, which underlies the amoeboid-like movements of its sperm. Studies in muscle and endothelial cells have reported on the role of myoferlin in membrane repair, endocytosis, myoblast fusion, and the proper expression of various plasma membrane receptors. In this study, using an in vitro human breast cancer cell model, we demonstrate that myoferlin is abundantly expressed in invasive breast tumor cells. Depletion of MYOF using lentiviral-driven shRNA expression revealed that MDA-MB-231 cells reverted to an epithelial morphology, suggesting at least some features of mesenchymal to epithelial transition (MET. These observations were confirmed by the down-regulation of some mesenchymal cell markers (e.g., fibronectin and vimentin and coordinate up-regulation of the E-cadherin epithelial marker. Cell invasion assays using Boyden chambers showed that loss of MYOF led to a significant diminution in invasion through Matrigel or type I collagen, while cell migration was unaffected. PCR array and screening of serum-free culture supernatants from shRNA(MYOF transduced MDA-MB-231 cells indicated a significant reduction in the steady-state levels of several matrix metalloproteinases. These data when considered in toto suggest a novel role of MYOF in breast tumor cell invasion and a potential reversion to an epithelial phenotype upon loss of MYOF.

  13. Laminin receptor 37/67LR regulates adhesion and proliferation of normal human intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Taoufik Khalfaoui

    Full Text Available Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.

  14. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    by inducible nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells. METHODS: A colonic cell line (HT29) was stimulated for 1-10 weeks with interferon...... stimulated cells had increased DNA instability (Pcytokine exposure induces an iNOS dependent up-regulation of ROS production and DNA instability. This mechanism...

  15. Reduced expression IRF7 in nasal epithelial cells from smokers as a potential mechanism mediating enhanced susceptibility to influenza

    Science.gov (United States)

    Rationale: Smokers are more susceptible to viral infections, including influenza virus, yet the mechanisms mediating this effect are not known. Methods: We have established an in vitro model of differentiated nasal epithelial cells from smokers, which maintain enhanced levels...

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

    Science.gov (United States)

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

    2013-03-01

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

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

    Science.gov (United States)

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

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel. PMID:23538640

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

    Science.gov (United States)

    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. ©2014 Poultry Science Association Inc.

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

    Directory of Open Access Journals (Sweden)

    Tomoyo Yoshinaga

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

  20. BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity.

    Science.gov (United States)

    Munye, Mustafa M; Shoemark, Amelia; Hirst, Robert A; Delhove, Juliette M; Sharp, Tyson V; McKay, Tristan R; O'Callaghan, Christopher; Baines, Deborah L; Howe, Steven J; Hart, Stephen L

    2017-02-01

    Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination. Copyright © 2017 the American Physiological Society.

  1. Sodium selectivity of semicircular canal duct epithelial cells

    Directory of Open Access Journals (Sweden)

    Harbidge Donald G

    2011-09-01

    Full Text Available Abstract Background Sodium absorption by semicircular canal duct (SCCD epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC, comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197, whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the

  2. Cancer Stem Cells, Epithelial to Mesenchymal Markers, and Circulating Tumor Cells in Small Cell Lung Cancer

    NARCIS (Netherlands)

    Pore, Milind; Meijer, Coby; de Bock, Geertruida H.; Boersma-van Ek, Wytske; Terstappen, Leon W. M. M.; Groen, Harry J. M.; Timens, Wim; Kruyt, Frank A. E.; Hiltermann, T. Jeroen N.

    2016-01-01

    The prognostic value of markers of cancer stem cells and epithelial to mesenchymal transition in small cell lung cancer is not known. We retrospectively studied these markers in the biopsy tissue of patients with small cell lung cancer and correlated them with overall survival and the strongest

  3. Nerve Invasion by Epithelial Cells in Benign Breast Diseases

    Directory of Open Access Journals (Sweden)

    Yu-Jan Chan

    2009-03-01

    Full Text Available Nerve invasion by glandular epithelial cells in a lesion is usually regarded as invasive carcinoma. However, some benign conditions in the pancreas, prostate, breast and other organs may show involvement of nerve bundles by benign epithelial cells. We report an 18-year-old female with nerve invasion in benign breast disease. The lesion in her right breast revealed fibrocystic changes with ductal hyperplasia and stromal sclerosis. Perineural and intraneural involvement by bland-looking small ducts lined by 2 layers of cells including an outer layer of myoepithelial cells were found, suggestive of benign nerve invasion. There was no evidence of malignant cells in any of the sections. The patient remains well after 31 months of follow-up. About 44 cases of nerve invasion in benign breast diseases have been reported in the literature. It is necessary to carefully evaluate nerve involvement in breast lesions to avoid over-diagnosis and inappropriate operation.

  4. Aluminium chloride promotes anchorage-independent growth in human mammary epithelial cells

    OpenAIRE

    Sappino, André-Pascal; Buser Llinares, Raphaële; Lesne, Laurence; Gimelli, Stefania; Bena, Frédérique; Belin, Dominique; Mandriota, Stefano Jacopo

    2012-01-01

    Aluminium salts used as antiperspirants have been incriminated as contributing to breast cancer incidence in Western societies. To date, very little or no epidemiological or experimental data confirm or infirm this hypothesis. We report here that in MCF-10A human mammary epithelial cells, a well-established normal human mammary epithelial cell model, long-term exposure to aluminium chloride (AlCl(3) ) concentrations of 10-300 µ m, i.e. up to 100 000-fold lower than those found in antiperspira...

  5. Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells.

    Directory of Open Access Journals (Sweden)

    Damien Maggiorani

    Full Text Available Tubular epithelial cells in the kidney are continuously exposed to urinary fluid shear stress (FSS generated by urine movement and recent in vitro studies suggest that changes of FSS could contribute to kidney injury. However it is unclear whether FSS alters the epithelial characteristics of the renal tubule. Here, we evaluated in vitro and in vivo the influence of FSS on epithelial characteristics of renal proximal tubular cells taking the organization of junctional complexes and the presence of the primary cilium as markers of epithelial phenotype. Human tubular cells (HK-2 were subjected to FSS (0.5 Pa for 48 h. Control cells were maintained under static conditions. Markers of tight junctions (Claudin-2, ZO-1, Par polarity complex (Pard6, adherens junctions (E-Cadherin, β-Catenin and the primary cilium (α-acetylated Tubulin were analysed by quantitative PCR, Western blot or immunocytochemistry. In response to FSS, Claudin-2 disappeared and ZO-1 displayed punctuated and discontinuous staining in the plasma membrane. Expression of Pard6 was also decreased. Moreover, E-Cadherin abundance was decreased, while its major repressors Snail1 and Snail2 were overexpressed, and β-Catenin staining was disrupted along the cell periphery. Finally, FSS subjected-cells exhibited disappeared primary cilium. Results were confirmed in vivo in a uninephrectomy (8 months mouse model where increased FSS induced by adaptive hyperfiltration in remnant kidney was accompanied by both decreased epithelial gene expression including ZO-1, E-cadherin and β-Catenin and disappearance of tubular cilia. In conclusion, these results show that proximal tubular cells lose an important number of their epithelial characteristics after long term exposure to FSS both in vitro and in vivo. Thus, the changes in urinary FSS associated with nephropathies should be considered as potential insults for tubular cells leading to disorganization of the tubular epithelium.

  6. [STR genotyping from trace epithelial cells on fountain pen].

    Science.gov (United States)

    Yang, Fan; Mei, Shan-Zong; Li, Yong-Hong; Feng, Yan; Yu, Wei-Dong; Zhang, Yue

    2008-02-01

    To evaluate the feasibility of STR genotyping from trace epithelial cells on fountain pen and to discuss the impact of conservation time on DNA typing. Seven fountain pens were separately used by each of the 17 volunteers 20 minutes per day for a month and then were preserved on day 1, 3, 5, 7, 14, 21, and 28. DNA was extracted from the epithelial cells on fountain pen by silicon bead and was genotyped by Identifier kit. The corresponding control samples were buccal swabs of the above volunteers. The detectable numbers of loci were counted for assessment. There were statistically significant differences in the DNA genotyping by detectable numbers of gene loci between buccal swabs and epithelial cells on fountain pen of different conservation times (P fountain pen preserved on day 1, 3, 5, 7, 14, 21, 28 and the corresponding oral swabs were also statistically significant (P fountain pen if the tests were performed within 24 hours. The trace epithelial cells on fountain pen can be used as biological samples for personal identification, but the conservation time would have influence on the results of DNA genotyping.

  7. Membrane dynamics and the regulation of epithelial cell polarity

    NARCIS (Netherlands)

    van der Wouden, JM; Maier, O; van IJzendoorn, SCD; Hoekstra, D

    2003-01-01

    Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains

  8. The Role of SnoN and Ski in Mammary Epithelial Cell Transformation

    National Research Council Canada - National Science Library

    Pan, Deng

    2007-01-01

    .... Higher level of Ski/SnoN is found in transformed mammary epithelial cells. Ski/SnoN might play a role in regulation of the transformation of mammary epithelial cell by antagonizing TGF signaling pathway...

  9. Nasal epithelial cells can act as a physiological surrogate for paediatric asthma studies.

    Directory of Open Access Journals (Sweden)

    Surendran Thavagnanam

    Full Text Available INTRODUCTION: Differentiated paediatric epithelial cells can be used to study the role of epithelial cells in asthma. Nasal epithelial cells are easier to obtain and may act as a surrogate for bronchial epithelium in asthma studies. We assessed the suitability of nasal epithelium from asthmatic children to be a surrogate for bronchial epithelium using air-liquid interface cultures. METHODS: Paired nasal and bronchial epithelial cells from asthmatic children (n = 9 were differentiated for 28 days under unstimulated and IL-13-stimulated conditions. Morphological and physiological markers were analysed using immunocytochemistry, transepithelial-electrical-resistance, Quantitative Real-time-PCR, ELISA and multiplex cytokine/chemokine analysis. RESULTS: Physiologically, nasal epithelial cells from asthmatic children exhibit similar cytokine responses to stimulation with IL-13 compared with paired bronchial epithelial cells. Morphologically however, nasal epithelial cells differed significantly from bronchial epithelial cells from asthmatic patients under unstimulated and IL-13-stimulated conditions. Nasal epithelial cells exhibited lower proliferation/differentiation rates and lower percentages of goblet and ciliated cells when unstimulated, while exhibiting a diminished and varied response to IL-13. CONCLUSIONS: We conclude that morphologically, nasal epithelial cells would not be a suitable surrogate due to a significantly lower rate of proliferation and differentiation of goblet and ciliated cells. Physiologically, nasal epithelial cells respond similarly to exogenous stimulation with IL-13 in cytokine production and could be used as a physiological surrogate in the event that bronchial epithelial cells are not available.

  10. IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells

    Science.gov (United States)

    Laoukili, Jamila; Perret, Eric; Willems, Tom; Minty, Adrian; Parthoens, Eef; Houcine, Odile; Coste, Andre; Jorissen, Mark; Marano, Francelyne; Caput, Daniel; Tournier, Frédéric

    2001-01-01

    In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor α subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13’s effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction. PMID:11748265

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

    Science.gov (United States)

    Takano, Mikihisa; Nagahiro, Machi; Yumoto, Ryoko

    2016-02-01

    Nicotine is absorbed from the lungs into the systemic circulation during cigarette smoking. However, there is little information concerning the transport mechanism of nicotine in alveolar epithelial cells. In this study, we characterized the uptake of nicotine in rat primary cultured type II (TII) and transdifferentiated type I-like (TIL) epithelial cells. In both TIL and TII cells, [(3)H]nicotine uptake was time and temperature-dependent, and showed saturation kinetics. [(3)H]Nicotine uptake in these cells was not affected by Na(+), but was sensitive to extracellular and intracellular pH, suggesting the involvement of a nicotine/proton antiport system. The uptake of [(3)H]nicotine in these cells was potently inhibited by organic cations such as clonidine, diphenhydramine, and pyrilamine, but was not affected by substrates and/or inhibitors of known organic cation transporters such as carnitine, 1-methyl-4-phenylpyridinium, and tetraethylammonium. In addition, the uptake of [(3)H]nicotine in TIL cells was stimulated by preloading the cells with unlabeled nicotine, pyrilamine, and diphenhydramine, but not with tetraethylammonium. These results suggest that a novel proton-coupled antiporter is involved in the uptake of nicotine in alveolar epithelial cells and its absorption from the lungs into the systemic circulation. Copyright © 2016. Published by Elsevier Inc.

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

    Directory of Open Access Journals (Sweden)

    Rahman Irfan

    2006-10-01

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

  13. Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney.

    Science.gov (United States)

    Vogetseder, Alexander; Palan, Thomas; Bacic, Desa; Kaissling, Brigitte; Le Hir, Michel

    2007-02-01

    We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus.

  14. Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhangyong1956@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China); Gao, Ming-Qing, E-mail: gaomingqing@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China)

    2016-11-15

    Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

  15. In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination

    Science.gov (United States)

    2015-12-01

    AWARD NUMBER: W81XWH-13-1-0184 TITLE: In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell Dissemination PRINCIPAL...Sep 2013 - 14 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER In Vivo Tagging of Lung Epithelial Cells To Define the Early Steps of Tumor Cell...understand the early events that accompany invasive behavior in vivo , we proposed to develop a lineage-labeling system to detect and isolate cells of lung

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang, E-mail: wenfang64@hotmail.com; Zhang, Yi, E-mail: syzi960@yahoo.com

    2013-11-01

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

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

    Science.gov (United States)

    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. © 2014 AlphaMed Press.

  19. Wave Propagation of Junctional Remodeling in Collective Cell Movement of Epithelial Tissue: Numerical Simulation Study

    Directory of Open Access Journals (Sweden)

    Tetsuya Hiraiwa

    2017-07-01

    Full Text Available During animal development, epithelial cells forming a monolayer sheet move collectively to achieve the morphogenesis of epithelial tissues. One driving mechanism of such collective cell movement is junctional remodeling, which is found in the process of clockwise rotation of Drosophila male terminalia during metamorphosis. However, it still remains unknown how the motions of cells are spatiotemporally organized for collective movement by this mechanism. Since these moving cells undergo elastic deformations, the influence of junctional remodeling may mechanically propagate among them, leading to spatiotemporal pattern formations. Here, using a numerical cellular vertex model, we found that the junctional remodeling in collective cell movement exhibits spatiotemporal self-organization without requiring spatial patterns of molecular signaling activity. The junctional remodeling propagates as a wave in a specific direction with a much faster speed than that of cell movement. Such propagation occurs in both the absence and presence of fluctuations in the contraction of cell boundaries.

  20. Cigarette smoke condensate modulates migration of human gingival epithelial cells and their interactions with Porphyromonas gingivalis.

    Science.gov (United States)

    Imamura, K; Kokubu, E; Kita, D; Ota, K; Ishihara, K; Saito, A

    2015-06-01

    Epithelial cells are recognized as the first line of defense against bacterial infection and environmental harmful stimuli such as cigarette smoke (CS). Although previous studies explored the effects of nicotine on host cells, mechanisms by which CS affects cellular functions remain uncertain. The present study investigated the effects of CS condensate (CSC) on in vitro wound closure of gingival epithelial cells and their potential interactions with a major periodontal pathogen, Porphyromonas gingivalis. Human gingival epithelial cells (Ca9-22) were treated with CSC for 24 h. Cell proliferation was determined using a WST-1 assay. Cell migration was assessed using a wound closure model. The expression of integrins was analyzed by confocal scanning laser microscopy and real-time PCR. Intracellular invasion of P. gingivalis was evaluated by confocal scanning laser microscopy and an antibiotic protection assay. Low concentrations (1-10 μg/mL) of CSC showed no significant effect on cell proliferation. CSC demonstrated dual effects on epithelial wound closure of Ca9-22 cells: high concentrations (i.e. 250 μg/mL) significantly inhibited the wound closure whereas low concentrations (i.e. 10 μg/mL) promoted it (p < 0.01). CSC induced distinct changes in cytoskeleton. When CSC-exposed cells were infected with P. gingivalis for 2 h, a significant inhibition of wound closure was observed concurrent with a decrease in integrin α3 expression near the wound area. A significantly increased P. gingivalis invasion into Ca9-22 was observed when exposed to low concentrations of CSC. Low concentrations of CSC increased invasion of human gingival epithelial cells by P. gingivalis and induced changes in cytoskeleton and integrin expression, thereby modulating the cell migration. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  1. File list: His.Lng.50.AllAg.Tracheal_epithelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Science.gov (United States)

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

    2011-02-21

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

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

    Science.gov (United States)

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

    2017-12-01

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

  5. Linking changes in epithelial morphogenesis to cancer mutations using computational modeling.

    Directory of Open Access Journals (Sweden)

    Katarzyna A Rejniak

    2010-08-01

    Full Text Available Most tumors arise from epithelial tissues, such as mammary glands and lobules, and their initiation is associated with the disruption of a finely defined epithelial architecture. Progression from intraductal to invasive tumors is related to genetic mutations that occur at a subcellular level but manifest themselves as functional and morphological changes at the cellular and tissue scales, respectively. Elevated proliferation and loss of epithelial polarization are the two most noticeable changes in cell phenotypes during this process. As a result, many three-dimensional cultures of tumorigenic clones show highly aberrant morphologies when compared to regular epithelial monolayers enclosing the hollow lumen (acini. In order to shed light on phenotypic changes associated with tumor cells, we applied the bio-mechanical IBCell model of normal epithelial morphogenesis quantitatively matched to data acquired from the non-tumorigenic human mammary cell line, MCF10A. We then used a high-throughput simulation study to reveal how modifications in model parameters influence changes in the simulated architecture. Three parameters have been considered in our study, which define cell sensitivity to proliferative, apoptotic and cell-ECM adhesive cues. By mapping experimental morphologies of four MCF10A-derived cell lines carrying different oncogenic mutations onto the model parameter space, we identified changes in cellular processes potentially underlying structural modifications of these mutants. As a case study, we focused on MCF10A cells expressing an oncogenic mutant HER2-YVMA to quantitatively assess changes in cell doubling time, cell apoptotic rate, and cell sensitivity to ECM accumulation when compared to the parental non-tumorigenic cell line. By mapping in vitro mutant morphologies onto in silico ones we have generated a means of linking the morphological and molecular scales via computational modeling. Thus, IBCell in combination with 3D acini

  6. Smad2 overexpression enhances adhesion of gingival epithelial cells.

    Science.gov (United States)

    Hongo, Shoichi; Yamamoto, Tadashi; Yamashiro, Keisuke; Shimoe, Masayuki; Tomikawa, Kazuya; Ugawa, Yuki; Kochi, Shinsuke; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

    2016-11-01

    Gingival epithelial cells play an important role in preventing the initiation of periodontitis, by their hemidesmosomal adhesion to the tooth root surface. Adhesion requires integrin-extracellular matrix (ECM) interactions that are intricately regulated by transforming growth factor-β (TGF-β) signaling. However, the mechanisms underlying the interplay between adhesion molecules and TGF-β, especially the respective roles of Smad2 and Smad3, remain elusive. In this study, we examined the effects of Smad overexpression on gingival epithelial cell adhesion and expression profiles of integrin and ECM-related genes. Human gingival epithelial cells immortalized by the SV40 T-antigen were transfected with Smad2- and Smad3-overexpression vectors. A cell adhesion assay involving fluorescence detection of attached cells was performed using the ArrayScan imaging system. Real-time PCR was performed to examine the kinetics of integrin and ECM gene expression. In vitro and in vivo localization of adhesion molecules was examined by immunofluorescence analysis. By using SB431542, a specific inhibitor of the TGF-β type I receptor, Smad2/3 signaling was confirmed to be dominant in TGF-β1-induced cell adhesion. The Smad2-transfectant demonstrated higher potency for cell adhesion and integrin expression (α2, α5, β4, and β6) than the Smad3-transfectant, whereas little or no change in ECM expression was observed in either transfectant. Moreover, the gingival epithelium of transgenic mice that overexpressed Smad2 driven by the keratin 14 promoter showed increased integrin α2 expression. These findings indicate the crucial role of Smad2 in increased adhesion of gingival epithelial cells via upregulation of integrin α2. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

    Science.gov (United States)

    Banerjee, Aditi; Basu, Malini; Blanchard, Thomas G; Chintalacharuvu, Subba R; Guang, Wei; Lillehoj, Erik P; Czinn, Steven J

    2016-10-01

    Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events. © 2016 John Wiley & Sons Ltd.

  8. Effect of TGF-β on ocular surface epithelial cells.

    Science.gov (United States)

    Benito, Maria Jesús; Calder, Virginia; Corrales, Rosa M; García-Vázquez, Carmen; Narayanan, Srihari; Herreras, José M; Stern, Michael E; Calonge, Margarita; Enríquez-de-Salamanca, Amalia

    2013-02-01

    A role for transforming growth factor (TGF)-β in the pathogenesis of some ocular surface diseases has been proposed. We determined if secretion of TGF-β and expression of TGF-β receptors RI, RII, and RIII by human ocular surface epithelial cells were modified under inflammatory conditions. We also determined how these cells responded to TGF-β. A human corneal epithelial (HCE) cell line and a conjunctival epithelial cell line (IOBA-NHC) were exposed to TGF-β1 and -β2 and to proinflammatory cytokines. TGF-β receptor mRNAs were analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) in both cell lines, and in conjunctival, limbal, and corneal epithelial cells from post-mortem human specimens. Expression of TGF-β receptors and pSMAD2/SMAD2 were determined by Western blot and immunofluorescence assays. Secretion of TGF-β isoforms, cytokine/chemokine, and metalloproteinases (MMPs) were analyzed in cell supernatants by immunobead-based assays. Secretory leukocyte proteinase inhibitor (SLPI) secretion was analyzed by enzyme-linked immunosorbent assay. TGF-β isoform and receptor gene expression was determined by RT-PCR in conjunctival epithelium of dry eye (DE) patients and healthy subjects. Our results showed that TGF-β RI expression was down-regulated with IL-4 exposure, whereas TGF-β RII and TGF-β2 were upregulated by TNF-α in HCE cells. TGF-β RIII receptor expression was upregulated in IOBA-NHC cells by TNF-α and IFN-γ. SMAD2 phosphorylation occurred in HCE and IOBA-NHC cells after TGF-β treatment. TGF-β significantly up- and down-regulated secretion of several cytokines/chemokines by both cell lines and MMP by HCE cells. TGF-β2 and TGF-β3 were upregulated and TGF-β RIII mRNA was down-regulated in DE conjunctival epithelium. These results show that TGF-β plays an important role in directing local inflammatory responses in ocular surface epithelial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Acrolein stimulates eicosanoid release from bovine airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Doupnik, C.A.; Leikauf, G.D. (Univ. of Cincinnati College of Medicine, OH (USA))

    1990-10-01

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

  10. Estradiol increases mucus synthesis in bronchial epithelial cells.

    Directory of Open Access Journals (Sweden)

    Anthony Tam

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

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  12. Development of a syngeneic mouse model of epithelial ovarian cancer

    Directory of Open Access Journals (Sweden)

    Quinn Bridget A

    2010-10-01

    Full Text Available Abstract Background Most cases of ovarian cancer are epithelial in origin and diagnosed at advanced stage when the cancer is widely disseminated in the peritoneal cavity. The objective of this study was to establish an immunocompetent syngeneic mouse model of disseminated epithelial ovarian cancer (EOC to facilitate laboratory-based studies of ovarian tumor biology and preclinical therapeutic strategies. Methods Individual lines of TgMISIIR-TAg transgenic mice were phenotypically characterized and backcrossed to inbred C57BL/6 mice. In addition to a previously described line of EOC-prone mice, two lines (TgMISIIR-TAg-Low were isolated that express the oncogenic transgene, but have little or no susceptibility to tumor development. Independent murine ovarian carcinoma (MOVCAR cell lines were established from the ascites of tumor-bearing C57BL/6 TgMISIIR-TAg transgenic mice, characterized and tested for engraftment in the following recipient mice: 1 severe immunocompromised immunodeficient (SCID, 2 wild type C57BL/6, 3 oophorectomized tumor-prone C57BL/6 TgMISIIR-TAg transgenic and 4 non-tumor prone C57BL/6 TgMISIIR-TAg-Low transgenic. Lastly, MOVCAR cells transduced with a luciferase reporter were implanted in TgMISIIR-TAg-Low mice and in vivo tumor growth monitored by non-invasive optical imaging. Results Engraftment of MOVCAR cells by i.p. injection resulted in the development of disseminated peritoneal carcinomatosis in SCID, but not wild type C57BL/6 mice. Oophorectomized tumor-prone TgMISIIR-TAg mice developed peritoneal carcinomas with high frequency, rendering them unsuitable as allograft recipients. Orthotopic or pseudo-orthotopic implantation of MOVCAR cells in TgMISIIR-TAg-Low mice resulted in the development of disseminated peritoneal tumors, frequently accompanied by the production of malignant ascites. Tumors arising in the engrafted mice bore histopathological resemblance to human high-grade serous EOC and exhibited a similar pattern

  13. Alveolar epithelial type II cell: defender of the alveolus revisited

    Directory of Open Access Journals (Sweden)

    Fehrenbach Heinz

    2001-01-01

    Full Text Available Abstract In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2 cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today.

  14. Epithelial cell-targeted transgene expression enables isolation of cyan fluorescent protein (CFP)-expressing prostate stem/progenitor cells.

    Science.gov (United States)

    Peng, Weidan; Bao, Yunhua; Sawicki, Janet A

    2011-10-01

    To establish a method for efficient and relatively easy isolation of a cell population containing epithelial prostate stem cells, we developed two transgenic mouse models, K5/CFP and K18/RFP. In these models, promoters of the cytokeratin 5 (Krt5) and the cytokeratin 18 (Krt18) genes regulate cyan and red fluorescent proteins (CFP and RFP), respectively. CFP and RFP reporter protein fluorescence allows for visualization of K5(+) and K18(+) epithelial cells within the cellular spatial context of the prostate gland and for their direct isolation by FACS. Using these models, it is possible to test directly the stem cell properties of prostate epithelial cell populations that are positively selected based on expression of cytoplasmic proteins, K5 and K18. After validating appropriate expression of the K5/CFP and K18/RFP transgenes in the developing and adult prostate, we demonstrate that a subset of CFP-expressing prostate cells exhibits stem cell proliferation potential and differentiation capabilities. Then, using prostate cells sorted from double transgenic mice (K5/CFP + K18/RFP), we compare RNA microarrays of sorted K5(+)K18(+) basal and K5(-)K18(+) luminal epithelial cells, and identify genes that are differentially expressed. Several genes that are over-expressed in K5(+) cells have previously been identified as potential stem cell markers. These results suggest that FACS isolation of prostate cells from these mice based on combining reporter gene fluorescence with expression of potential stem cell surface marker proteins will yield populations of cells enriched for stem cells to a degree that has not been attained by using cell surface markers alone.

  15. On the Sulfation and Methylation of Catecholestrogens in Human Mammary Epithelial Cells and Breast Cancer Cells

    National Research Council Canada - National Science Library

    Hui, Ying; Yasuda, Shin; Liu, Ming-Yih; Wu, Yi-yong; Liu, Ming-Cheh

    2008-01-01

    .... The present study was designed to examine the role of sulfation in the metabolism of CEs. MCF-7 breast cancer cells and MCF 10A human mammary epithelial cells were metabolically labeled with [35S...

  16. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Satoru Yoshida

    Full Text Available 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.

  17. Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced Pluripotent Stem Cells

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Yu Qin

    2015-05-01

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

  19. Profibrotic potential of Prominin-1+ epithelial progenitor cells in pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Lüscher Thomas F

    2011-09-01

    Full Text Available Abstract Background In idiopathic pulmonary fibrosis loss of alveolar epithelium induces inflammation of the pulmonary tissue followed by accumulation of pathogenic myofibroblasts leading eventually to respiratory failures. In animal models inflammatory and resident cells have been demonstrated to contribute to pulmonary fibrosis. Regenerative potential of pulmonary and extra-pulmonary stem and progenitor cells raised the hope for successful treatment option against pulmonary fibrosis. Herein, we addressed the contribution of lung microenvironment and prominin-1+ bone marrow-derived epithelial progenitor cells in the mouse model of bleomycin-induced experimental pulmonary fibrosis. Methods Prominin-1+ bone marrow-derived epithelial progenitors were expanded from adult mouse lungs and differentiated in vitro by cytokines and growth factors. Pulmonary fibrosis was induced in C57Bl/6 mice by intratracheal instillation of bleomycin. Prominin-1+ progenitors were administered intratracheally at different time points after bleomycin challenge. Green fluorescence protein-expressing cells were used for cell tracking. Cell phenotypes were characterized by immunohistochemistry, flow cytometry and quantitative reverse transcription-polymerase chain reaction. Results Prominin-1+ cells expanded from healthy lung represent common progenitors of alveolar type II epithelial cells, myofibroblasts, and macrophages. Administration of prominin-1+ cells 2 hours after bleomycin instillation protects from pulmonary fibrosis, and some of progenitors differentiate into alveolar type II epithelial cells. In contrast, prominin-1+ cells administered at day 7 or 14 lose their protective effects and differentiate into myofibroblasts and macrophages. Bleomycin challenge enhances accumulation of bone marrow-derived prominin-1+ cells within inflamed lung. In contrast to prominin-1+ cells from healthy lung, prominin-1+ precursors isolated from inflamed organ lack regenerative

  20. Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers.

    Science.gov (United States)

    Min, Kyoung Ah; Rosania, Gus R; Kim, Chong-Kook; Shin, Meong Cheol

    2016-03-01

    To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly different with the NHBE or Calu-3 cells, showing low paracellular permeability and high TEER values, while the NL-20 cells showed high paracellular permeability and low TEER. Quantitative image analysis of the confocal microscope sections further confirmed that the Calu-3 cells formed intact cell monolayers in contrast to the NHBE and NL-20 cells with multilayers. Among three lung epithelial cell types, the Calu-3 cell cultures under the ALI condition showed optimal cytometric features for mimicking the biophysical characteristics of in vivo airway epithelium. Therefore, the Calu-3 cell monolayers could be used as functional cell barriers for the lung-targeted drug transport studies.

  1. Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells

    Directory of Open Access Journals (Sweden)

    Schedin Pepper J

    2010-07-01

    Full Text Available Abstract Background Recent research has yielded a wealth of data underscoring the key role of the cancer microenvironment, especially immune and stromal cells, in the progression of cancer and the development of metastases. However, the role of adjacent benign epithelial cells, which provide initial cell-cell contacts with cancer cells, in tumor progression has not been thoroughly examined. In this report we addressed the question whether benign MECs alter the transformed phenotype of human breast cancer cells. Methods We used both in vitro and in vivo co-cultivation approaches, whereby we mixed GFP-tagged MCF-10A cells (G2B-10A, as a model of benign mammary epithelial cells (MECs, and RFP-tagged MDA-MB-231-TIAS cells (R2-T1AS, as a model of breast cancer cells. Results The in vitro studies showed that G2B-10A cells increase the colony formation of R2-T1AS cells in both soft agar and clonogenicity assays. Conditioned media derived from G2B-10A cells enhanced colony formation of R2-T1AS cells, whereas prior paraformaldehyde (PFA fixation of G2B-10A cells abrogated this enhancement effect. Moreover, two other models of benign MECs, MCF-12A and HuMECs, also enhanced R2-T1AS colony growth in soft agar and clonogenicity assays. These data reveal that factors secreted by benign MECs are responsible for the observed enhancement of the R2-T1AS transformed phenotype. To determine whether G2B-10A cells enhance the tumorigenic growth of co-injected R2-T1AS cells in vivo, we used the nude mouse xenograft assay. Co-injecting R2-T1AS cells with G2B-10A cells ± PFA-fixation, revealed that G2B-10A cells promoted a ~3-fold increase in tumor growth, irrespective of PFA pre-treatment. These results indicate that soluble factors secreted by G2B-10A cells play a less important role in promoting R2-T1AS tumorigenesis in vivo, and that additional components are operative in the nude mouse xenograft assay. Finally, using array analysis, we found that both live and PFA

  2. Two-layer tissue engineered urethra using oral epithelial and muscle derived cells.

    Science.gov (United States)

    Mikami, Hiroshi; Kuwahara, Go; Nakamura, Nobuyuki; Yamato, Masayuki; Tanaka, Masatoshi; Kodama, Shohta

    2012-05-01

    We fabricated novel tissue engineered urethral grafts using autologously harvested oral cells. We report their viability in a canine model. Oral tissues were harvested by punch biopsy and divided into mucosal and muscle sections. Epithelial cells from mucosal sections were cultured as epithelial cell sheets. Simultaneously muscle derived cells were seeded on collagen mesh matrices to form muscle cell sheets. At 2 weeks the sheets were joined and tubularized to form 2-layer tissue engineered urethras, which were autologously grafted to surgically induced urethral defects in 10 dogs in the experimental group. Tissue engineered grafts were not applied to the induced urethral defect in control dogs. The dogs were followed 12 weeks postoperatively. Urethrogram and histological examination were done to evaluate the grafting outcome. We successfully fabricated 2-layer tissue engineered urethras in vitro and transplanted them in dogs in the experimental group. The 12-week complication-free rate was significantly higher in the experimental group than in controls. Urethrogram confirmed urethral patency without stricture in the complication-free group at 12 weeks. Histologically urethras in the transplant group showed a stratified epithelial layer overlying well differentiated submucosa. In contrast, urethras in controls showed severe fibrosis without epithelial layer formation. Two-layer tissue engineered urethras were engineered using cells harvested by minimally invasive oral punch biopsy. Results suggest that this technique can encourage regeneration of a functional urethra. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  3. The podocyte and parietal epithelial cell in proteinuria and glomerulosclerosis

    NARCIS (Netherlands)

    Dijkman, H.B.P.M.

    2006-01-01

    FSGS has become one of the most common glomerular diseases and is characterized by focal and segmental occurrence of lesions. Proteinuria is an important hallmark of glomerular diseases. Based on findings in a mouse model of FSGS we questioned if PECs play a role in human FSGS. Until now epithelial

  4. Malignant transformation of human benign prostate epithelial cells by high linear energy transfer alpha-particles.

    Science.gov (United States)

    Li, Hongzhen; Gu, Yongpeng; Miki, Jun; Hukku, Bharati; McLeod, David G; Hei, Tom K; Rhim, Johng S

    2007-09-01

    Although epidemiological studies have suggested a positive correlation between environmental radon exposure and prostate cancer, the mechanism involved is not clear. In the present study, we examined the oncogenic transforming potency of alpha-particles using non-tumorigenic, telomerase-immortalized human benign prostate epithelial cells. We report the malignant transformation of human benign prostate epithelial cells after a single exposure to 0.6 Gy dose of alpha-particles. Transformed cells showed anchorage-independent growth in soft agar and induced progressively growing tumors when transplanted into SCID mice. The tumors were characterized histologically as poorly differentiated adenocarcinomas. The cell line derived from tumor (SCID 5015), like the unirradiated cells, expressed cytokeratin 5, 8 and 18, NKX3.1 and AMACR. The malignant cells showed increased secretion of MMP2. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Chromosome abnormalities were identified in both irradiated and tumorigenic cells relative to the non-irradiated control cells. Prominent changes in chromosomes 6, 11 and 16, as well as mutations and deletions of the p53 gene were observed in the tumor outgrowth and tumor cells. These findings provide the first evidence of malignant transformation of human benign prostate epithelial cells exposed to a single dose of alpha-particles. This model provides an opportunity to study the cellular and molecular alterations that occur in radiation carcinogenesis in human prostate cells.

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

    Science.gov (United States)

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

    2015-09-01

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

  6. Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

    Science.gov (United States)

    Herceg, Zdenko

    Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the

  7. Osmoregulation of chloride channels in epithelial cells

    NARCIS (Netherlands)

    C.H. Lim (Christina)

    2008-01-01

    markdownabstract__Abstract__ The plasma membrane of mammalian cells is formed by two layers of lipids (lipid bilayer), primarily phospholipids, glycolipids and cholesterol, in which many different proteins are embedded. Phospholipid consists of a glycerol backbone esterified to fatty acids

  8. Transforming growth factor beta-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions

    National Research Council Canada - National Science Library

    Peinado, Hector; Quintanilla, Miguel; Cano, Amparo

    2003-01-01

    .... We show here that transforming growth factor beta-1 (TGFbeta1) induces Snail expression in Madin-Darby canine kidney cells and triggers epithelial-mesenchymal transitions by a mechanism dependent on the MAPK signaling pathway...

  9. Current Concepts and Occurrence of Epithelial Odontogenic Tumors: II. Calcifying Epithelial Odontogenic Tumor Versus Ghost Cell Odontogenic Tumors Derived from Calcifying Odontogenic Cyst

    OpenAIRE

    Lee, Suk Keun; Kim, Yeon Sook

    2014-01-01

    Calcifying epithelial odontogenic tumors (CEOTs) and ghost cell odontogenic tumors (GCOTs) are characteristic odontogenic origin epithelial tumors which produce calcifying materials from transformed epithelial tumor cells. CEOT is a benign odontogenic tumor composed of polygonal epithelial tumor cells that show retrogressive calcific changes, amyloid-like deposition, and clear cytoplasm. Differentially, GCOTs are a group of transient tumors characterized by ghost cell presence, which comprise...

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

    Directory of Open Access Journals (Sweden)

    Taylor S Cohen

    2010-06-01

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

  11. Immunopathology of childhood celiac disease-Key role of intestinal epithelial cells.

    Science.gov (United States)

    Pietz, Grzegorz; De, Rituparna; Hedberg, Maria; Sjöberg, Veronika; Sandström, Olof; Hernell, Olle; Hammarström, Sten; Hammarström, Marie-Louise

    2017-01-01

    Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria. Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells. A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease.

  12. Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both?

    Science.gov (United States)

    2013-01-01

    Intact intercellular junctions and cellular matrix contacts are crucial structural components for the formation and maintenance of epithelial barrier functions in humans to control the commensal flora and protect against intruding microbes. Campylobacter jejuni is one of the most important zoonotic pathogens causing food-borne gastroenteritis and potentially more severe diseases such as reactive arthritis or Guillain–Barré syndrome. Crossing the intestinal epithelial barrier and host cell invasion by C. jejuni are considered to represent the primary reasons of gut tissue damage in humans and various animal model systems including monkeys, piglets, rabbits, hamsters and ferrets. C. jejuni is also able to invade underlying tissues such as the lamina propria, can enter the bloodstream, and possibly reach distinct organs such as spleen, liver or mesenteric lymph nodes. However, the molecular mechanisms as well as major bacterial and host cell factors involved in these activities are poorly understood. Various models exist by which the pathogen can trigger its own transmigration across polarized intestinal epithelial cells in vitro, the paracellular and/or transcellular mechanism. Recent studies suggest that bacterial factors such as flagellum, serine protease HtrA and lipooligosaccharide LOS may play an active role in bacterial transmigration. Here we review our knowledge on transmigration of C. jejuni as well as some other Campylobacter species, and discuss the pros and cons for the route(s) taken to travel across polarized epithelial cell monolayers. These studies provide fresh insights into the infection strategies employed by this important pathogen. PMID:24079544

  13. Early Alterations in Ovarian Surface Epithelial Cells and Induction of Ovarian Epithelial Tumors Triggered by Loss of FSH Receptor

    Directory of Open Access Journals (Sweden)

    Xinlei Chen

    2007-06-01

    Full Text Available Little is known about the behavior of the ovarian surface epithelium (OSE, which plays a central role in ovarian cancer etiology. It has been suggested that incessant ovulation causes OSE changes leading to transformation and that high gonadotropin levels during postmenopause activate OSE receptors, inducing proliferation. We examined the chronology of OSE changes, including tumor appearance, in a mouse model where ovulation never occurs due to deletion of follitropin receptor. Changes in epithelial cells were marked by pan-cytokeratin (CK staining. Histologic changes and CK staining in the OSE increased from postnatal day 2. CK staining was observed inside the ovary by 24 days and increased thereafter in tumor-bearing animals. Ovaries from a third of aged (1 year mutant mice showed CK deep inside, indicating cell migration. These tumors resembled serous papillary adenoma of human ovaries. Weak expression of GATA-4 and elevation of PCNA, cyclooxygenase-1, cyclooxygenase-2, and plateletderived growth factor receptors α and β in mutants indicated differences in cell proliferation, differentiation, and inflammation. Thus, we report that OSE changes occur long before epithelial tumors appear in FORKO mice. Our results suggest that neither incessant ovulation nor follicle-stimulating hormone receptor presence in the OSE is required for inducing ovarian tumors; thus, other mechanisms must contribute to ovarian tumorigenesis.

  14. Serratia marcescens is injurious to intestinal epithelial cells.

    Science.gov (United States)

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

    2014-01-01

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

  15. Molecular cloning, sequence analysis, and function of the intestinal epithelial stem cell marker Bmi1 in pig intestinal epithelial cells.

    Science.gov (United States)

    Li, C-M; Yan, H-C; Fu, H-L; Xu, G-F; Wang, X-Q

    2014-01-01

    In the present work, we cloned the full-length cDNA of the pig Bmi1 gene (BMI1 polycomb ring finger oncogene), which has been indicated as an intestinal epithelial stem cell (IESC) marker in other mammals. This paper provides the first report of the function of Bmi1 in pig intestinal epithelial cells and a brief description of its underlying mechanism. Rapid amplification of cDNA ends technology was used to clone the complete pig Bmi1 sequence, and a Bmi1-pcDNA3.1 vector was constructed for transfection into an intestinal porcine epithelial cell line (IPEC-1). The proliferation ability of the cells was estimated using the MTT assay and the EdU incorporation method at different time points after seeding. Cell cycle information was detected by flow cytometry. The mRNA abundances of cell cycle-related genes were also measured. The results indicated that the pig Bmi1 cDNA is 3,193 bp in length and consists of a 981 bp open reading frame, a 256 bp 5´ untranslated region (UTR), and a 1,956 bp 3' UTR. The transcript contains no signal peptides, and there are no transmembrane regions in the pig Bmi1 coded protein, which has a total of 326 AA. The overexpression of the pig Bmi1 in the IPEC-1 cells led to increased cell proliferation and a lower percentage of cells in the G1 and S phases (P cells in the G2 phase (P 0.05). Our data suggested that pig Bmi1 can increase the proliferation of IPEC-1 cells by promoting the G1/S transition and the overall cell cycle process.

  16. CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.

    Science.gov (United States)

    Rogers, Natasha M; Zhang, Zheng J; Wang, Jiao-Jing; Thomson, Angus W; Isenberg, Jeffrey S

    2016-08-01

    Defects in renal tubular epithelial cell repair contribute to renal ischemia reperfusion injury, cause acute kidney damage, and promote chronic renal disease. The matricellular protein thrombospondin-1 and its receptor CD47 are involved in experimental renal ischemia reperfusion injury, although the role of this interaction in renal recovery is unknown. We found upregulation of self-renewal genes (transcription factors Oct4, Sox2, Klf4 and cMyc) in the kidney of CD47(-/-) mice after ischemia reperfusion injury. Wild-type animals had minimal self-renewal gene expression, both before and after injury. Suggestive of cell autonomy, CD47(-/-) renal tubular epithelial cells were found to increase expression of the self-renewal genes. This correlated with enhanced proliferative capacity compared with cells from wild-type mice. Exogenous thrombospondin-1 inhibited self-renewal gene expression in renal tubular epithelial cells from wild-type but not CD47(-/-) mice, and this was associated with decreased proliferation. Treatment of renal tubular epithelial cells with a CD47 blocking antibody or CD47-targeting small interfering RNA increased expression of some self-renewal transcription factors and promoted cell proliferation. In a syngeneic kidney transplant model, treatment with a CD47 blocking antibody increased self-renewal transcription factor expression, decreased tissue damage, and improved renal function compared with that in control mice. Thus, thrombospondin-1 via CD47 inhibits renal tubular epithelial cell recovery after ischemia reperfusion injury through inhibition of proliferation/self-renewal. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Protective Effects of Trehalose on the Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Pasquale Aragona

    2014-01-01

    Full Text Available Purpose. Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Methods. Twelve patients undergoing laser subepithelial keratomileusis (LASEK were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. Results. In both trehalose-untreated eyes (TUE and trehalose-treated eyes (TTE, the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Conclusions. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

  18. The origin of stroma surrounding epithelial ovarian cancer cells.

    Science.gov (United States)

    Akahane, Tomoko; Hirasawa, Akira; Tsuda, Hiroshi; Kataoka, Fumio; Nishimura, Sadako; Tanaka, Hideo; Tominaga, Eiichiro; Nomura, Hiroyuki; Chiyoda, Tatsuyuki; Iguchi, Yoko; Yamagami, Wataru; Susumu, Nobuyuki; Aoki, Daisuke

    2013-01-01

    Cancer stroma is thought to play an important role in tumor behavior, including invasion or metastasis and response to therapy. Cancer stroma is generally thought either to be non-neoplastic cells, including tissue-marrow or bone-marrow-derived fibroblasts, or to originate in epithelial mesenchymal transition of cancer cells. In this study, we evaluated the status of the p53 gene in both the cancer cells and the cancer stroma in epithelial ovarian cancer (EOC) to elucidate the origin of the stroma. Samples from 16 EOC patients were included in this study. Tumor cells and adjacent nontumor stromal cells were microdissected and DNA was extracted separately. We analyzed p53 sequences (exons 5-8) of both cancer and stromal tissues in all cases. Furthermore, we examined p53 protein expression in all cases. Mutations in p53 were detected in 9 of the 16 EOCs: in 8 of these cases, the mutations were detected only in cancer cells. In 1 case, the same mutation (R248Q) was detected in both cancer and stromal tissues, and p53 protein expression was detected in both the cancer cells and the cancer stroma. Most cancer stroma in EOC is thought to originate from non-neoplastic cells, but some parts of the cancer stroma might originate from cancer cells.

  19. Side Population Cells From an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties.

    Science.gov (United States)

    Tokiwa, Takayoshi; Yamazaki, Taisuke; Enosawa, Shin

    2012-01-01

    The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2015-04-01

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

  2. Primary mouse small intestinal epithelial cell cultures

    NARCIS (Netherlands)

    Sato, T.; Clevers, H.

    2013-01-01

    The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have

  3. [Adhesion of clinical Candida albicans isolate to buccal epithelial cells].

    Science.gov (United States)

    Wellmer, A

    1999-01-01

    Mucosal adherence and germ tube formation are considered to be important virulence factors of C. albicans. Adherence is a precondition for colonisation and invasion. We investigated 11 clinical isolates (among them 5 cases recovered from oesophageal thrush) for quantification of the two characteristics and correlated the results with clinical data. Adherence was measured on buccal epithelial cells and the continuous flow culture was used for quantification of germ tube formation. Adherence of strains recovered from clinically, culturally and serologically confirmed oesophageal thrush adhered stronger to buccal epithelial cells than isolates from patients with heavy colonisation without signs of candidosis. Strains with stronger adherence showed a significantly faster and an increased germ tube formation in the continuous flow culture. Strains from oesophageal thrush therefore show a more marked expression of the investigated virulence factors. Therefore a good adherence is a necessity for infection of the oesophagus by C. albicans. The preferential isolation of C. albicans from oesophageal thrush (> 90%) supports this assumption.

  4. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

    NARCIS (Netherlands)

    Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

  5. An in vitro model that recapitulates the epithelial to mesenchymal transition (EMT in human breast cancer.

    Directory of Open Access Journals (Sweden)

    Elad Katz

    Full Text Available The epithelial to mesenchymal transition (EMT is a developmental program in which epithelial cells down-regulate their cell-cell junctions, acquire spindle cell morphology and exhibit cellular motility. In human breast cancer, invasion into surrounding tissue is the first step in metastatic progression. Here, we devised an in vitro model using selected cell lines, which recapitulates many features of EMT as observed in human breast cancer. By comparing the gene expression profiles of claudin-low breast cancers with the experimental model, we identified a 9-gene signature characteristic of EMT. This signature was found to distinguish a series of breast cancer cell lines that have demonstrable, classical EMT hallmarks, including loss of E-cadherin protein and acquisition of N-cadherin and vimentin expression. We subsequently developed a three-dimensional model to recapitulate the process of EMT with these cell lines. The cells maintain epithelial morphology when encapsulated in a reconstituted basement membrane, but undergo spontaneous EMT and invade into surrounding collagen in the absence of exogenous cues. Collectively, this model of EMT in vitro reveals the behaviour of breast cancer cells beyond the basement membrane breach and recapitulates the in vivo context for further investigation into EMT and drugs that may interfere with it.

  6. The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model.

    Science.gov (United States)

    Kreft, Mateja Erdani; Jerman, Urška Dragin; Lasič, Eva; Lanišnik Rižner, Tea; Hevir-Kene, Neli; Peternel, Luka; Kristan, Katja

    2015-02-01

    The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa. Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters. Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface. The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.

  7. Ivermectin inhibits growth of Chlamydia trachomatis in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Matthew A Pettengill

    Full Text Available Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia.

  8. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  9. Epithelial Cell-Neutrophil Interactions in the Alimentary Tract: A Complex Dialog in Mucosal Surveillance and Inflammation

    Directory of Open Access Journals (Sweden)

    Sean P. Colgan

    2002-01-01

    Full Text Available Inflammatory diseases of mucosal organs as diverse as the lung, kidney, and intestine, inevitably require the intimate interactions of neutrophils with columnar epithelia. The physiologic consequences of such interactions often determine endpoint organ function, and for this reason, much recent interest has developed in identifying mechanisms and novel targets for the treatment of mucosal inflammation. Elegant in vitro model systems incorporating purified human neutrophils and human epithelial cells grown in physiologic orientations have aided in discovery of new and insightful pathways to define basic inflammatory pathways. Here, we will review the recent literature regarding the interactions between columnar epithelial cells and neutrophils, with an emphasis on intestinal epithelial cells, structural aspects of neutrophil transepithelial migration, molecular determinants of neutrophil-epithelial cell interactions, as well as modulation of these pathways. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

  10. Reversible effect of dextran sodium sulfate on mucus secreting intestinal epithelial cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, V

    2016-01-01

    Development of animal models of inflammatory bowel disease (IBD) is important to provide insights into the pathogenesis of IBD and to evaluate modalities to prevent or ameliorate inflammation or enhance mucosal healing. However, the use of in vitro models of the inflamed gastrointestinal tract can...... investigated effects of increasing doses of DSS on viability and integrity of these intestinal epithelial cells. For cell viability studies, cells were treated with DSS solutions for 24 or 48 h and viability was measured fluorometrically by PicoGreen double-stranded DNA quantitation. HT29-MTX-E12 cells were...

  11. Stretch induces cytokine release by alveolar epithelial cells in vitro.

    Science.gov (United States)

    Vlahakis, N E; Schroeder, M A; Limper, A H; Hubmayr, R D

    1999-07-01

    Mechanical ventilation can injure the lung, causing edema and alveolar inflammation. Interleukin-8 (IL-8) plays an important role in this inflammatory response. We postulated that cyclic cell stretch upregulates the production and release of IL-8 by human alveolar epithelium in the absence of structural cell damage or paracrine stimulation. To test this hypothesis, alveolar epithelial cells (A549 cells) were cultured on a deformable silicoelastic membrane. When stretched by 30% for up to 48 h, the cells released 49 +/- 34% more IL-8 (P static controls. Smaller deformations (20% stretch) produced no consistent increase in IL-8. Stretch of 4 h duration increased IL-8 gene transcription fourfold above baseline. Stretch had no effect on cell proliferation, cell viability as assessed by (51)Cr release assay, or the release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha. We conclude that deformation per se can trigger inflammatory signaling and that alveolar epithelial cells may be active participants in the alveolitis associated with ventilator-induced lung injury.

  12. Receptor-mediated endocytosis of macromolecules and strategy to enhance their transport in alveolar epithelial cells.

    Science.gov (United States)

    Takano, Mikihisa; Kawami, Masashi; Aoki, Ayako; Yumoto, Ryoko

    2015-05-01

    Pulmonary delivery is an attractive administration route for therapeutic proteins and peptides. In this context, endocytosis/transcytosis at the distal lung epithelial barrier is an important process in the pulmonary absorption of therapeutic macromolecules. The alveolar epithelium is comprised of type I and type II cells. Understanding the transport mechanisms in these cells is essential for the development of efficient pulmonary delivery systems of therapeutic macromolecules. Endocytic pathways for albumin and insulin in alveolar epithelial cells and possible receptors for the endocytosis are discussed. Strategies to enhance the endocytosis and pulmonary absorption of macromolecules are also discussed, by focusing on the effects of cationic poly(amino acid)s. Although the surface area occupied by type II cells in alveoli is much smaller than that covered by type I cells, type II cells may significantly contribute to the endocytosis/transcytosis of macromolecules such as albumin. Identification of the receptors involved in the cellular uptake of each macromolecule is prerequisite for the understanding and regulation of its transport into and across alveolar epithelial cells. Establishment of novel in-vitro culture cell models of type I and type II cells would be a great help for the future advance of this research field.

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

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

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

  14. Isolation, separation, and characterization of epithelial and connective cells from rat palate

    Energy Technology Data Exchange (ETDEWEB)

    Terranova, Victor Paul [Univ. of Rochester, NY (United States)

    1979-01-01

    Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means of labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.

  15. Airway Epithelial Cell Cilia and Obstructive Lung Disease

    Directory of Open Access Journals (Sweden)

    Asma Yaghi

    2016-11-01

    Full Text Available Airway epithelium is the first line of defense against exposure of the airway and lung to various inflammatory stimuli. Ciliary beating of airway epithelial cells constitutes an important part of the mucociliary transport apparatus. To be effective in transporting secretions out of the lung, the mucociliary transport apparatus must exhibit a cohesive beating of all ciliated epithelial cells that line the upper and lower respiratory tract. Cilia function can be modulated by exposures to endogenous and exogenous factors and by the viscosity of the mucus lining the epithelium. Cilia function is impaired in lung diseases such as COPD and asthma, and pharmacologic agents can modulate cilia function and mucus viscosity. Cilia beating is reduced in COPD, however, more research is needed to determine the structural-functional regulation of ciliary beating via all signaling pathways and how this might relate to the initiation or progression of obstructive lung diseases. Additionally, genotypes and how these can influence phenotypes and epithelial cell cilia function and structure should be taken into consideration in future investigations.

  16. Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

    Science.gov (United States)

    Ghosh, Moumita; Ahmad, Shama; White, Carl W; Reynolds, Susan D

    2017-01-01

    Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

  17. Epithelial cell adhesion molecule-1 (ECAM1) is required in the maintenance of corneal epithelial barrier integrity.

    Science.gov (United States)

    Zhou, Jinzi; Jiang, Jian; Wang, Shuhong; Xia, Xiaobo

    2016-01-01

    Corneal epithelial barrier integrity is critical in the maintenance of the corneal homeostasis. The corneal barrier dysfunction may be associated with the pathogenesis of a number of eye diseases. In this study, we assessed the expression of epithelial cell adhesion molecule-1 (ECAM1) in human corneal epithelial cells (HCE). The epithelial barrier function of the corneal epithelial monolayer was determined in Transwells. We found that the HCE cells expressed ECAM1. Knockdown of ECAM1 markedly compromised the HCE monolayer barrier function. A complex of ECAM1, claudin1, and occludin was detected in the HCE monolayers, which was not detected in the ECAM1-null HCE monolayers. Exposure to the proinflammatory cytokine, interleukin-13, inhibited the expression of ECAM1 in HCE cells and compromised the barrier function, which was prevented in the HCE monolayer with the ECAM1 overexpression. In conclusion, ECAM1 is required in the formation of the tight junction complex and maintaining the corneal epithelial barrier function. © 2015 International Federation for Cell Biology.

  18. Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hou

    Full Text Available PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.

  19. Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

    Science.gov (United States)

    Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

    2017-12-01

    In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5–3 µm were used and a shear stress of ~0.03 dyne cm‑2 was created by applying a low flow rate of 20 nl s‑1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

  20. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J. [and others

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

  1. Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

    Science.gov (United States)

    Wöllert, Torsten; Langford, George M

    2016-01-01

    Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live

  2. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    Directory of Open Access Journals (Sweden)

    Laura Lafon-Hughes

    2014-10-01

    Full Text Available Poly-ADP-ribose (PAR is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs and degraded by poly-ADP-ribose-glycohydrolase (PARG. Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair. Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt. In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO. PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  3. Foxp3+Regulatory T Cell Expression of Keratinocyte Growth Factor Enhances Lung Epithelial Proliferation.

    Science.gov (United States)

    Dial, Catherine F; Tune, Miriya K; Doerschuk, Claire M; Mock, Jason R

    2017-08-01

    Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3 + regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.

  4. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Tang, J.; Jiang, Y. [Southern Medical University, Nanfang Hospital, Department of Anesthesia, Guangzhou, China, Department of Anesthesia, Nanfang Hospital, Southern Medical University, Guangzhou (China); Tang, Y.; Chen, B. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China); Sun, X. [Laboratory of Traditional Chinese Medicine Syndrome, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou (China); Su, L.; Liu, Z. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China)

    2013-06-25

    Gut-derived endotoxin and pathogenic bacteria have been proposed as important causative factors of morbidity and death during heat stroke. However, it is still unclear what kind of damage is induced by heat stress. In this study, the rat intestinal epithelial cell line (IEC-6) was treated with heat stress or a combination of heat stress and lipopolysaccharide (LPS). In addition, propofol, which plays an important role in anti-inflammation and organ protection, was applied to study its effects on cellular viability and apoptosis. Heat stress, LPS, or heat stress combined with LPS stimulation can all cause intestinal epithelial cell damage, including early apoptosis and subsequent necrosis. However, propofol can alleviate injuries caused by heat stress, LPS, or the combination of heat stress and LPS. Interestingly, propofol can only mitigate LPS-induced intestinal epithelial cell apoptosis, and has no protective role in heat-stress-induced apoptosis. This study developed a model that can mimic the intestinal heat stress environment. It demonstrates the effects on intestinal epithelial cell damage, and indicated that propofol could be used as a therapeutic drug for the treatment of heat-stress-induced intestinal injuries.

  5. Prion infection of epithelial Rov cells is a polarized event.

    Science.gov (United States)

    Paquet, Sophie; Sabuncu, Elifsu; Delaunay, Jean-Louis; Laude, Hubert; Vilette, Didier

    2004-07-01

    During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells.

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

    OpenAIRE

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

    2017-01-01

    Isolation and characterization of lung epithelial stem and progenitor cells and understanding of their specific role in lung physiopathology are critical for preventing and controlling lung diseases including lung cancer. In this review, we summarized recent advances in classification and research methods of lung epithelial stem and progenitor cells. Lung epithelial stem and progenitor cells were region-specific, which primarily included basal cells and duct cells in proximal airway, Clara ce...

  7. Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state.

    Science.gov (United States)

    Soady, Kelly; Smalley, Matthew J

    2012-09-10

    Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells.

  8. Loss of Glis2/NPHP7 causes kidney epithelial cell senescence and suppresses cyst growth in the Kif3a mouse model of cystic kidney disease.

    Science.gov (United States)

    Lu, Dongmei; Rauhauser, Alysha; Li, Binghua; Ren, Chongyu; McEnery, Kayla; Zhu, Jili; Chaki, Moumita; Vadnagara, Komal; Elhadi, Sarah; Jetten, Anton M; Igarashi, Peter; Attanasio, Massimo

    2016-06-01

    Enlargement of kidney tubules is a common feature of multiple cystic kidney diseases in humans and mice. However, while some of these pathologies are characterized by cyst expansion and organ enlargement, in others, progressive interstitial fibrosis and kidney atrophy prevail. The Kif3a knockout mouse is an established non-orthologous mouse model of cystic kidney disease. Conditional inactivation of Kif3a in kidney tubular cells results in loss of primary cilia and rapid cyst growth. Conversely, loss of function of the gene GLIS2/NPHP7 causes progressive kidney atrophy, interstitial inflammatory infiltration, and fibrosis. Kif3a null tubular cells have unrestrained proliferation and reduced stabilization of p53 resulting in a loss of cell cycle arrest in the presence of DNA damage. In contrast, loss of Glis2 is associated with activation of checkpoint kinase 1, stabilization of p53, and induction of cell senescence. Interestingly, the cystic phenotype of Kif3a knockout mice is partially rescued by genetic ablation of Glis2 and pharmacological stabilization of p53. Thus, Kif3a is required for cell cycle regulation and the DNA damage response, whereas cell senescence is significantly enhanced in Glis2 null cells. Hence, cell senescence is a central feature in nephronophthisis type 7 and Kif3a is unexpectedly required for efficient DNA damage response and cell cycle arrest. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  9. The extracellular microenvironment explains variations in passive drug transport across different airway epithelial cell types.

    Science.gov (United States)

    Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A; Yu, Jing-Yu; Lim, Dong Hyun; Rosania, Gus R

    2013-08-01

    We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in the extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.

  10. Transdifferentiation of Peripheral Blood Mononuclear Cells into Epithelial-Like Cells

    Science.gov (United States)

    Medina, Abelardo; Kilani, Ruhangiz T.; Carr, Nicholas; Brown, Erin; Ghahary, Aziz

    2007-01-01

    Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3ς for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3ς and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P ≤ 0.01). The depletion of 14-3-3ς from these conditioned media by immunoprecipitation reduced the effect by 39.5% (P value, 0.05). Therefore, the releasable 14-3-3ς from PBMC-derived epithelial-like cells is involved in this process. Our findings provide new insights into the PBMC transdifferentiation to generate epithelial-like cells and subsequently release of 14-3-3ς that will disclose new therapeutic alternatives for different dermal clinical settings. PMID:17717137

  11. Epithelial cell mitochondrial dysfunction and PINK1 are induced by transforming growth factor-beta1 in pulmonary fibrosis.

    Directory of Open Access Journals (Sweden)

    Avignat S Patel

    Full Text Available Epithelial cell death is a major contributor to fibrogenesis in the lung. In this study, we sought to determine the function of mitochondria and their clearance (mitophagy in alveolar epithelial cell death and fibrosis.We studied markers of mitochondrial injury and the mitophagy marker, PTEN-induced putative kinase 1 (PINK1, in IPF lung tissues by Western blotting, transmission electron microscopy (TEM, and immunofluorescence. In vitro experiments were carried out in lung epithelial cells stimulated with transforming growth factor-β1 (TGF-β1. Changes in cell function were measured by Western blotting, flow cytometry and immunofluorescence. In vivo experiments were performed using the murine bleomycin model of lung fibrosis.Evaluation of IPF lung tissue demonstrated increased PINK1 expression by Western blotting and immunofluorescence and increased numbers of damaged mitochondria by TEM. In lung epithelial cells, TGF-β1 induced mitochondrial depolarization, mitochondrial ROS, and PINK1 expression; all were abrogated by mitochondrial ROS scavenging. Finally, Pink1-/- mice were more susceptible than control mice to bleomycin induced lung fibrosis.TGF-β1 induces lung epithelial cell mitochondrial ROS and depolarization and stabilizes the key mitophagy initiating protein, PINK1. PINK1 ameliorates epithelial cell death and may be necessary to limit fibrogenesis.

  12. Adipose-derived mesenchymal stem cells promote cell proliferation and invasion of epithelial ovarian cancer

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    Chu, Yijing; Tang, Huijuan; Guo, Yan; Guo, Jing; Huang, Bangxing; Fang, Fang; Cai, Jing, E-mail: caijingmmm@hotmail.com; Wang, Zehua, E-mail: zehuawang@163.net

    2015-09-10

    Adipose-derived mesenchymal stem cell (ADSC) is an important component of tumor microenvironment. However, whether ADSCs have a hand in ovarian cancer progression remains unclear. In this study, we investigated the impact of human ADSCs derived from the omentum of normal donors on human epithelial ovarian cancer (EOC) cells in vitro and in vivo. Direct and indirect co-culture models including ADSCs and human EOC cell lines were established and the effects of ADSCs on EOC cell proliferation were evaluated by EdU incorporation and flow cytometry. Transwell migration assays and detection of MMPs were performed to assess the invasion activity of EOC cells in vitro. Mouse models were established by intraperitoneal injection of EOC cells with or without concomitant ADSCs to investigate the role of ADSCs in tumor progression in vivo. We found that ADSCs significantly promoted proliferation and invasion of EOC cells in both direct and indirect co-culture assays. In addition, after co-culture with ADSCs, EOC cells secreted higher levels of matrix metalloproteinases (MMPs), and inhibition of MMP2 and MMP9 partially relieved the tumor-promoting effects of ADSCs in vitro. In mouse xenograft models, we confirmed that ADSCs promoted EOC growth and metastasis and elevated the expression of MMP2 and MMP9. Our findings indicate that omental ADSCs play a promotive role during ovarian cancer progression. - Highlights: • Omental adipose derived stem cells enhanced growth and invasion properties of ovarian cancer cells. • Adipose derived stem cells promoted the growth and metastasis of ovarian cancer in mice models. • Adipose derived stem cells promoted MMPs expression and secretion of ovarian cancer cells. • Elevated MMPs mediated the tumor promoting effects of ADSCs.

  13. Reciprocal epithelial:endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation.

    Science.gov (United States)

    Hick, Anne-Christine; Delmarcelle, Anne-Sophie; Bouquet, Mahé; Klotz, Sabrina; Copetti, Tamara; Forez, Celine; Van Der Smissen, Patrick; Sonveaux, Pierre; Collet, Jean-François; Feron, Olivier; Courtoy, Pierre J; Pierreux, Christophe E

    2013-09-01

    The thyroid is a highly vascularized endocrine gland, displaying a characteristic epithelial organization in closed spheres, called follicles. Here we investigate how endothelial cells are recruited into the developing thyroid and if they control glandular organization as well as thyrocytes and C-cells differentiation. We show that endothelial cells closely surround, and then invade the expanding thyroid epithelial cell mass to become closely associated with nascent polarized follicles. This close and sustained endothelial:epithelial interaction depends on epithelial production of the angiogenic factor, Vascular Endothelial Growth Factor-A (VEGF-A), as its thyroid-specific genetic inactivation reduced the endothelial cell pool of the thyroid by > 90%. Vegfa KO also displayed decreased C-cells differentiation and impaired organization of the epithelial cell mass into follicles. We developed an ex vivo model of thyroid explants that faithfully mimicks bilobation of the thyroid anlagen, endothelial and C-cells invasion, folliculogenesis and differentiation. Treatment of thyroid explants at e12.5 with a VEGFR2 inhibitor ablated the endothelial pool and reproduced ex vivo folliculogenesis defects observed in conditional Vegfa KO. In the absence of any blood supply, rescue by embryonic endothelial progenitor cells restored folliculogenesis, accelerated lumen expansion and stimulated calcitonin expression by C-cells. In conclusion, our data demonstrate that, in developing mouse thyroid, epithelial production of VEGF-A is necessary for endothelial cells recruitment and expansion. In turn, endothelial cells control epithelial reorganization in follicles and C-cells differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Effects in cigarette smoke stimulated bronchial epithelial cells of a corticosteroid entrapped into nanostructured lipid carriers.

    Science.gov (United States)

    Bondì, Maria Luisa; Ferraro, Maria; Di Vincenzo, Serena; Gerbino, Stefania; Cavallaro, Gennara; Giammona, Gaetano; Botto, Chiara; Gjomarkaj, Mark; Pace, Elisabetta

    2014-11-29

    Nanomedicine studies have showed a great potential for drug delivery into the lung. In this manuscript nanostructured lipid carriers (NLC) containing Fluticasone propionate (FP) were prepared and their biocompatibility and effects in a human bronchial epithelial cell line (16-HBE) stimulated with cigarette smoke extracts (CSE) were tested. Biocompatibility studies showed that the NLC did not induce cell necrosis or apoptosis. Moreover, it was confirmed that CSE increased intracellular ROS production and TLR4 expression in bronchial epithelial cells and that FP-loaded NLC were more effective than free drug in modulating these processes. Finally, the nanoparticles increased GSH levels improving cell protection against oxidative stress. The present study shows that NLC may be considered a promising strategy to improve corticosteroid mediated effects in cellular models associated to corticosteroid resistance. The NLC containing FP can be considered good systems for dosage forms useful for increasing the effectiveness of fluticasone decreasing its side effects.

  15. Establishment of a human conjunctival epithelial cell line lacking the functional TACSTD2 gene (an American Ophthalmological Society thesis).

    Science.gov (United States)

    Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

    2012-12-01

    To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction-related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD.

  16. In vitro generation of renal tubular epithelial cells from fibroblasts: implications for precision and regenerative medicine in nephrology.

    Science.gov (United States)

    Wyatt, Christina M; Dubois, Nicole

    2017-02-01

    Prior efforts to generate renal epithelial cells in vitro have relied on pluripotent or bone marrow-derived mesenchymal stem cells. A recent publication in Nature Cell Biology describes the generation of induced tubular epithelial cells from fibroblasts, potentially offering a novel platform for personalized drug toxicity screening and in vitro disease modeling. This report serves as a promising proof of principle study and opens future research directions, including the optimization of the reprogramming process, efficient translation to adult human fibroblasts, and the generation of highly specific functional renal cell types. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles.

    Directory of Open Access Journals (Sweden)

    Giulia Chiabotto

    Full Text Available 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.

  18. Low Temperature Plasma for the Treatment of Epithelial Cancer Cells

    Science.gov (United States)

    Mohades, Soheila

    Biomedical applications of low temperature plasmas (LTP) may lead to a paradigm shift in treating various diseases by conducting fundamental research on the effects of LTP on cells, tissues, organisms (plants, insects, and microorganisms). This is a rapidly growing interdisciplinary research field that involves engineering, physics, life sciences, and chemistry to find novel solutions for urgent medical needs. Effects of different LTP sources have shown the anti-tumor properties of plasma exposure; however, there are still many unknowns about the interaction of plasma with eukaryotic cells which must be elucidated in order to evaluate the practical potential of plasma in cancer treatment. Plasma, the fourth state of matter, is composed of electrons, ions, reactive molecules (radicals and non-radicals), excited species, radiation, and heat. A sufficient dose (time) of plasma exposure can induce death in cancer cells. The plasma pencil is employed to study the anti-tumor properties of this treatment on epithelial cells. The plasma pencil has been previously used for the inactivation of bacteria, destroying amyloid fibrils, and the killing of various cancer cells. Bladder cancer is the 9th leading cause of cancer. In this dissertation, human urinary bladder tissue with the squamous cell carcinoma disease (SCaBER cells) is treated with LTP utilizing two different approaches: direct plasma exposure and Plasma Activated Media (PAM) as an advancement to the treatment. PAM is produced by exposing a liquid cell culture medium to the plasma pencil. Direct LTP treatment of cancer cells indicates a dose-dependent killing effect at post-treatment times. Similarly, PAM treatment shows an anti-cancer effect by inducing substantial cell death. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have an important role in the biomedical effects of LTP treatment. This study demonstrates the capability of the plasma pencil to transport ROS/RNS into cell culture media

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

    Science.gov (United States)

    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

  20. Inflammation and the Intestinal Barrier: Leukocyte–Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair

    Science.gov (United States)

    Luissint, Anny-Claude; Parkos, Charles A.; Nusrat, Asma

    2017-01-01

    The intestinal tract is lined by a single layer of columnar epithelial cells that forms a dynamic, permeable barrier allowing for selective absorption of nutrients, while restricting access to pathogens and food-borne antigens. Precise regulation of epithelial barrier function is therefore required for maintaining mucosal homeostasis and depends, in part, on barrier-forming elements within the epithelium and a balance between pro- and anti-inflammatory factors in the mucosa. Pathologic states, such as inflammatory bowel disease, are associated with a leaky epithelial barrier, resulting in excessive exposure to microbial antigens, recruitment of leukocytes, release of soluble mediators, and ultimately mucosal damage. An inflammatory microenvironment affects epithelial barrier properties and mucosal homeostasis by altering the structure and function of epithelial intercellular junctions through direct and indirect mechanisms. We review our current understanding of complex interactions between the intestinal epithelium and immune cells, with a focus on pathologic mucosal inflammation and mechanisms of epithelial repair. We discuss leukocyte–epithelial interactions, as well as inflammatory mediators that affect the epithelial barrier and mucosal repair. Increased knowledge of communication networks between the epithelium and immune system will lead to tissue-specific strategies for treating pathologic intestinal inflammation. PMID:27436072

  1. GM-CSF produced by non-hematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa1,2

    Science.gov (United States)

    Egea, Laia; McAllister, Christopher S.; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F.

    2012-01-01

    GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, and dendritic cell (DC) differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn’s disease in humans and colitis in murine models has been considered mainly to reflect its activity on myeloid cells. We used GM-CSF deficient (GM-CSF−/−) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS) at doses that resulted in little epithelial damage and mucosal ulceration in wild type (WT) mice resulted in marked colon ulceration and delayed ulcer healing in GM-CSF−/− mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF−/− mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF−/− mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Non-hematopoietic cells and not myeloid cells produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury as revealed by bone marrow chimera and DC depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell produced GM-CSF has a novel non-redundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium. PMID:23325885

  2. Stroma regulates increased epithelial lateral cell adhesion in 3D culture: a role for actin/cadherin dynamics.

    Directory of Open Access Journals (Sweden)

    Karen F Chambers

    2011-04-01

    Full Text Available Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures.The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability.In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult

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

    Science.gov (United States)

    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).

  4. Staphylococcus aureus hemolysin A disrupts cell-matrix adhesions in human airway epithelial cells.

    Science.gov (United States)

    Hermann, Ina; Räth, Susann; Ziesemer, Sabine; Volksdorf, Thomas; Dress, Regine J; Gutjahr, Melanie; Müller, Christian; Beule, Achim G; Hildebrandt, Jan-Peter

    2015-01-01

    Treatment of primary or immortalized human airway epithelial cells (16HBE14o-, S9) or alveolar cancer cells (A549) with recombinant hemolysin A (rHla), a major virulence-associated factor of Staphylococcus aureus, induces alterations in cell shape and formation of paracellular gaps in the cell layer. Semiquantitative Western blotting using extracts of freshly isolated airway tissue (nasal epithelium) or 16HBE14o- model cells revealed that phosphorylation levels of focal adhesion kinase (Fak) and paxillin were altered upon treatment of tissue or cells with rHla. Immune fluorescence analyses showed that rHla treatment of 16HBE14o- cells results in losses of vinculin and paxillin from focal contacts and a net reduction in the number of focal contacts. The actin cytoskeleton was strongly remodeled. We concluded that treatment of cells with rHla activates Fak signaling, which accelerates focal contact turnover and prevents newly formed focal contacts (focal complexes) from maturation to focal adhesions. The inability of rHla-treated cells to form stable focal adhesions may be one factor that contributes to gap formation in the cell layer. In vivo, such changes may disturb the defensive barrier function of the airway epithelium and may facilitate lung infections by S. aureus.

  5. Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells.

    Science.gov (United States)

    Shannon-Lowe, C D; Neuhierl, B; Baldwin, G; Rickinson, A B; Delecluse, H-J

    2006-05-02

    Epstein-Barr virus (EBV), an orally transmitted herpesvirus, efficiently targets B lymphocytes through binding of the viral envelope glycoprotein gp350 to the complement receptor CD21. How the virus accesses epithelial cells is less well understood, because such cells are largely resistant to infection with cell-free virus in vitro. Here, we show that, after binding to primary B cells, most Epstein-Barr virions are not internalized but remain on the B cell surface and from there can transfer efficiently to CD21-negative epithelial cells, increasing epithelial infection by 10(3)- to 10(4)-fold compared with cell-free virus. Transfer infection is associated with the formation of B cell-epithelial conjugates with gp350/CD21 complexes focused at the intercellular synapse; transfer involves the gp85 and gp110 viral glycoproteins but is independent of gp42, the HLA class II ligand that is essential for B cell entry. Therefore, through efficient binding to the B cell surface, EBV has developed a means of simultaneously accessing both lymphoid and epithelial compartments; in particular, infection of pharyngeal epithelium by orally transmitted virus becomes independent of initial virus replication in the B cell system.

  6. Quiescent epithelial cell rests of Malassez can differentiate into ameloblast-like cells.

    Science.gov (United States)

    Shinmura, Yuka; Tsuchiya, Shuhei; Hata, Ken-Ichiro; Honda, Masaki J

    2008-12-01

    Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. After completion of crown formation, HERS are converted from cervical loop cells, which have the potential to generate enamel for tooth crown formation. Cervical loop cells have the potential to differentiate into ameloblasts. Generally, no new ameloblasts can be generated from HERS, however this study demonstrated that subcultured ERM can differentiate into ameloblast-like cells and generate enamel-like tissues in combination with dental pulp cells at the crown formation stage. Porcine ERM were obtained from periodontal ligament tissue by explant culture and were subcultured with non-serum medium. Thereafter, subcultured ERM were expanded on 3T3-J2 feeder cell layers until the tenth passage. The in vitro mRNA expression pattern of the subcultured ERM after four passages was found to be different from that of enamel organ epithelial cells and oral gingival epithelial cells after the fourth passage using the same expansion technique. When subcultured ERM were combined with subcultured dental pulp cells, ERM expressed cytokeratin14 and amelogenin proteins in vitro. In addition, subcultured ERM combined with primary dental pulp cells seeded onto scaffolds showed enamel-like tissues at 8 weeks post-transplantation. Moreover, positive staining for amelogenin was observed in the enamel-like tissues, indicating the presence of well-developed ameloblasts in the implants. These results suggest that ERM can differentiate into ameloblast-like cells.

  7. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

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    Sidjanin, D. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences; Grdina, D. [Argonne National Lab., IL (United States); Woloschak, G.E. [Northern Illinois Univ., De Kalb, IL (United States). Dept. of Biological Sciences

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  8. IL-17C regulates the innate immune function of epithelial cells in an autocrine manner.

    Science.gov (United States)

    Ramirez-Carrozzi, Vladimir; Sambandam, Arivazhagan; Luis, Elizabeth; Lin, Zhongua; Jeet, Surinder; Lesch, Justin; Hackney, Jason; Kim, Janice; Zhou, Meijuan; Lai, Joyce; Modrusan, Zora; Sai, Tao; Lee, Wyne; Xu, Min; Caplazi, Patrick; Diehl, Lauri; de Voss, Jason; Balazs, Mercedesz; Gonzalez, Lino; Singh, Harinder; Ouyang, Wenjun; Pappu, Rajita

    2011-10-12

    Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the T(H)17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate-induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

  9. Only Two Can Tango: Mast Cells Displace Epithelial Cells to Dance with ILC2s.

    Science.gov (United States)

    Bouchery, Tiffany; Harris, Nicola L

    2017-05-16

    Mast cells have been implicated in protective immunity to helminth infection, but the precise mechanism remains unclear. In this issue of Immunity, Shimokawa et al., 2017 report that mast cells are a bridge linking dying epithelial cells with effector type 2 innate lymphoid cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Sulphate-reducing bacteria from ulcerative colitis patients induce apoptosis of gastrointestinal epithelial cells.

    Science.gov (United States)

    Coutinho, Cláudia Mara Lara Melo; Coutinho-Silva, Robson; Zinkevich, Vitally; Pearce, Callum B; Ojcius, David M; Beech, Iwona

    2017-11-01

    The human microbiome consists of a multitude of bacterial genera and species which continuously interact with one another and their host establishing a metabolic equilibrium. The dysbiosis can lead to the development of pathology, such as inflammatory bowel diseases. Sulfide-producing prokaryotes, including sulphate-reducing bacteria (SRB) constituting different genera, including the Desulfovibrio, are commonly detected within the human microbiome recovered from fecal material or colonic biopsy samples. It has been proposed that SRB likely contribute to colonic pathology, for example ulcerative colitis (UC). The interaction of SRB with the human colon and intestinal epithelial cell lines has been investigated using Desulfovibrio indonesiensis as a model mono-culture and in a co-culture with E. coli isolate, and with SRB consortia from human biopsy samples. We find that D. indonesiensis, whether as a mono- or co-culture, was internalized and induced apoptosis in colon epithelial cells. This effect was enhanced in the presence of E. coli. The SRB combination obtained through enrichment of biopsies from UC patients induced apoptosis of a human intestinal epithelial cell line. This response was not observed in SRB enrichments from healthy (non-UC) controls. Importantly, apoptosis was detected in epithelial cells from UC patients and was not seen in epithelial cells of healthy donors. Furthermore, the antibody raised against exopolysaccharides (EPS) of D. indonesiensis cross reacted with the SRB population from UC patients but not with the SRB combination from non-UC controls. This study reinforces a correlation between the presence of sulphate-reducing bacteria and an inflammatory response in UC sufferers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Andrea C. Lei-Leston

    2017-09-01

    Full Text Available Pattern recognition recep