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Sample records for cell dynamic phenotype

  1. Phenotypic equilibrium as probabilistic convergence in multi-phenotype cell population dynamics.

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    Da-Quan Jiang

    Full Text Available We consider the cell population dynamics with n different phenotypes. Both the Markovian branching process model (stochastic model and the ordinary differential equation (ODE system model (deterministic model are presented, and exploited to investigate the dynamics of the phenotypic proportions. We will prove that in both models, these proportions will tend to constants regardless of initial population states ("phenotypic equilibrium" under weak conditions, which explains the experimental phenomenon in Gupta et al.'s paper. We also prove that Gupta et al.'s explanation is the ODE model under a special assumption. As an application, we will give sufficient and necessary conditions under which the proportion of one phenotype tends to 0 (die out or 1 (dominate. We also extend our results to non-Markovian cases.

  2. Nonequilibrium population dynamics of phenotype conversion of cancer cells.

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    Joseph Xu Zhou

    Full Text Available Tumorigenesis is a dynamic biological process that involves distinct cancer cell subpopulations proliferating at different rates and interconverting between them. In this paper we proposed a mathematical framework of population dynamics that considers both distinctive growth rates and intercellular transitions between cancer cell populations. Our mathematical framework showed that both growth and transition influence the ratio of cancer cell subpopulations but the latter is more significant. We derived the condition that different cancer cell types can maintain distinctive subpopulations and we also explain why there always exists a stable fixed ratio after cell sorting based on putative surface markers. The cell fraction ratio can be shifted by changing either the growth rates of the subpopulations (Darwinism selection or by environment-instructed transitions (Lamarckism induction. This insight can help us to understand the dynamics of the heterogeneity of cancer cells and lead us to new strategies to overcome cancer drug resistance.

  3. Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

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    Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

    2016-01-01

    Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

  4. Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

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    Li, Bobby W. S.; Stadhouders, Ralph; de Bruijn, Marjolein J. W.; Lukkes, Melanie; Beerens, Dior M. J. M.; Brem, Maarten D.; KleinJan, Alex; Bergen, Ingrid; Vroman, Heleen; Kool, Mirjam; van IJcken, Wilfred F. J.; Rao, Tata Nageswara; Fehling, Hans Jörg; Hendriks, Rudi W.

    2017-01-01

    Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought

  5. Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation

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    Bobby W. S. Li

    2017-12-01

    Full Text Available Group 2 innate lymphoid cells (ILC2 are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than

  6. A mathematical model of cancer cells with phenotypic plasticity

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    Da Zhou

    2015-12-01

    Full Text Available Purpose: The phenotypic plasticity of cancer cells is recently becoming a cutting-edge research area in cancer, which challenges the cellular hierarchy proposed by the conventional cancer stem cell theory. In this study, we establish a mathematical model for describing the phenotypic plasticity of cancer cells, based on which we try to find some salient features that can characterize the dynamic behavior of the phenotypic plasticity especially in comparison to the hierarchical model of cancer cells. Methods: We model cancer as population dynamics composed of different phenotypes of cancer cells. In this model, not only can cancer cells divide (symmetrically and asymmetrically and die, but they can also convert into other cellular phenotypes. According to the Law of Mass Action, the cellular processes can be captured by a system of ordinary differential equations (ODEs. On one hand, we can analyze the long-term stability of the model by applying qualitative method of ODEs. On the other hand, we are also concerned about the short-term behavior of the model by studying its transient dynamics. Meanwhile, we validate our model to the cell-state dynamics in published experimental data.Results: Our results show that the phenotypic plasticity plays important roles in both stabilizing the distribution of different phenotypic mixture and maintaining the cancer stem cells proportion. In particular, the phenotypic plasticity model shows decided advantages over the hierarchical model in predicting the phenotypic equilibrium and cancer stem cells’ overshoot reported in previous biological experiments in cancer cell lines.Conclusion: Since the validity of the phenotypic plasticity paradigm and the conventional cancer stem cell theory is still debated in experimental biology, it is worthy of theoretically searching for good indicators to distinguish the two models through quantitative methods. According to our study, the phenotypic equilibrium and overshoot

  7. Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas

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    2017-09-01

    AWARD NUMBER: W81XWH-14-1-0115 TITLE: Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas PRINCIPAL INVESTIGATOR: Kyuson Yun...CA130273 - Cell of Origin and Cancer Stem Cell Phenotype in Medulloblastomas 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0115 5c. PROGRAM...hypothesis, we originally proposed to transform neural stem cells (NSCs) and neural progenitor cells (NPCs) in vivo by expressing an activated form

  8. Expanding the phenotypic spectrum of ARID1B-mediated disorders and identification of altered cell-cycle dynamics due to ARID1B haploinsufficiency.

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    Sim, Joe C H; White, Susan M; Fitzpatrick, Elizabeth; Wilson, Gabrielle R; Gillies, Greta; Pope, Kate; Mountford, Hayley S; Torring, Pernille M; McKee, Shane; Vulto-van Silfhout, Anneke T; Jhangiani, Shalini N; Muzny, Donna M; Leventer, Richard J; Delatycki, Martin B; Amor, David J; Lockhart, Paul J

    2014-03-27

    Mutations in genes encoding components of the Brahma-associated factor (BAF) chromatin remodeling complex have recently been shown to contribute to multiple syndromes characterised by developmental delay and intellectual disability. ARID1B mutations have been identified as the predominant cause of Coffin-Siris syndrome and have also been shown to be a frequent cause of nonsyndromic intellectual disability. Here, we investigate the molecular basis of a patient with an overlapping but distinctive phenotype of intellectual disability, plantar fat pads and facial dysmorphism. High density microarray analysis of the patient demonstrated a heterozygous deletion at 6q25.3, which resulted in the loss of four genes including AT Rich Interactive Domain 1B (ARID1B). Subsequent quantitative real-time PCR analysis revealed ARID1B haploinsufficiency in the patient. Analysis of both patient-derived and ARID1B knockdown fibroblasts after serum starvation demonstrated delayed cell cycle re-entry associated with reduced cell number in the S1 phase. Based on the patient's distinctive phenotype, we ascertained four additional patients and identified heterozygous de novo ARID1B frameshift or nonsense mutations in all of them. This study broadens the spectrum of ARID1B associated phenotypes by describing a distinctive phenotype including plantar fat pads but lacking the hypertrichosis or fifth nail hypoplasia associated with Coffin-Siris syndrome. We present the first direct evidence in patient-derived cells that alterations in cell cycle contribute to the underlying pathogenesis of syndromes associated with ARID1B haploinsufficiency.

  9. Cell Phenotype Transitions in Cardiovascular Calcification

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    Luis Hortells

    2018-03-01

    Full Text Available Cardiovascular calcification was originally considered a passive, degenerative process, however with the advance of cellular and molecular biology techniques it is now appreciated that ectopic calcification is an active biological process. Vascular calcification is the most common form of ectopic calcification, and aging as well as specific disease states such as atherosclerosis, diabetes, and genetic mutations, exhibit this pathology. In the vessels and valves, endothelial cells, smooth muscle cells, and fibroblast-like cells contribute to the formation of extracellular calcified nodules. Research suggests that these vascular cells undergo a phenotypic switch whereby they acquire osteoblast-like characteristics, however the mechanisms driving the early aspects of these cell transitions are not fully understood. Osteoblasts are true bone-forming cells and differentiate from their pluripotent precursor, the mesenchymal stem cell (MSC; vascular cells that acquire the ability to calcify share aspects of the transcriptional programs exhibited by MSCs differentiating into osteoblasts. What is unknown is whether a fully-differentiated vascular cell directly acquires the ability to calcify by the upregulation of osteogenic genes or, whether these vascular cells first de-differentiate into an MSC-like state before obtaining a “second hit” that induces them to re-differentiate down an osteogenic lineage. Addressing these questions will enable progress in preventative and regenerative medicine strategies to combat vascular calcification pathologies. In this review, we will summarize what is known about the phenotypic switching of vascular endothelial, smooth muscle, and valvular cells.

  10. Aberrant phenotypes in peripheral T cell lymphomas.

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    Hastrup, N; Ralfkiaer, E; Pallesen, G

    1989-01-01

    Seventy six peripheral T cell lymphomas were examined immunohistologically to test their reactivity with a panel of monoclonal antibodies against 11 T cell associated antigens (CD1-8, CD27, UCHL1, and the T cell antigen receptor). Sixty two (82%) lymphomas showed aberrant phenotypes, and four main categories were distinguished as follows: (i) lack of one or several pan-T cell antigens (49, 64% of the cases); (ii) loss of both the CD4 and CD8 antigens (11, 15% of the cases); (iii) coexpression of the CD4 and CD8 antigens (13, 17% of the cases); and (iv) expression of the CD1 antigen (eight, 11% of the cases). No correlation was seen between the occurrence of aberrant phenotypes and the histological subtype. It is concluded that the demonstration of an aberrant phenotype is a valuable supplement to histological assessment in the diagnosis of peripheral T cell lymphomas. It is recommended that the panel of monoclonal antibodies against T cell differentiation antigens should be fairly large, as apparently any antigen may be lost in the process of malignant transformation. Images Figure PMID:2469701

  11. Smooth muscle cell phenotypic switching in stroke.

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    Poittevin, Marine; Lozeron, Pierre; Hilal, Rose; Levy, Bernard I; Merkulova-Rainon, Tatiana; Kubis, Nathalie

    2014-06-01

    Disruption of cerebral blood flow after stroke induces cerebral tissue injury through multiple mechanisms that are not yet fully understood. Smooth muscle cells (SMCs) in blood vessel walls play a key role in cerebral blood flow control. Cerebral ischemia triggers these cells to switch to a phenotype that will be either detrimental or beneficial to brain repair. Moreover, SMC can be primarily affected genetically or by toxic metabolic molecules. After stroke, this pathological phenotype has an impact on the incidence, pattern, severity, and outcome of the cerebral ischemic disease. Although little research has been conducted on the pathological role and molecular mechanisms of SMC in cerebrovascular ischemic diseases, some therapeutic targets have already been identified and could be considered for further pharmacological development. We examine these different aspects in this review.

  12. Phenotypic switching of populations of cells in a stochastic environment

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    Hufton, Peter G.; Lin, Yen Ting; Galla, Tobias

    2018-02-01

    In biology phenotypic switching is a common bet-hedging strategy in the face of uncertain environmental conditions. Existing mathematical models often focus on periodically changing environments to determine the optimal phenotypic response. We focus on the case in which the environment switches randomly between discrete states. Starting from an individual-based model we derive stochastic differential equations to describe the dynamics, and obtain analytical expressions for the mean instantaneous growth rates based on the theory of piecewise-deterministic Markov processes. We show that optimal phenotypic responses are non-trivial for slow and intermediate environmental processes, and systematically compare the cases of periodic and random environments. The best response to random switching is more likely to be heterogeneity than in the case of deterministic periodic environments, net growth rates tend to be higher under stochastic environmental dynamics. The combined system of environment and population of cells can be interpreted as host-pathogen interaction, in which the host tries to choose environmental switching so as to minimise growth of the pathogen, and in which the pathogen employs a phenotypic switching optimised to increase its growth rate. We discuss the existence of Nash-like mutual best-response scenarios for such host-pathogen games.

  13. Phenotype heterogeneity in cancer cell populations

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    Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean; Escargueil, Alexandre; Lorenzi, Tommaso; Lorz, Alexander; Trélat, Emmanuel

    2016-01-01

    Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as “bet hedging” of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

  14. Phenotype heterogeneity in cancer cell populations

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    Almeida, Luis [CNRS UMR 7598, LJLL, & INRIA MAMBA team, Sorbonne Universités, UPMC Univ Paris 06, Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, luis@ann.jussieu.fr (France); Chisholm, Rebecca [School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia, rebecca.chisholm@gmail.com (Australia); Clairambault, Jean [INRIA MAMBA team & LJLL, UMR 7598, Sorbonne Universités, UPMC Univ Paris 06, Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, jean.clairambault@inria.fr, Corresponding author (France); Escargueil, Alexandre [INSERM “Cancer Biology and Therapeutics”, Sorbonne Universités, UPMC Univ Paris 06, UMR-S 938, CDR St Antoine, Hôpital St Antoine, 184 Fbg. St Antoine, 75571 Paris cedex 12, France, alexandre.escargueil@upmc.fr (France); Lorenzi, Tommaso [CMLA, ENS Cachan, 61, Av. du Président Wilson, 94230 Cachan cedex & INRIA MAMBA team, & LJLL, UMR 7598, UPMC Univ Paris 06, Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, tommaso.lorenzi@gmail.com (France); Lorz, Alexander [Sorbonne Universités, UPMC Univ Paris 06, LJLL, UMR 7598 & INRIA Boîte courrier 187, 4 Pl. Jussieu, 75252 Paris cedex 05, France, alex.lorz@ann.jussieu.fr (France); Trélat, Emmanuel [Institut Universitaire de France, Sorbonne Universités, UPMC Univ Paris 06, LJLL, UMR 7598, Boîte courrier 187, UPMC Univ Paris 06, 4 Pl. Jussieu, 75252 Paris cedex 05, France, emmanuel.trelat@upmc.fr (France)

    2016-06-08

    Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as “bet hedging” of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

  15. Phenotype heterogeneity in cancer cell populations

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    Almeida, Luis; Chisholm, Rebecca; Clairambault, Jean; Escargueil, Alexandre; Lorenzi, Tommaso; Lorz, Alexander; Trélat, Emmanuel

    2016-06-01

    Phenotype heterogeneity in cancer cell populations, be it of genetic, epigenetic or stochastic origin, has been identified as a main source of resistance to drug treatments and a major source of therapeutic failures in cancers. The molecular mechanisms of drug resistance are partly understood at the single cell level (e.g., overexpression of ABC transporters or of detoxication enzymes), but poorly predictable in tumours, where they are hypothesised to rely on heterogeneity at the cell population scale, which is thus the right level to describe cancer growth and optimise its control by therapeutic strategies in the clinic. We review a few results from the biological literature on the subject, and from mathematical models that have been published to predict and control evolution towards drug resistance in cancer cell populations. We propose, based on the latter, optimisation strategies of combined treatments to limit emergence of drug resistance to cytotoxic drugs in cancer cell populations, in the monoclonal situation, which limited as it is still retains consistent features of cell population heterogeneity. The polyclonal situation, that may be understood as "bet hedging" of the tumour, thus protecting itself from different sources of drug insults, may lie beyond such strategies and will need further developments. In the monoclonal situation, we have designed an optimised therapeutic strategy relying on a scheduled combination of cytotoxic and cytostatic treatments that can be adapted to different situations of cancer treatments. Finally, we review arguments for biological theoretical frameworks proposed at different time and development scales, the so-called atavistic model (diachronic view relying on Darwinian genotype selection in the coursof billions of years) and the Waddington-like epigenetic landscape endowed with evolutionary quasi-potential (synchronic view relying on Lamarckian phenotype instruction of a given genome by reversible mechanisms), to

  16. VEGF-A, cytoskeletal dynamics, and the pathological vascular phenotype

    International Nuclear Information System (INIS)

    Nagy, Janice A.; Senger, Donald R.

    2006-01-01

    Normal angiogenesis is a complex process involving the organization of proliferating and migrating endothelial cells (ECs) into a well-ordered and highly functional vascular network. In contrast, pathological angiogenesis, which is a conspicuous feature of tumor growth, ischemic diseases, and chronic inflammation, is characterized by vessels with aberrant angioarchitecture and compromised barrier function. Herein we review the subject of pathological angiogenesis, particularly that driven by vascular endothelial growth factor (VEGF-A), from a new perspective. We propose that the serious structural and functional anomalies associated with VEGF-A-elicited neovessels, reflect, at least in part, imbalances in the internal molecular cues that govern the ordered assembly of ECs into three dimensional vascular networks and preserve vessel barrier function. Adopting such a viewpoint widens the focus from solely on specific pro-angiogenic stimuli such as VEGF-A to include a key set of cytoskeletal regulatory molecules, the Rho GTPases, which are known to direct multiple aspects of vascular morphogenesis including EC motility, alignment, multi-cellular organization, as well as intercellular junction integrity. We offer this perspective to draw attention to the importance of endothelial cytoskeletal dynamics for proper neovascularization and to suggest new therapeutic strategies with the potential to improve the pathological vascular phenotype

  17. 3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

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    Hatta, Mitsutoki; Naganuma, Kaori; Kato, Kenichi; Yamazaki, Jun

    2015-01-01

    In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

  18. 3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

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    Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan); Naganuma, Kaori [Department of Oral and Maxillofacial Surgery, Fukuoka Dental College, Fukuoka (Japan); Kato, Kenichi; Yamazaki, Jun [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan)

    2015-12-04

    In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

  19. Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

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    Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

    2009-11-01

    Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

  20. Multiparametric classification links tumor microenvironments with tumor cell phenotype.

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    Bojana Gligorijevic

    2014-11-01

    Full Text Available While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which

  1. Structural phenotyping of stem cell-derived cardiomyocytes.

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    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-03-10

    Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Motion as a phenotype: the use of live-cell imaging and machine visual screening to characterize transcription-dependent chromosome dynamics

    Directory of Open Access Journals (Sweden)

    Silver Pamela A

    2006-04-01

    Full Text Available Abstract Background Gene transcriptional activity is well correlated with intra-nuclear position, especially relative to the nuclear periphery, which is a region classically associated with gene silencing. Recently however, actively transcribed genes have also been found localized to the nuclear periphery in the yeast Saccharomyces cerevisiae. When genes are activated, they become associated with the nuclear pore complex (NPC at the nuclear envelope. Furthermore, chromosomes are not static structures, but exhibit constrained diffusion in real-time, live-cell studies of particular loci. The relationship of chromosome motion with transcriptional activation and active-gene recruitment to the nuclear periphery has not yet been investigated. Results We have generated a yeast strain that enables us to observe the motion of the galactose-inducible GAL gene locus relative to the nuclear periphery in real-time under transcriptionally active and repressed conditions. Using segmented geometric particle tracking, we show that the repressed GAL locus undergoes constrained diffusive movement, and that transcriptional induction with galactose is associated with an enrichment in cells with GAL loci that are both associated with the nuclear periphery and much more constrained in their movement. Furthermore, we report that the mRNA export factor Sac3 is involved in this galactose-induced enrichment of GAL loci at the nuclear periphery. In parallel, using a novel machine visual screening technique, we find that the motion of constrained GAL loci correlates with the motion of the cognate nuclei in galactose-induced cells. Conclusion Transcriptional activation of the GAL genes is associated with their tethering and motion constraint at the nuclear periphery. We describe a model of gene recruitment to the nuclear periphery involving gene diffusion and the mRNA export factor Sac3 that can be used as a framework for further experimentation. In addition, we applied to

  3. Discrimination of meniscal cell phenotypes using gene expression profiles

    Directory of Open Access Journals (Sweden)

    M Son

    2012-03-01

    Full Text Available The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus. The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

  4. Identifying niche-mediated regulatory factors of stem cell phenotypic state: a systems biology approach.

    Science.gov (United States)

    Ravichandran, Srikanth; Del Sol, Antonio

    2017-02-01

    Understanding how the cellular niche controls the stem cell phenotype is often hampered due to the complexity of variegated niche composition, its dynamics, and nonlinear stem cell-niche interactions. Here, we propose a systems biology view that considers stem cell-niche interactions as a many-body problem amenable to simplification by the concept of mean field approximation. This enables approximation of the niche effect on stem cells as a constant field that induces sustained activation/inhibition of specific stem cell signaling pathways in all stem cells within heterogeneous populations exhibiting the same phenotype (niche determinants). This view offers a new basis for the development of single cell-based computational approaches for identifying niche determinants, which has potential applications in regenerative medicine and tissue engineering. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  5. Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity.

    Science.gov (United States)

    Binder, Dennis; Drepper, Thomas; Jaeger, Karl-Erich; Delvigne, Frank; Wiechert, Wolfgang; Kohlheyer, Dietrich; Grünberger, Alexander

    2017-07-01

    In natural habitats, microbes form multispecies communities that commonly face rapidly changing and highly competitive environments. Thus, phenotypic heterogeneity has evolved as an innate and important survival strategy to gain an overall fitness advantage over cohabiting competitors. However, in defined artificial environments such as monocultures in small- to large-scale bioreactors, cell-to-cell variations are presumed to cause reduced production yields as well as process instability. Hence, engineering microbial production toward phenotypic homogeneity is a highly promising approach for synthetic biology and bioprocess optimization. In this review, we discuss recent studies that have unraveled the cell-to-cell heterogeneity observed during bacterial gene expression and metabolite production as well as the molecular mechanisms involved. In addition, current single-cell technologies are briefly reviewed with respect to their applicability in exploring cell-to-cell variations. We highlight emerging strategies and tools to reduce phenotypic heterogeneity in biotechnological expression setups. Here, strain or inducer modifications are combined with cell physiology manipulations to achieve the ultimate goal of equalizing bacterial populations. In this way, the majority of cells can be forced into high productivity, thus reducing less productive subpopulations that tend to consume valuable resources during production. Modifications in uptake systems, inducer molecules or nutrients represent valuable tools for diminishing heterogeneity. Finally, we address the challenge of transferring homogeneously responding cells into large-scale bioprocesses. Environmental heterogeneity originating from extrinsic factors such as stirring speed and pH, oxygen, temperature or nutrient distribution can significantly influence cellular physiology. We conclude that engineering microbial populations toward phenotypic homogeneity is an increasingly important task to take biotechnological

  6. Stem Cell Microencapsulation for Phenotypic Control, Bioprocessing, and Transplantation

    Science.gov (United States)

    Wilson, Jenna L.

    2014-01-01

    Cell microencapsulation has been utilized for decades as a means to shield cells from the external environment while simultaneously permitting transport of oxygen, nutrients, and secretory molecules. In designing cell therapies, donor primary cells are often difficult to obtain and expand to appropriate numbers, rendering stem cells an attractive alternative due to their capacities for self-renewal, differentiation, and trophic factor secretion. Microencapsulation of stem cells offers several benefits, namely the creation of a defined microenvironment which can be designed to modulate stem cell phenotype, protection from hydrodynamic forces and prevention of agglomeration during expansion in suspension bioreactors, and a means to transplant cells behind a semi-permeable barrier, allowing for molecular secretion while avoiding immune reaction. This review will provide an overview of relevant microencapsulation processes and characterization in the context of maintaining stem cell potency, directing differentiation, investigating scalable production methods, and transplanting stem cells for clinically relevant disorders. PMID:23239279

  7. Sickle cell disease clinical phenotypes in children from South ...

    African Journals Online (AJOL)

    2014-07-20

    Jul 20, 2014 ... Background:The clinical phenotypes of children with sickle cell disease (SCD) are poorly described in many sub-Saharan countries ..... World Health Organization. ... apps.who.int/gb/ebwha/pdf_files/WHA59/A59_9‑en.pdf.

  8. Antithymocyte Globulin Induces a Tolerogenic Phenotype in Human Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Tobias Roider

    2016-12-01

    Full Text Available Antithymocyte globulin (ATG is used in the prevention of graft-versus-host disease during allogeneic hematopoietic stem cell transplantation. It is generally accepted that ATG mediates its immunosuppressive effect primarily via depletion of T cells. Here, we analyzed the impact of ATG-Fresenius (now Grafalon® on human monocyte-derived dendritic cells (DC. ATG induced a semi-mature phenotype in DC with significantly reduced expression of CD14, increased expression of HLA-DR, and intermediate expression of CD54, CD80, CD83, and CD86. ATG-DC showed an increase in IL-10 secretion but no IL-12 production. In line with this tolerogenic phenotype, ATG caused a significant induction of indoleamine 2,3-dioxygenase expression and a concomitant increase in levels of tryptophan metabolites in the supernatants of DC. Further, ATG-DC did not induce the proliferation of allogeneic T cells in a mixed lymphocyte reaction but actively suppressed the T cell proliferation induced by mature DC. These data suggest that besides its well-known effect on T cells, ATG modulates the phenotype of DC in a tolerogenic way, which might constitute an essential part of its immunosuppressive action in vivo.

  9. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils

    DEFF Research Database (Denmark)

    Galli, Stephen J; Borregaard, Niels; Wynn, Thomas A

    2011-01-01

    Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function......). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially...... and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents...

  10. Phenotypic and functional plasticity of cells of innate immunity: macrophages, mast cells and neutrophils

    DEFF Research Database (Denmark)

    Galli, Stephen J; Borregaard, Niels; Wynn, Thomas A

    2011-01-01

    ). Here we focus on the occurrence of phenotypically distinct subpopulations in three lineages of myeloid cells with important roles in innate and acquired immunity: macrophages, mast cells and neutrophils. Cytokine signals, epigenetic modifications and other microenvironmental factors can substantially......Hematopoietic cells, including lymphoid and myeloid cells, can develop into phenotypically distinct 'subpopulations' with different functions. However, evidence indicates that some of these subpopulations can manifest substantial plasticity (that is, undergo changes in their phenotype and function...... and, in some cases, rapidly and reversibly alter the phenotype of these cells and influence their function. This suggests that regulation of the phenotype and function of differentiated hematopoietic cells by microenvironmental factors, including those generated during immune responses, represents...

  11. Characterizing visible and invisible cell wall mutant phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Carpita, Nicholas C.; McCann, Maureen C.

    2015-04-06

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with ‘invisible’ phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall.

  12. Heterogeneity of functional properties of Clone 66 murine breast cancer cells expressing various stem cell phenotypes.

    Science.gov (United States)

    Mukhopadhyay, Partha; Farrell, Tracy; Sharma, Gayatri; McGuire, Timothy R; O'Kane, Barbara; Sharp, J Graham

    2013-01-01

    Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. The proportion of cells expressing CD44(high)CD24(low/neg), side population (SP) cells, ALDH1(+), CD49f(high), CD133(high), and CD34(high) differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1(+), CD34(low), and CD49f(high) suggested properties of transit amplifying cells. Colony formation was higher from ALDH1(-) and non-SP cells than ALDH1(+) and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than "non-stem" cells. Fewer SP cells were needed to form tumors than ALDH1(+) cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

  13. Cell and small animal models for phenotypic drug discovery

    Directory of Open Access Journals (Sweden)

    Szabo M

    2017-06-01

    Full Text Available Mihaly Szabo,1 Sara Svensson Akusjärvi,1 Ankur Saxena,1 Jianping Liu,2 Gayathri Chandrasekar,1 Satish S Kitambi1 1Department of Microbiology Tumor, and Cell Biology, 2Department of Biochemistry and Biophysics, Karolinska Institutet, Solna, Sweden Abstract: The phenotype-based drug discovery (PDD approach is re-emerging as an alternative platform for drug discovery. This review provides an overview of the various model systems and technical advances in imaging and image analyses that strengthen the PDD platform. In PDD screens, compounds of therapeutic value are identified based on the phenotypic perturbations produced irrespective of target(s or mechanism of action. In this article, examples of phenotypic changes that can be detected and quantified with relative ease in a cell-based setup are discussed. In addition, a higher order of PDD screening setup using small animal models is also explored. As PDD screens integrate physiology and multiple signaling mechanisms during the screening process, the identified hits have higher biomedical applicability. Taken together, this review highlights the advantages gained by adopting a PDD approach in drug discovery. Such a PDD platform can complement target-based systems that are currently in practice to accelerate drug discovery. Keywords: phenotype, screening, PDD, discovery, zebrafish, drug

  14. Migration Phenotype of Brain-Cancer Cells Predicts Patient Outcomes

    Directory of Open Access Journals (Sweden)

    Chris L. Smith

    2016-06-01

    Full Text Available Glioblastoma multiforme is a heterogeneous and infiltrative cancer with dismal prognosis. Studying the migratory behavior of tumor-derived cell populations can be informative, but it places a high premium on the precision of in vitro methods and the relevance of in vivo conditions. In particular, the analysis of 2D cell migration may not reflect invasion into 3D extracellular matrices in vivo. Here, we describe a method that allows time-resolved studies of primary cell migration with single-cell resolution on a fibrillar surface that closely mimics in vivo 3D migration. We used this platform to screen 14 patient-derived glioblastoma samples. We observed that the migratory phenotype of a subset of cells in response to platelet-derived growth factor was highly predictive of tumor location and recurrence in the clinic. Therefore, migratory phenotypic classifiers analyzed at the single-cell level in a patient-specific way can provide high diagnostic and prognostic value for invasive cancers.

  15. The therapeutic implications of plasticity of the cancer stem cell phenotype.

    Directory of Open Access Journals (Sweden)

    Kevin Leder

    2010-12-01

    Full Text Available The cancer stem cell hypothesis suggests that tumors contain a small population of cancer cells that have the ability to undergo symmetric self-renewing cell division. In tumors that follow this model, cancer stem cells produce various kinds of specified precursors that divide a limited number of times before terminally differentiating or undergoing apoptosis. As cells within the tumor mature, they become progressively more restricted in the cell types to which they can give rise. However, in some tumor types, the presence of certain extra- or intracellular signals can induce committed cancer progenitors to revert to a multipotential cancer stem cell state. In this paper, we design a novel mathematical model to investigate the dynamics of tumor progression in such situations, and study the implications of a reversible cancer stem cell phenotype for therapeutic interventions. We find that higher levels of dedifferentiation substantially reduce the effectiveness of therapy directed at cancer stem cells by leading to higher rates of resistance. We conclude that plasticity of the cancer stem cell phenotype is an important determinant of the prognosis of tumors. This model represents the first mathematical investigation of this tumor trait and contributes to a quantitative understanding of cancer.

  16. Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Hamada, Shin; Masamune, Atsushi; Takikawa, Tetsuya; Suzuki, Noriaki; Kikuta, Kazuhiro; Hirota, Morihisa; Hamada, Hirofumi; Kobune, Masayoshi; Satoh, Kennichi; Shimosegawa, Tooru

    2012-01-01

    Highlights: ► Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. ► Pancreatic cancer cells co-cultured with PSCs showed enhanced spheroid formation. ► Expression of stem cell-related genes ABCG2, Nestin and LIN28 was increased. ► Co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. ► This study suggested a novel role of PSCs as a part of the cancer stem cell niche. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Recent studies have identified that a portion of cancer cells, called “cancer stem cells”, within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the “stemness” of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

  17. A basal stem cell signature identifies aggressive prostate cancer phenotypes

    Science.gov (United States)

    Smith, Bryan A.; Sokolov, Artem; Uzunangelov, Vladislav; Baertsch, Robert; Newton, Yulia; Graim, Kiley; Mathis, Colleen; Cheng, Donghui; Stuart, Joshua M.; Witte, Owen N.

    2015-01-01

    Evidence from numerous cancers suggests that increased aggressiveness is accompanied by up-regulation of signaling pathways and acquisition of properties common to stem cells. It is unclear if different subtypes of late-stage cancer vary in stemness properties and whether or not these subtypes are transcriptionally similar to normal tissue stem cells. We report a gene signature specific for human prostate basal cells that is differentially enriched in various phenotypes of late-stage metastatic prostate cancer. We FACS-purified and transcriptionally profiled basal and luminal epithelial populations from the benign and cancerous regions of primary human prostates. High-throughput RNA sequencing showed the basal population to be defined by genes associated with stem cell signaling programs and invasiveness. Application of a 91-gene basal signature to gene expression datasets from patients with organ-confined or hormone-refractory metastatic prostate cancer revealed that metastatic small cell neuroendocrine carcinoma was molecularly more stem-like than either metastatic adenocarcinoma or organ-confined adenocarcinoma. Bioinformatic analysis of the basal cell and two human small cell gene signatures identified a set of E2F target genes common between prostate small cell neuroendocrine carcinoma and primary prostate basal cells. Taken together, our data suggest that aggressive prostate cancer shares a conserved transcriptional program with normal adult prostate basal stem cells. PMID:26460041

  18. Comparison Of Liver Cell Models Using The Basel Phenotyping Cocktail

    Directory of Open Access Journals (Sweden)

    Benjamin Berger

    2016-11-01

    Full Text Available Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH cultures. We investigated the suit-ability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4] in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6 and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least 4-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20µM rifampicin, mRNA increased 3 to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6 and 17-fold (CYP3A4 for 2D-PHH and 4-fold (CYP3A4 for HepG2. In 3D-PHH at least a 2-fold in-crease in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model.Our studies show that 3D-PHH and (with some limitations HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to as-sess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro.

  19. Microbial Cell Dynamics Lab (MCDL)

    Data.gov (United States)

    Federal Laboratory Consortium — The Microbial Cell Dynamics Laboratory at PNNL enables scientists to study the molecular details of microbes under relevant environmental conditions. The MCDL seeks...

  20. HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution.

    Science.gov (United States)

    Stepanenko, A A; Dmitrenko, V V

    2015-09-15

    293 cell line (widely known as the Human Embryonic Kidney 293 cells) and its derivatives were the most used cells after HeLa in cell biology studies and after CHO in biotechnology as a vehicle for the production of adenoviral vaccines and recombinant proteins, for analysis of the neuronal synapse formation, in electrophysiology and neuropharmacology. Despite the historically long-term productive exploitation, the origin, phenotype, karyotype, and tumorigenicity of 293 cells are still debated. 293 cells were considered the kidney epithelial cells or even fibroblasts. However, 293 cells demonstrate no evident tissue-specific gene expression signature and express the markers of renal progenitor cells, neuronal cells and adrenal gland. This complicates efforts to reveal the authentic cell type/tissue of origin. On the other hand, the potential to propagate the highly neurotropic viruses, inducible synaptogenesis, functionality of the endogenous neuron-specific voltage-gated channels, and response to the diverse agonists implicated in neuronal signaling give credibility to consider 293 cells of neuronal lineage phenotype. The compound phenotype of 293 cells can be due to heterogeneous, unstable karyotype. The mean chromosome number and chromosome aberrations differ between 293 cells and derivatives as well as between 293 cells from the different cell banks/labs. 293 cells are tumorigenic, whereas acute changes of expression of the cancer-associated genes aggravate tumorigenicity by promoting chromosome instability. Importantly, the procedure of a stable empty vector transfection can also impact karyotype and phenotype. The discussed issues caution against misinterpretations and pitfalls during the different experimental manipulations with 293 cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

    Science.gov (United States)

    Okubo-Kurihara, Emiko; Matsui, Minami

    2018-01-01

    The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

  2. A probabilistic model for cell population phenotyping using HCS data.

    Directory of Open Access Journals (Sweden)

    Edouard Pauwels

    Full Text Available High Content Screening (HCS platforms allow screening living cells under a wide range of experimental conditions and give access to a whole panel of cellular responses to a specific treatment. The outcome is a series of cell population images. Within these images, the heterogeneity of cellular response to the same treatment leads to a whole range of observed values for the recorded cellular features. Consequently, it is difficult to compare and interpret experiments. Moreover, the definition of phenotypic classes at a cell population level remains an open question, although this would ease experiments analyses. In the present work, we tackle these two questions. The input of the method is a series of cell population images for which segmentation and cellular phenotype classification has already been performed. We propose a probabilistic model to represent and later compare cell populations. The model is able to fully exploit the HCS-specific information: "dependence structure of population descriptors" and "within-population variability". The experiments we carried out illustrate how our model accounts for this specific information, as well as the fact that the model benefits from considering them. We underline that these features allow richer HCS data analysis than simpler methods based on single cellular feature values averaged over each well. We validate an HCS data analysis method based on control experiments. It accounts for HCS specificities that were not taken into account by previous methods but have a sound biological meaning. Biological validation of previously unknown outputs of the method constitutes a future line of work.

  3. Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype.

    Science.gov (United States)

    Esteves, Cristina L; Sheldrake, Tara A; Dawson, Lucy; Menghini, Timothy; Rink, Burgunde Elisabeth; Amilon, Karin; Khan, Nusrat; Péault, Bruno; Donadeu, Francesc Xavier

    2017-07-01

    Mesenchymal stem/stromal cells (MSCs) have been used in human and equine regenerative medicine, and interest in exploiting their potential has increased dramatically over the years. Despite significant effort to characterize equine MSCs, the actual origin of these cells and how much of their native phenotype is maintained in culture have not been determined. In this study, we investigated the relationship between MSCs, derived from adipose tissue (AT) and bone marrow (BM), and pericytes in the horse. Both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD90, and CD73) markers were detected in equine AT and colocalized around blood vessels. Importantly, as assessed by flow cytometry, both pericyte (CD146, NG2, and αSMA) and MSC (CD29, CD44, CD90, and CD105) markers were present in a majority (≥90%) of cells in cultures of AT-MSCs and BM-MSCs; however, levels of pericyte markers were variable within each of those populations. Moreover, the expression of pericyte markers was maintained for at least eight passages in both AT-MSCs and BM-MSCs. Hematopoietic (CD45) and endothelial (CD144) markers were also detected at low levels in MSCs by quantitative polymerase chain reaction (qPCR). Finally, in coculture experiments, AT-MSCs closely associated with networks produced by endothelial cells, resembling the natural perivascular location of pericytes in vivo. Our results indicate that equine MSCs originate from perivascular cells and moreover maintain a pericyte-like phenotype in culture. Therefore, we suggest that, in addition to classical MSC markers, pericyte markers such as CD146 could be used when assessing and characterizing equine MSCs.

  4. Uncovering cancer cell behavioral phenotype in 3-D in vitro metastatic landscapes

    Science.gov (United States)

    Liu, Liyu; Sun, Bo; Duclos, Guillaume; Kam, Yoonseok; Gatenby, Robert; Stone, Howard; Austin, Robert

    2012-02-01

    One well-known fact is that cancer cell genetics determines cell metastatic potentials. However, from a physics point of view, genetics as cell properties cannot directly act on metastasis. An agent is needed to unscramble the genetics first before generating dynamics for metastasis. Exactly this agent is cell behavioral phenotype, which is rarely studied due to the difficulties of real-time cell tracking in in vivo tissue. Here we have successfully constructed a micro in vitro environment with collagen based Extracellular Matrix (ECM) structures for cell 3-D metastasis. With stable nutrition (glucose) gradient inside, breast cancer cell MDA-MB-231 is able to invade inside the collagen from the nutrition poor site towards the nutrition rich site. Continuous confocal microscopy captures images of the cells every 12 hours and tracks their positions in 3-D space. The micro fluorescent beads pre-mixed inside the ECM demonstrate that invasive cells have altered the structures through mechanics. With the observation and the analysis of cell collective behaviors, we argue that game theory may exist between the pioneering cells and their followers in the metastatic cell group. The cell collaboration may explain the high efficiency of metastasis.

  5. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    International Nuclear Information System (INIS)

    Yang, Qiwei; Tian, Yufeng; Ostler, Kelly R; Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Godley, Lucy A; Cohn, Susan L

    2010-01-01

    Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation

  6. Volatility of Mutator Phenotypes at Single Cell Resolution.

    Directory of Open Access Journals (Sweden)

    Scott R Kennedy

    2015-04-01

    Full Text Available Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.

  7. Human T Cell Memory: A Dynamic View

    Directory of Open Access Journals (Sweden)

    Derek C. Macallan

    2017-02-01

    Full Text Available Long-term T cell-mediated protection depends upon the formation of a pool of memory cells to protect against future pathogen challenge. In this review we argue that looking at T cell memory from a dynamic viewpoint can help in understanding how memory populations are maintained following pathogen exposure or vaccination. For example, a dynamic view resolves the apparent paradox between the relatively short lifespans of individual memory cells and very long-lived immunological memory by focussing on the persistence of clonal populations, rather than individual cells. Clonal survival is achieved by balancing proliferation, death and differentiation rates within and between identifiable phenotypic pools; such pools correspond broadly to sequential stages in the linear differentiation pathway. Each pool has its own characteristic kinetics, but only when considered as a population; single cells exhibit considerable heterogeneity. In humans, we tend to concentrate on circulating cells, but memory T cells in non-lymphoid tissues and bone marrow are increasingly recognised as critical for immune defence; their kinetics, however, remain largely unexplored. Considering vaccination from this viewpoint shifts the focus from the size of the primary response to the survival of the clone and enables identification of critical system pinch-points and opportunities to improve vaccine efficacy.

  8. Manual for Dynamic Triaxial Cell

    DEFF Research Database (Denmark)

    Pedersen, Thomas Schmidt; Ibsen, Lars Bo

    This report is a test report that describes the test setup for a dynamic triaxial cell at the Laboratory for Geotechnique at Aalborg University.......This report is a test report that describes the test setup for a dynamic triaxial cell at the Laboratory for Geotechnique at Aalborg University....

  9. Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells.

    Science.gov (United States)

    Guidi, Novella; Sacma, Mehmet; Ständker, Ludger; Soller, Karin; Marka, Gina; Eiwen, Karina; Weiss, Johannes M; Kirchhoff, Frank; Weil, Tanja; Cancelas, Jose A; Florian, Maria Carolina; Geiger, Hartmut

    2017-04-03

    Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging. © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  10. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  11. Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype.

    Science.gov (United States)

    Ngo, Kevin; Patil, Prashanti; McGowan, Sara J; Niedernhofer, Laura J; Robbins, Paul D; Kang, James; Sowa, Gwendolyn; Vo, Nam

    2017-09-01

    Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1 -/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss. Published by Elsevier B.V.

  12. Induction of appropriate Th-cell phenotypes: cellular decision-making in heterogeneous environments.

    Science.gov (United States)

    van den Ham, H-J; Andeweg, A C; de Boer, R J

    2013-11-01

    Helper T (Th)-cell differentiation is a key event in the development of the adaptive immune response. By the production of a range of cytokines, Th cells determine the type of immune response that is raised against an invading pathogen. Th cells can adopt many different phenotypes, and Th-cell phenotype decision-making is crucial in mounting effective host responses. This review discusses the different Th-cell phenotypes that have been identified and how Th cells adopt a particular phenotype. The regulation of Th-cell phenotypes has been studied extensively using mathematical models, which have explored the role of regulatory mechanisms such as autocrine cytokine signalling and cross-inhibition between self-activating transcription factors. At the single cell level, Th responses tend to be heterogeneous, but corrections can be made soon after T-cell activation. Although pathogens and the innate immune system provide signals that direct the induction of Th-cell phenotypes, these instructive mechanisms could be easily subverted by pathogens. We discuss that a model of success-driven feedback would select the most appropriate phenotype for clearing a pathogen. Given the heterogeneity in the induction phase of the Th response, such a success-driven feedback loop would allow the selection of effective Th-cell phenotypes while terminating incorrect responses. © 2013 John Wiley & Sons Ltd.

  13. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  14. Phenotypic resistance and the dynamics of bacterial escape from phage control

    DEFF Research Database (Denmark)

    Bull, James J.; Vegge, Christina Skovgaard; Schmerer, Matthew

    2014-01-01

    The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages...... mathematical models of these processes and suggest how different types of this 'phenotypic' resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may...

  15. A Phenotypic Cell-Binding Screen Identifies a Novel Compound Targeting Triple-Negative Breast Cancer.

    Science.gov (United States)

    Chen, Luxi; Long, Chao; Youn, Jonghae; Lee, Jiyong

    2018-06-11

    We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug targets. Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelial-mesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

  16. [Notochord cells enhance proliferation and phenotype-keeping of intervertebral disc chondroid cells].

    Science.gov (United States)

    Zhao, Xianfeng; Liu, Hao; Feng, Ganjun; Deng, Li; Li, Xiuqun; Liang, Tao

    2008-08-01

    To isolate and culture the chondroid cells and notochord cells from New Zealand rabbit immature nucleus pulposus (NP) in monolayer, and to evaluate the responsiveness of rabbit disc-derived chondroid cells to notochord cells with respect to cell proliferation and phenotype. The NP cells were released from the minced immature NP of 6 New Zealand rabbits (4-week-old) by 0.2% collagenase II digestion. The chondroid cells and notochord cells were purified by discontinuous gradient density centrifugation. The chondroid cells were cultured alone (group A) and co-cultured with notochord cells (group B) (1:1), and cell proliferation and phenotype including proteoglycan and collagen II were evaluated. The cells in both groups were observed by the inverted microscope, and the survival rates of the primary and passage cells were detected by toluidine blue staining. The growth curves of the second passage cells in both groups were determined by MTT. Besides, the expressions of proteoglycan and collagen II of the primary and passage cells were examined by toluidine blue and immunocytochemistry staining. The notochord cells and chondroid cells were isolated and purified. With the diameter of 10-15 microm, the notochord cell had abundant intracytoplasmic vesicles, while the chondroid cell, with the diameter of 4-6 microm, had no intracytoplasmic vesicle. The cell survival rate was 89.0%-95.3% in group A and 91.3%-96.3% in group B. There was no significant difference between the same passages in both groups (P > 0.05). The co-cultured cells (group B) increased in cell proliferation compared with the chondroid cells alone (group A) in repeated experiments. The cells in group A reached their logarithmic growth phase after 3-4 days of culture, while the cells in group B did after 2 days of culture. The cell proliferation in group B was more than that in group A after 4-day culture (P notochord cells are conducive for the proliferation and phenotype-keeping of the chondroid cells and

  17. Capillary regeneration in scleroderma: stem cell therapy reverses phenotype?

    Directory of Open Access Journals (Sweden)

    Jo N Fleming

    2008-01-01

    Full Text Available Scleroderma is an autoimmune disease with a characteristic vascular pathology. The vasculopathy associated with scleroderma is one of the major contributors to the clinical manifestations of the disease.We used immunohistochemical and mRNA in situ hybridization techniques to characterize this vasculopathy and showed with morphometry that scleroderma has true capillary rarefaction. We compared skin biopsies from 23 scleroderma patients and 24 normal controls and 7 scleroderma patients who had undergone high dose immunosuppressive therapy followed by autologous hematopoietic cell transplant. Along with the loss of capillaries there was a dramatic change in endothelial phenotype in the residual vessels. The molecules defining this phenotype are: vascular endothelial cadherin, a supposedly universal endothelial marker required for tube formation (lost in the scleroderma tissue, antiangiogenic interferon alpha (overexpressed in the scleroderma dermis and RGS5, a signaling molecule whose expression coincides with the end of branching morphogenesis during development and tumor angiogenesis (also overexpressed in scleroderma skin. Following high dose immunosuppressive therapy, patients experienced clinical improvement and 5 of the 7 patients with scleroderma had increased capillary counts. It was also observed in the same 5 patients, that the interferon alpha and vascular endothelial cadherin had returned to normal as other clinical signs in the skin regressed, and in all 7 patients, RGS5 had returned to normal.These data provide the first objective evidence for loss of vessels in scleroderma and show that this phenomenon is reversible. Coordinate changes in expression of three molecules already implicated in angiogenesis or anti-angiogenesis suggest that control of expression of these three molecules may be the underlying mechanism for at least the vascular component of this disease. Since rarefaction has been little studied, these data may have

  18. Phenotypic and functional characteristics of blood natural killer cells from melanoma patients at different clinical stages.

    Directory of Open Access Journals (Sweden)

    Giulia Fregni

    Full Text Available Melanomas are aggressive skin tumors characterized by high metastatic potential. Immunotherapy is a valuable alternative for metastatic melanoma patients resistant to chemotherapy. Natural Killer (NK cells are efficient anti-tumor cytotoxic effectors. We previously showed that blood NK cells from stage IV metastatic melanoma patients display decreased NK receptors and that chemotherapy modifies the functional status of blood NK cells. To investigate the role of NK cells along melanoma progression, we have here studied NK cells from patients at different stages of the disease. First, we showed that ex vivo NK cells from certain stage III-IV patients displayed low degranulation potential. Using a dynamic label-free assay, we found that immunoselected IL-2 activated blood NK cells from patients efficiently lysed melanoma cells through NKp46 and NKG2D receptors, independently to the clinical stage. Moreover, the ex vivo phenotype of circulating NK cells from 33 patients (stage I to IV was extensively analyzed. NK cells from patients displayed higher variability in the percentages of Natural Cytotoxicity Receptors (NCR and Natural Killer Group 2D (NKG2D receptor expression compared to donor NK cells. The main defect was the decreased expression of NCR1 (NKp46 by NK cells from metastatic patients. Interestingly, we found a positive correlation between the NK cell percentages of NKp46 and the duration of stage IV in melanoma patients. Finally, we showed that NK cells infiltrated primary melanomas and displayed a predominant peritumoral distribution. These results are new arguments for the development of NK-based therapies in melanoma patients.

  19. A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes

    Directory of Open Access Journals (Sweden)

    Etchells J

    2012-11-01

    Full Text Available Abstract Background Homeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR, one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5, a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp, a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev, required for radial patterning of the stem. Results Plants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening. Conclusions Our data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly

  20. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    International Nuclear Information System (INIS)

    Yamagishi, Naoko; Teshima-Kondo, Shigetada; Masuda, Kiyoshi; Nishida, Kensei; Kuwano, Yuki; Dang, Duyen T; Dang, Long H; Nikawa, Takeshi; Rokutan, Kazuhito

    2013-01-01

    Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF

  1. T Cell Phenotype and T Cell Receptor Repertoire in Patients with Major Depressive Disorder

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    Kostas Patas

    2018-02-01

    Full Text Available While a link between inflammation and the development of neuropsychiatric disorders, including major depressive disorder (MDD is supported by a growing body of evidence, little is known about the contribution of aberrant adaptive immunity in this context. Here, we conducted in-depth characterization of T cell phenotype and T cell receptor (TCR repertoire in MDD. For this cross-sectional case–control study, we recruited antidepressant-free patients with MDD without any somatic or psychiatric comorbidities (n = 20, who were individually matched for sex, age, body mass index, and smoking status to a non-depressed control subject (n = 20. T cell phenotype and repertoire were interrogated using a combination of flow cytometry, gene expression analysis, and next generation sequencing. T cells from MDD patients showed significantly lower surface expression of the chemokine receptors CXCR3 and CCR6, which are known to be central to T cell differentiation and trafficking. In addition, we observed a shift within the CD4+ T cell compartment characterized by a higher frequency of CD4+CD25highCD127low/− cells and higher FOXP3 mRNA expression in purified CD4+ T cells obtained from patients with MDD. Finally, flow cytometry-based TCR Vβ repertoire analysis indicated a less diverse CD4+ T cell repertoire in MDD, which was corroborated by next generation sequencing of the TCR β chain CDR3 region. Overall, these results suggest that T cell phenotype and TCR utilization are skewed on several levels in patients with MDD. Our study identifies putative cellular and molecular signatures of dysregulated adaptive immunity and reinforces the notion that T cells are a pathophysiologically relevant cell population in this disorder.

  2. Contribution of neural cell death to depressive phenotypes of streptozotocin-induced diabetic mice

    Directory of Open Access Journals (Sweden)

    Cheng Chen

    2014-06-01

    Full Text Available Major depression disorder (MDD or depression is highly prevalent in individuals with diabetes, and the depressive symptoms are more severe and less responsive to antidepressant therapies in these patients. The underlying mechanism is little understood. We hypothesized that the pathophysiology of comorbid depression was more complex than that proposed for MDD and that neural cell death played a role in the disease severity. To test this hypothesis, we generated streptozotocin (STZ-induced diabetic mice. These mice had blood glucose levels threefold above controls and exhibited depressive phenotypes as judged by a battery of behavioral tests, thus confirming the comorbidity in mice. Immunohistological studies showed markedly increased TUNEL-positive cells in the frontal cortex and hippocampus of the comorbid mice, indicating apoptosis. This finding was supported by increased caspase-3 and decreased Bcl-2 proteins in these brain regions. In addition, the serum brain-derived neurotrophic factor (BDNF level of comorbid mice was reduced compared with controls, further supporting the neurodegenerative change. Mechanistic analyses showed an increased expression of mitochondrial fission genes fission protein 1 (Fis1 and dynamin-related protein 1 (Drp1, and a decreased expression of mitochondrial fusion genes mitofusin 1 (Mfn1, mitofusin 2 (Mfn2 and optical atrophy 1 (Opa1. Representative assessment of the proteins Drp1 and Mfn2 mirrored the mRNA changes. The data demonstrated that neural cell death was associated with the depressive phenotype of comorbid mice and that a fission-dominant expression of genes and proteins mediating mitochondrial dynamics played a role in the hyperglycemia-induced cell death. The study provides new insight into the disease mechanism and could aid the development of novel therapeutics aimed at providing neuroprotection by modulating mitochondrial dynamics to treat comorbid depression with diabetes.

  3. When stem cells grow old: phenotypes and mechanisms of stem cell aging

    Science.gov (United States)

    Schultz, Michael B.; Sinclair, David A.

    2016-01-01

    All multicellular organisms undergo a decline in tissue and organ function as they age. An attractive theory is that a loss in stem cell number and/or activity over time causes this decline. In accordance with this theory, aging phenotypes have been described for stem cells of multiple tissues, including those of the hematopoietic system, intestine, muscle, brain, skin and germline. Here, we discuss recent advances in our understanding of why adult stem cells age and how this aging impacts diseases and lifespan. With this increased understanding, it is feasible to design and test interventions that delay stem cell aging and improve both health and lifespan. PMID:26732838

  4. Odontoblast-Like Cells Differentiated from Dental Pulp Stem Cells Retain Their Phenotype after Subcultivation

    Directory of Open Access Journals (Sweden)

    Paula A. Baldión

    2018-01-01

    Full Text Available Odontoblasts, the main cell type in teeth pulp tissue, are not cultivable and they are responsible for the first line of response after dental restauration. Studies on dental materials cytotoxicity and odontoblast cells physiology require large quantity of homogenous cells retaining most of the phenotype characteristics. Odontoblast-like cells (OLC were differentiated from human dental pulp stem cells using differentiation medium (containing TGF-β1, and OLC expanded after trypsinization (EXP-21 were evaluated and compared. Despite a slower cell growth curve, EXP-21 cells express similarly the odontoblast markers dentinal sialophosphoprotein and dentin matrix protein-1 concomitantly with RUNX2 transcripts and low alkaline phosphatase activity as expected. Both OLC and EXP-21 cells showed similar mineral deposition activity evidenced by alizarin red and von Kossa staining. These results pointed out minor changes in phenotype of subcultured EXP-21 regarding the primarily differentiated OLC, making the subcultivation of these cells a useful strategy to obtain odontoblasts for biocompatibility or cell physiology studies in dentistry.

  5. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  6. PHENOTYPIC FEATURES OF T REGULATORY CELLS IN EARLY RHEUMATOID ARTHRITIS

    Directory of Open Access Journals (Sweden)

    A. S. Avdeeva

    2016-01-01

    Full Text Available Objective: to investigate the count and characteristics of the phenotype of T regulatory cells (Treg in the peripheral blood of healthy donors and patients with early rheumatoid arthritis (RA, by using multicolor flow cytometry.Subjects and methods. The investigation enrolled 39 patients with early RA. The percentage and absolute count of Treg (FoxP3+CD25+, surface CD152+, intracellular CD152+, FoxP3+CD127, CD25+CD127, FoxP3+ICOS+, FoxP3+CD154+; and FoxP3+CD274+ was determined by multicolor flow-cytometry. A control group consisted of 20 healthy donors matched for sex and age with the examined patients.Results and discussion. In the patients included in the study, the median [25th; 75th percentiles] DAS28 was 5.01 [4.2; 5.8]; high, moderate, and low activity showed 22 (48.9%, 20 (44.4%, and 3 (6.7% patients, respectively. The patients with early RA had a lower percentage of FoxP3+CD25+ cells and a lower percentage and absolute count of FoxP3+ICOS+, FoxP3+CD154+, and FoxP3+CD274+ T cells than the healthy donors (p<0.05 in all cases. There was a negative correlation of the percentage of FoxP3+CD25+ cells with C-reactive protein (CRP (r = -0.4, that of intracellular CD152+ with DAS28 (r = -0.35, erythrocyte sedimentation rate (ESR (r = -0.46, and CRP (r=-0.54; that of FoxP3+CD127 with CRP (r = -0.42; that of CD25+CD127 with DAS28 (r = -0.38, Simplified Disease Activity Index (r = -0.41, Clinical Disease Activity Index (r = -0.36, ESR (r = -0.39, and CRP (r = -0.47 (p < 0.05 in all cases.Conclusion. The findings suggest that the functional activity of Treg is impaired in early RA, which has an impact on the activity of the inflammatory process.

  7. Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture.

    Science.gov (United States)

    Zhou, Haibo; Shi, Jianmin; Zhang, Chao; Li, Pei

    2018-02-28

    Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis. © 2018 The Author(s).

  8. Glioma Cells in the Tumor Periphery Have a Stem Cell Phenotype

    DEFF Research Database (Denmark)

    Munthe, Sune; Petterson, Stine Asferg; Dahlrot, Rikke Hedegaard

    2016-01-01

    and a panel of markers was used. The panel comprised of six stem cell-related markers (CD133, Musashi-1, Bmi-1, Sox-2, Nestin and Glut-3), a proliferation marker (Ki-67) as well as a chemo-resistance marker (MGMT). Computer-based automated classifiers were designed to measure the mIDH1 positive nucleus area......-fraction of the chosen markers. Moreover, orthotopic glioblastoma xenografts from five different patient-derived spheroid cultures were obtained and the tumor cells identified by human specific immunohistochemical markers. The results showed that tumor cells in the periphery of patient gliomas expressed stem cell...... in the periphery of patient gliomas have a stem cell phenotype, although it is less pronounced than in the tumor core. Novel therapies aiming at preventing recurrence should therefore take tumor stemness into account. Migrating cells in orthotopic glioblastoma xenografts preserve expression and stem cell markers...

  9. Dynamic genetic linkage of intermediate blood pressure phenotypes during postural adaptations in a founder population

    Science.gov (United States)

    Arenas, I. A.; Tremblay, J.; Deslauriers, B.; Sandoval, J.; Šeda, O.; Gaudet, D.; Merlo, E.; Kotchen, T.; Cowley, A. W.

    2013-01-01

    Blood pressure (BP) is a dynamic phenotype that varies rapidly to adjust to changing environmental conditions. Standing upright is a recent evolutionary trait, and genetic factors that influence postural adaptations may contribute to BP variability. We studied the effect of posture on the genetics of BP and intermediate BP phenotypes. We included 384 sib-pairs in 64 sib-ships from families ascertained by early-onset hypertension and dyslipidemia. Blood pressure, three hemodynamic and seven neuroendocrine intermediate BP phenotypes were measured with subjects lying supine and standing upright. The effect of posture on estimates of heritability and genetic covariance was investigated in full pedigrees. Linkage was conducted on 196 candidate genes by sib-pair analyses, and empirical estimates of significance were obtained. A permutation algorithm was implemented to study the postural effect on linkage. ADRA1A, APO, CAST, CORIN, CRHR1, EDNRB, FGF2, GC, GJA1, KCNB2, MMP3, NPY, NR3C2, PLN, TGFBR2, TNFRSF6, and TRHR showed evidence of linkage with any phenotype in the supine position and not upon standing, whereas AKR1B1, CD36, EDNRA, F5, MMP9, PKD2, PON1, PPARG, PPARGC1A, PRKCA, and RET were specifically linked to standing phenotypes. Genetic profiling was undertaken to show genetic interactions among intermediate BP phenotypes and genes specific to each posture. When investigators perform genetic studies exclusively on a single posture, important genetic components of BP are missed. Supine and standing BPs have distinct genetic signatures. Standardized maneuvers influence the results of genetic investigations into BP, thus reflecting its dynamic regulation. PMID:23269701

  10. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    Energy Technology Data Exchange (ETDEWEB)

    Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

    2014-04-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

  11. Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

    International Nuclear Information System (INIS)

    Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

    2014-01-01

    Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM TGF , FCM PDGF ) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM B ). FCM TGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM TGF ≫FCM PDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM TGF >FCM PDGF ) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify

  12. Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells

    International Nuclear Information System (INIS)

    Mulvey, Hillary E.; Chang, Audrey; Adler, Jason; Del Tatto, Michael; Perez, Kimberly; Quesenberry, Peter J.; Chatterjee, Devasis

    2015-01-01

    Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype. EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB. This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs. Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer

  13. Phenotypic plasticity despite source-sink population dynamics in a long-lived perennial plant.

    Science.gov (United States)

    Anderson, Jill T; Sparks, Jed P; Geber, Monica A

    2010-11-01

    • Species that exhibit adaptive plasticity alter their phenotypes in response to environmental conditions, thereby maximizing fitness in heterogeneous landscapes. However, under demographic source-sink dynamics, selection should favor traits that enhance fitness in the source habitat at the expense of fitness in the marginal habitat. Consistent with source-sink dynamics, the perennial blueberry, Vaccinium elliottii (Ericaceae), shows substantially higher fitness and population sizes in dry upland forests than in flood-prone bottomland forests, and asymmetrical gene flow occurs from upland populations into bottomland populations. Here, we examined whether this species expresses plasticity to these distinct environments despite source-sink dynamics. • We assessed phenotypic responses to a complex environmental gradient in the field and to water stress in the glasshouse. • Contrary to expectations, V. elliottii exhibited a high degree of plasticity in foliar and root traits (specific leaf area, carbon isotope ratios, foliar nitrogen content, root : shoot ratio, root porosity and root architecture). • We propose that plasticity can be maintained in source-sink systems if it is favored within the source habitat and/or a phylogenetic artifact that is not costly. Additionally, plasticity could be advantageous if habitat-based differences in fitness result from incipient niche expansion. Our results illuminate the importance of evaluating phenotypic traits and fitness components across heterogeneous landscapes. © The Authors (2010). Journal compilation © New Phytologist Trust (2010).

  14. Increased memory phenotypes of CD4+ and CD8+ T cells in ...

    African Journals Online (AJOL)

    Leonard Mboera

    Tanzania Journal of Health Research ... In this study, we investigated the phenotype and activation ... Children with Sickle Cell Anaemia (SCA), the homozygous form of Sickle Cell ..... J.L., Fenton, P., Blumberg, N. & Walters, M.C. (2005) Hematopoietic stem cell ... cell anaemia patients and effects of hydroxyurea therapy.

  15. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion

    DEFF Research Database (Denmark)

    Haack-Sørensen, Mandana; Hansen, Susanne Kofoed; Hansen, Louise

    2013-01-01

    BACKGROUND: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit...... differentiation. This phenotype persisted independent of increasing cell densities. DISCUSSION: These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which...... persists independent of cell density can be used for clinical therapies....

  16. Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

    International Nuclear Information System (INIS)

    Spangrude, G.J.; Scollay, R.

    1990-01-01

    To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes

  17. Spontaneous transformation of human granulosa cell tumours into an aggressive phenotype: a metastasis model cell line

    International Nuclear Information System (INIS)

    Imai, Misa; Muraki, Miho; Takamatsu, Kiyoshi; Saito, Hidekazu; Seiki, Motoharu; Takahashi, Yuji

    2008-01-01

    Granulosa cell tumours (GCTs) are frequently seen in menopausal women and are relatively indolent. Although the physiological properties of normal granulosa cells have been studied extensively, little is known about the molecular mechanism of GCT progression. Here, we characterise the unique behavioural properties of a granulosa tumour cell line, KGN cells, for the molecular analysis of GCT progression. Population doubling was carried out to examine the proliferation capacity of KGN cells. Moreover, the invasive capacity of these cells was determined using the in vitro invasion assay. The expression level of tumour markers in KGN cells at different passages was then determined by Western blot analysis. Finally, the growth and metastasis of KGN cells injected subcutaneously (s.c.) into nude mice was observed 3 months after injection. During in vitro culture, the advanced passage KGN cells grew 2-fold faster than the early passage cells, as determined by the population doubling assay. Moreover, we found that the advanced passage cells were 2-fold more invasive than the early passage cells. The expression pattern of tumour markers, such as p53, osteopontin, BAX and BAG-1, supported the notion that with passage, KGN cells became more aggressive. Strikingly, KGN cells at both early and advanced passages metastasized to the bowel when injected s.c. into nude mice. In addition, more tumour nodules were formed when the advanced passage cells were implanted. KGN cells cultured in vitro acquire an aggressive phenotype, which was confirmed by the analysis of cellular activities and the expression of biomarkers. Interestingly, KGN cells injected s.c. are metastatic with nodule formation occurring mostly in the bowel. Thus, this cell line is a good model for analysing GCT progression and the mechanism of metastasis in vivo

  18. Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells

    DEFF Research Database (Denmark)

    Fornara, O; Bartek, J; Rahbar, A

    2016-01-01

    Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express...... human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary...... GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co...

  19. Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

    Science.gov (United States)

    Li, Pei; Zhang, Ruijie; Wang, Liyuan; Gan, Yibo; Xu, Yuan; Song, Lei; Luo, Lei; Zhao, Chen; Zhang, Chengmin; Ouyang, Bin; Tu, Bing; Zhou, Qiang

    2017-04-30

    Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells. © 2017 The Author(s).

  20. Stem Cell Plasticity and Niche Dynamics in Cancer Progression.

    Science.gov (United States)

    Picco, Noemi; Gatenby, Robert A; Anderson, Alexander R A

    2017-03-01

    Cancer stem cells (CSCs) have been hypothesized to initiate and drive tumor growth and recurrence due to their self-renewal ability. If correct, this hypothesis implies that successful therapy must focus primarily on eradication of this CSC fraction. However, recent evidence suggests stemness is niche dependent and may represent one of many phenotypic states that can be accessed by many cancer genotypes when presented with specific environmental cues. A better understanding of the relationship of stemness to niche-related phenotypic plasticity could lead to alternative treatment strategies. Here, we investigate the role of environmental context in the expression of stem-like cell properties through in-silico simulation of ductal carcinoma. We develop a two-dimensional hybrid discrete-continuum cellular automata model to describe the single-cell scale dynamics of multicellular tissue formation. Through a suite of simulations, we investigate interactions between a phenotypically heterogeneous cancer cell population and a dynamic environment. We generate homeostatic ductal structures that consist of a mixture of stem and differentiated cells governed by both intracellular and environmental dynamics. We demonstrate that a wide spectrum of tumor-like histologies can result from these structures by varying microenvironmental parameters. Niche driven phenotypic plasticity offers a simple first-principle explanation for the diverse ductal structures observed in histological sections from breast cancer. Conventional models of carcinogenesis largely focus on mutational events. We demonstrate that variations in the environmental niche can produce intraductal cancers independent of genetic changes in the resident cells. Therapies targeting the microenvironmental niche may offer an alternative cancer prevention strategy.

  1. Functional and phenotypical analysis of IL-6-secreting CD4+ T cells in human adipose tissue.

    Science.gov (United States)

    de Jong, Anja J; Pollastro, Sabrina; Kwekkeboom, Joanneke C; Andersen, Stefan N; Dorjée, Annemarie L; Bakker, Aleida M; Alzaid, Fawaz; Soprani, Antoine; Nelissen, Rob G H H; Mullers, Jan B; Venteclef, Nicolas; de Vries, Niek; Kloppenburg, Margreet; Toes, René E M; Ioan-Facsinay, Andreea

    2018-03-01

    Emerging evidence indicates that a dynamic interplay between the immune system and adipocytes contributes to the disturbed homeostasis in adipose tissue of obese subjects. Recently, we observed IL-6-secretion by CD4 + T cells from the stromal vascular fraction (SVF) of the infrapatellar fat pad (IFP) of knee osteoarthritis patients directly ex vivo. Here we show that human IL-6 + CD4 + T cells from SVF display a more activated phenotype than the IL-6 - T cells, as evidenced by the expression of the activation marker CD69. Analysis of cytokines secretion, as well as expression of chemokine receptors and transcription factors associated with different Th subsets (Treg, Th1, Th2, Th17 and Tfh) revealed that IL-6-secreting CD4 + T cells cannot be assigned to a conventional Th subset. TCRβ gene analysis revealed that IL-6 + and IL-6 - CD4 + T cells appear clonally unrelated to each other, suggesting a different specificity of these cells. In line with these observations, adipocytes are capable of enhancing IL-6 production by CD4 + T cells. Thus, IL-6 + CD4 + T cells are TCRαβ T cells expressing an activated phenotype potentially resulting from an interplay with adipocytes that could be involved in the inflammatory processes in the OA joint. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Oxygen-related Differences in Cellular and Vesicular Phenotypes Observed for Ovarian Cell Cancer Lines

    Directory of Open Access Journals (Sweden)

    Evo K. Lindersson Søndergaard

    2016-01-01

    The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.

  3. Delayed reproductive death as a dominant phenotype in cell clones surviving X-irradiation

    International Nuclear Information System (INIS)

    Chang, W.P.; Little, J.B.

    1992-01-01

    Residual damage manifested as reduced cloning efficiency was observed in many of the cloned progeny of Chinese hamster ovary (CHO) cells and human carcinoma SQ-20B cells surviving X-irradiation. This stable phenotype, which we have termed delayed reproductive death, persisted for >50 generations of cell replication post-irradiation. Clones showing this phenotype were aneuploid, and formed colonies with a high proportion of giant cells. By somatic cell hybridization of CHO clones, the delayed reproductive death phenotype was found to be a dominant trait; the cloning efficiency of hybrid clones was persistently depressed, as compared with that of control hybrid cells. These results suggest that delayed reproductive death represents a specific cellular response that may persist in some of the progeny of mammalian cells for long periods after X-irradiation. (author)

  4. Mesenchymal Stem Cells Adopt Lung Cell Phenotype in Normal and Radiation-induced Lung Injury Conditions.

    Science.gov (United States)

    Maria, Ola M; Maria, Ahmed M; Ybarra, Norma; Jeyaseelan, Krishinima; Lee, Sangkyu; Perez, Jessica; Shalaby, Mostafa Y; Lehnert, Shirley; Faria, Sergio; Serban, Monica; Seuntjens, Jan; El Naqa, Issam

    2016-04-01

    Lung tissue exposure to ionizing irradiation can invariably occur during the treatment of a variety of cancers leading to increased risk of radiation-induced lung disease (RILD). Mesenchymal stem cells (MSCs) possess the potential to differentiate into epithelial cells. However, cell culture methods of primary type II pneumocytes are slow and cannot provide a sufficient number of cells to regenerate damaged lungs. Moreover, effects of ablative radiation doses on the ability of MSCs to differentiate in vitro into lung cells have not been investigated yet. Therefore, an in vitro coculture system was used, where MSCs were physically separated from dissociated lung tissue obtained from either healthy or high ablative doses of 16 or 20 Gy whole thorax irradiated rats. Around 10±5% and 20±3% of cocultured MSCs demonstrated a change into lung-specific Clara and type II pneumocyte cells when MSCs were cocultured with healthy lung tissue. Interestingly, in cocultures with irradiated lung biopsies, the percentage of MSCs changed into Clara and type II pneumocytes cells increased to 40±7% and 50±6% at 16 Gy irradiation dose and 30±5% and 40±8% at 20 Gy irradiation dose, respectively. These data suggest that MSCs to lung cell differentiation is possible without cell fusion. In addition, 16 and 20 Gy whole thorax irradiation doses that can cause varying levels of RILD, induced different percentages of MSCs to adopt lung cell phenotype compared with healthy lung tissue, providing encouraging outlook for RILD therapeutic intervention for ablative radiotherapy prescriptions.

  5. When stem cells grow old: phenotypes and mechanisms of stem cell aging.

    Science.gov (United States)

    Schultz, Michael B; Sinclair, David A

    2016-01-01

    All multicellular organisms undergo a decline in tissue and organ function as they age. An attractive theory is that a loss in stem cell number and/or activity over time causes this decline. In accordance with this theory, aging phenotypes have been described for stem cells of multiple tissues, including those of the hematopoietic system, intestine, muscle, brain, skin and germline. Here, we discuss recent advances in our understanding of why adult stem cells age and how this aging impacts diseases and lifespan. With this increased understanding, it is feasible to design and test interventions that delay stem cell aging and improve both health and lifespan. © 2016. Published by The Company of Biologists Ltd.

  6. Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Adrian Biddle

    2016-02-01

    Full Text Available Cancer stem cells (CSCs drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT and mesenchymal-to-epithelial transition (MET to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC, we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44highEpCAMlow/−CD24+ cell surface marker profile. Treatment with TGFβ and retinoic acid (RA enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

  7. Phenotypic Plasticity Determines Cancer Stem Cell Therapeutic Resistance in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Biddle, Adrian; Gammon, Luke; Liang, Xiao; Costea, Daniela Elena; Mackenzie, Ian C

    2016-02-01

    Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44(high)EpCAM(low/-) CD24(+) cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

  8. CD4 T-helper cell cytokine phenotypes and antibody response following tetanus toxoid booster immunization

    Science.gov (United States)

    Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10+ cells. Eight healthy subjects were given a TT booster vacci...

  9. The phenotypic equilibrium of cancer cells: From average-level stability to path-wise convergence.

    Science.gov (United States)

    Niu, Yuanling; Wang, Yue; Zhou, Da

    2015-12-07

    The phenotypic equilibrium, i.e. heterogeneous population of cancer cells tending to a fixed equilibrium of phenotypic proportions, has received much attention in cancer biology very recently. In the previous literature, some theoretical models were used to predict the experimental phenomena of the phenotypic equilibrium, which were often explained by different concepts of stabilities of the models. Here we present a stochastic multi-phenotype branching model by integrating conventional cellular hierarchy with phenotypic plasticity mechanisms of cancer cells. Based on our model, it is shown that: (i) our model can serve as a framework to unify the previous models for the phenotypic equilibrium, and then harmonizes the different kinds of average-level stabilities proposed in these models; and (ii) path-wise convergence of our model provides a deeper understanding to the phenotypic equilibrium from stochastic point of view. That is, the emergence of the phenotypic equilibrium is rooted in the stochastic nature of (almost) every sample path, the average-level stability just follows from it by averaging stochastic samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Phenotype Clustering of Breast Epithelial Cells in Confocal Imagesbased on Nuclear Protein Distribution Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Long, Fuhui; Peng, Hanchuan; Sudar, Damir; Levievre, Sophie A.; Knowles, David W.

    2006-09-05

    Background: The distribution of the chromatin-associatedproteins plays a key role in directing nuclear function. Previously, wedeveloped an image-based method to quantify the nuclear distributions ofproteins and showed that these distributions depended on the phenotype ofhuman mammary epithelial cells. Here we describe a method that creates ahierarchical tree of the given cell phenotypes and calculates thestatistical significance between them, based on the clustering analysisof nuclear protein distributions. Results: Nuclear distributions ofnuclear mitotic apparatus protein were previously obtained fornon-neoplastic S1 and malignant T4-2 human mammary epithelial cellscultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 andthe number of days in cultured. A probabilistic ensemble approach wasused to define a set of consensus clusters from the results of multipletraditional cluster analysis techniques applied to the nucleardistribution data. Cluster histograms were constructed to show how cellsin any one phenotype were distributed across the consensus clusters.Grouping various phenotypes allowed us to build phenotype trees andcalculate the statistical difference between each group. The resultsshowed that non-neoplastic S1 cells could be distinguished from malignantT4-2 cells with 94.19 percent accuracy; that proliferating S1 cells couldbe distinguished from differentiated S1 cells with 92.86 percentaccuracy; and showed no significant difference between the variousphenotypes of T4-2 cells corresponding to increasing tumor sizes.Conclusion: This work presents a cluster analysis method that canidentify significant cell phenotypes, based on the nuclear distributionof specific proteins, with high accuracy.

  11. Alterations in Mesenteric Lymph Node T Cell Phenotype and Cytokine Secretion are Associated with Changes in Thymocyte Phenotype after LP-BM5 Retrovirus Infection

    Directory of Open Access Journals (Sweden)

    Maria C. Lopez

    2005-01-01

    Full Text Available In this study, mouse MLN cells and thymocytes from advanced stages of LP-BM5 retrovirus infection were studied. A decrease in the percentage of IL-7+ cells and an increase in the percentage of IL-16+ cells in the MLN indicated that secretion of these cytokines was also altered after LP-BM5 infection. The percentage of MLN T cells expressing IL-7 receptors was significantly reduced, while the percentage of MLN T cells expressing TNFR-p75 and of B cells expressing TNFR-p55 increased. Simultaneous analysis of surface markers and cytokine secretion was done in an attempt to understand whether the deregulation of IFN-Υ secretion could be ascribed to a defined cell phenotype, concluding that all T cell subsets studied increased IFN-Υ secretion after retrovirus infection. Finally, thymocyte phenotype was further analyzed trying to correlate changes in thymocyte phenotype with MLN cell phenotype. The results indicated that the increase in single positive either CD4+CD8- or CD4- CD8+ cells was due to accumulation of both immature (CD3- and mature (CD3+ single positive thymocytes. Moreover, single positive mature thymocytes presented a phenotype similar to the phenotype previously seen on MLN T cells. In summary, we can conclude that LP-BM5 uses the immune system to reach the thymus where it interferes with the generation of functionally mature T cells, favoring the development of T cells with an abnormal phenotype. These new T cells are activated to secrete several cytokines that in turn will favor retrovirus replication and inhibit any attempt of the immune system to control infection.

  12. The Phenotypic Fate of Bone Marrow-Derived Stem Cells in Acute Kidney Injury

    Directory of Open Access Journals (Sweden)

    Guowei Feng

    2013-11-01

    Full Text Available Background: Despite increasing attention on the role of bone marrow derived stem cells in repair or rejuvenation of tissues and organs, cellular mechanisms of such cell-based therapy remain poorly understood. Methods: We reconstituted hematopoiesis in recipient C57BL/6J mice by transplanting syngeneic GFP+ bone marrow (BM cells. Subsequently, the recipients received subcutaneous injection of granulocyte-colony stimulating factor (G-CSF and were subjected to acute renal ischemic injury. Flow cytometry and immunostaining were performed at various time points to assess engraftment and phenotype of BM derived stem cells. Results: Administration of G-CSF increased the release of BM derived stem cells into circulation and enhanced the ensuing recruitment of BM derived stem cells into injured kidney. During the second month post injury, migrated BM derived stem cells lost hematopoietic phenotype (CD45 but maintained the expression of other markers (Sca-1, CD133 and CD44, suggesting their potential of transdifferentiation into renal stem cells. Moreover, G-CSF treatment enhanced the phenotypic conversion. Conclusion: Our work depicted a time-course dependent transition of phenotypic characteristics of BM derived stem cells, demonstrated the existence of BM derived stem cells in damaged kidney and revealed the effects of G-CSF on cell transdifferentiation.

  13. Red blood cell phenotype prevalence in blood donors who self-identify as Hispanic

    DEFF Research Database (Denmark)

    Sheppard, Chelsea A; Bolen, Nicole L; Eades, Beth

    2017-01-01

    CONCLUSIONS: Molecular genotyping platforms provide a quick, high-throughput method for identifying red blood cell units for patients on extended phenotype-matching protocols, such as those with sickle cell disease or thalassemia. Most of the antigen prevalence data reported are for non-Hispanic ......CONCLUSIONS: Molecular genotyping platforms provide a quick, high-throughput method for identifying red blood cell units for patients on extended phenotype-matching protocols, such as those with sickle cell disease or thalassemia. Most of the antigen prevalence data reported are for non...

  14. The emerging phenotype of the testicular carcinoma in situ germ cell

    DEFF Research Database (Denmark)

    Rajpert-De Meyts, Ewa; Bartkova, Jirina; Samson, Michel

    2003-01-01

    This review summarises the existing knowledge on the phenotype of the carcinoma in situ (CIS) cell. CIS is a common pre-invasive precursor of testicular germ cell tumours of adolescents and young adults. These tumours display a variety of histological forms. Classical seminoma proliferates along...... of differentiation and pluripotency, CIS cells found in adult patients seem to be predestined for further malignant progression into one or the other of the two main types of overt tumours. A new concept of phenotypic continuity of differentiation of germ cells along germinal lineage with a gradual loss of embryonic...

  15. Arnold Tongues in Cell Dynamics

    Science.gov (United States)

    Jensen, Mogens

    In a recent work with Leo Kadanoff we studied the synchronization between an internal and an external frequency. One obtains a highly structured diagram with details that in essence are related to the difference between rational and irrational number. The synchronized regions appear as Arnold tongues that widen as the coupling between the frequencies increases. Such tongues have been observed in many physical systems, like in the Libchaber convective cell in the basement of the University of Chicago. In biological systems, where oscillators appear in in a broad variety, very little research on Arnold tongues has been performed. We discuss single cell oscillating dynamics triggered by an external cytokine signal. When this signal is overlaid by an oscillating variation, the two oscillators might couple leading to Arnold tongue diagram. When the tongues overlap, the cell dynamics can shift between the tongues eventually leading to a chaotic response. We quantify such switching in single cell experiments and in model systems based on Gillespie simulations. Kadanoff session.

  16. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

    Science.gov (United States)

    Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

    2015-12-01

    The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  17. Noise genetics: inferring protein function by correlating phenotype with protein levels and localization in individual human cells.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    2014-03-01

    Full Text Available To understand gene function, genetic analysis uses large perturbations such as gene deletion, knockdown or over-expression. Large perturbations have drawbacks: they move the cell far from its normal working point, and can thus be masked by off-target effects or compensation by other genes. Here, we offer a complementary approach, called noise genetics. We use natural cell-cell variations in protein level and localization, and correlate them to the natural variations of the phenotype of the same cells. Observing these variations is made possible by recent advances in dynamic proteomics that allow measuring proteins over time in individual living cells. Using motility of human cancer cells as a model system, and time-lapse microscopy on 566 fluorescently tagged proteins, we found 74 candidate motility genes whose level or localization strongly correlate with motility in individual cells. We recovered 30 known motility genes, and validated several novel ones by mild knockdown experiments. Noise genetics can complement standard genetics for a variety of phenotypes.

  18. Loss of end-differentiated β-cell phenotype following pancreatic islet transplantation.

    Science.gov (United States)

    Anderson, S J; White, M G; Armour, S L; Maheshwari, R; Tiniakos, D; Muller, Y D; Berishvili, E; Berney, T; Shaw, J A M

    2018-03-01

    Replacement of pancreatic β-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β-cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress-induced β-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated β-cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin + /urocortin-3 - cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites, and ultimately fully vascularized bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

  19. Dynamics of sexual populations structured by a space variable and a phenotypical trait

    KAUST Repository

    Mirrahimi, Sepideh

    2013-03-01

    We study sexual populations structured by a phenotypic trait and a space variable, in a non-homogeneous environment. Departing from an infinitesimal model, we perform an asymptotic limit to derive the system introduced in Kirkpatrick and Barton (1997). We then perform a further simplification to obtain a simple model. Thanks to this simpler equation, we can describe rigorously the dynamics of the population. In particular, we provide an explicit estimate of the invasion speed, or extinction speed of the species. Numerical computations show that this simple model provides a good approximation of the original infinitesimal model, and in particular describes quite well the evolution of the species\\' range. © 2013 Elsevier Inc.

  20. Dynamics of sexual populations structured by a space variable and a phenotypical trait

    KAUST Repository

    Mirrahimi, Sepideh; Raoul, Gaë l

    2013-01-01

    We study sexual populations structured by a phenotypic trait and a space variable, in a non-homogeneous environment. Departing from an infinitesimal model, we perform an asymptotic limit to derive the system introduced in Kirkpatrick and Barton (1997). We then perform a further simplification to obtain a simple model. Thanks to this simpler equation, we can describe rigorously the dynamics of the population. In particular, we provide an explicit estimate of the invasion speed, or extinction speed of the species. Numerical computations show that this simple model provides a good approximation of the original infinitesimal model, and in particular describes quite well the evolution of the species' range. © 2013 Elsevier Inc.

  1. Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells

    International Nuclear Information System (INIS)

    Yang, Min; Ma, Bo; Shao, Hanshuang; Clark, Amanda M.; Wells, Alan

    2016-01-01

    Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due

  2. Microendophenotypes of psychiatric disorders: phenotypes of psychiatric disorders at the level of molecular dynamics, synapses, neurons, and neural circuits.

    Science.gov (United States)

    Kida, S; Kato, T

    2015-01-01

    Psychiatric disorders are caused not only by genetic factors but also by complicated factors such as environmental ones. Moreover, environmental factors are rarely quantitated as biological and biochemical indicators, making it extremely difficult to understand the pathological conditions of psychiatric disorders as well as their underlying pathogenic mechanisms. Additionally, we have actually no other option but to perform biological studies on postmortem human brains that display features of psychiatric disorders, thereby resulting in a lack of experimental materials to characterize the basic biology of these disorders. From these backgrounds, animal, tissue, or cell models that can be used in basic research are indispensable to understand biologically the pathogenic mechanisms of psychiatric disorders. In this review, we discuss the importance of microendophenotypes of psychiatric disorders, i.e., phenotypes at the level of molecular dynamics, neurons, synapses, and neural circuits, as targets of basic research on these disorders.

  3. Salivary gland NK cells are phenotypically and functionally unique.

    Directory of Open Access Journals (Sweden)

    Marlowe S Tessmer

    2011-01-01

    Full Text Available Natural killer (NK cells and CD8(+ T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV. However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or T(reg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

  4. Surprisal analysis of Glioblastoma Multiform (GBM) microRNA dynamics unveils tumor specific phenotype.

    Science.gov (United States)

    Zadran, Sohila; Remacle, Francoise; Levine, Raphael

    2014-01-01

    Gliomablastoma multiform (GBM) is the most fatal form of all brain cancers in humans. Currently there are limited diagnostic tools for GBM detection. Here, we applied surprisal analysis, a theory grounded in thermodynamics, to unveil how biomolecule energetics, specifically a redistribution of free energy amongst microRNAs (miRNAs), results in a system deviating from a non-cancer state to the GBM cancer -specific phenotypic state. Utilizing global miRNA microarray expression data of normal and GBM patients tumors, surprisal analysis characterizes a miRNA system response capable of distinguishing GBM samples from normal tissue biopsy samples. We indicate that the miRNAs contributing to this system behavior is a disease phenotypic state specific to GBM and is therefore a unique GBM-specific thermodynamic signature. MiRNAs implicated in the regulation of stochastic signaling processes crucial in the hallmarks of human cancer, dominate this GBM-cancer phenotypic state. With this theory, we were able to distinguish with high fidelity GBM patients solely by monitoring the dynamics of miRNAs present in patients' biopsy samples. We anticipate that the GBM-specific thermodynamic signature will provide a critical translational tool in better characterizing cancer types and in the development of future therapeutics for GBM.

  5. Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

    Science.gov (United States)

    Behzad, A R; Morimoto, K; Gosselink, J; Green, J; Hogg, J C; Hayashi, S

    2006-12-01

    Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.

  6. Uremia modulates the phenotype of aortic smooth muscle cells

    DEFF Research Database (Denmark)

    Madsen, Marie; Pedersen, Annemarie Aarup; Albinsson, Sebastian

    2017-01-01

    the phenotype of aortic SMCs in vivo. METHODS: Moderate uremia was induced by 5/6 nephrectomy in apolipoprotein E knockout (ApoE(-/-)) and wildtype C57Bl/6 mice. Plasma analysis, gene expression, histology, and myography were used to determine uremia-mediated changes in the arterial wall. RESULTS: Induction...... of moderate uremia in ApoE(-/-) mice increased atherosclerosis in the aortic arch en face 1.6 fold (p = 0.04) and induced systemic inflammation. Based on histological analyses of aortic root sections, uremia increased the medial area, while there was no difference in the content of elastic fibers or collagen...... in the aortic media. In the aortic arch, mRNA and miRNA expression patterns were consistent with a uremia-mediated phenotypic modulation of SMCs; e.g. downregulation of myocardin, α-smooth muscle actin, and transgelin; and upregulation of miR146a. Notably, these expression patterns were observed after acute (2...

  7. Phenotypic instability of Saos-2 cells in long-term culture

    International Nuclear Information System (INIS)

    Hausser, Heinz-Juergen; Brenner, Rolf E.

    2005-01-01

    The human osteosarcoma cell line Saos-2 is widely used as a model system for human osteoblastic cells, though its phenotypic stability has not been ascertained. We therefore propagated these cells over 100 passages and compared relevant phenotypic properties. In general, higher passage cells exhibited higher proliferation rates and lower specific alkaline phosphatase activities, though mineralization was significantly more pronounced in cultures of late passage cells. Whereas expression of most genes investigated did not vary profoundly, some genes exhibited remarkable differences. Decorin, for instance, that has been discussed as a regulator of proliferation and mineralization, is strongly expressed only in early passage cells, and two receptors for pleiotrophin and midkine exhibited an almost mutually exclusive expression pattern in early and late passage cells, respectively. Our observations indicate that special care is required when results obtained with Saos-2 cells with different culture history are to be compared

  8. Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping.

    Science.gov (United States)

    Augustsson, Per; Karlsen, Jonas T; Su, Hao-Wei; Bruus, Henrik; Voldman, Joel

    2016-05-16

    Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic focusing of cell lines and leukocytes, showing that acoustic properties provide phenotypic information independent of size.

  9. Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype

    International Nuclear Information System (INIS)

    Puente, Pilar de la; Ludeña, Dolores; López, Marta; Ramos, Jennifer; Iglesias, Javier

    2013-01-01

    Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12 pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering. -- Highlights: ► Low fibrinogen concentration provides a suitable matrix for cell migration and differentiation. ► Autologous fibrin scaffolds is a promising technique in tissue engineering. ► Dermal cells are an easily accessible mesenchymal stem cell source. ► Fibrin scaffolds afforded adipogenic, osteogenic and chondrogenic differentiation.

  10. Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

    Science.gov (United States)

    Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

    2012-10-01

    Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

  11. MicroRNAs define distinct human neuroblastoma cell phenotypes and regulate their differentiation and tumorigenicity

    International Nuclear Information System (INIS)

    Samaraweera, Leleesha; Grandinetti, Kathryn B; Huang, Ruojun; Spengler, Barbara A; Ross, Robert A

    2014-01-01

    Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Thus, miRNAs define distinct NB cell phenotypes

  12. Chronic congestive heart failure is associated with a phenotypic shift of intramyocardial endothelial cells

    NARCIS (Netherlands)

    Marijianowski, M. M.; van Laar, M.; Bras, J.; Becker, A. E.

    1995-01-01

    There is evidence that patients with chronic congestive heart failure have endothelial cell-related abnormalities of the peripheral circulation and the coronary microvasculature. For that reason, we have studied the phenotypic expression of endothelial cells in hearts of patients with congestive

  13. Radiation-induced senescence-like phenotype in proliferating and plateau-phase vascular endothelial cells

    International Nuclear Information System (INIS)

    Igarashi, Kaori; Sakimoto, Ippei; Kataoka, Keiko; Ohta, Keisuke; Miura, Masahiko

    2007-01-01

    The effects of ionizing radiation (IR) on tumor angiogenesis still remain largely unknown. In this study, we found that IR (8 Gy) induces a high-frequency (80-90%) senescence-like phenotype in vascular endothelial cells (ECs) undergoing exponential growth. This finding allowed us to characterize the IR-induced senescence-like (IRSL) phenotype by examining the gene expression profiles and in vitro angiogenic activities of these ECs. The expression levels of genes associated with cell cycle progression and DNA replication were remarkably reduced in the IRSL ECs. Additionally, the in vitro invasion and migration activities of these cells through Matrigel were significantly suppressed. We also found that confluent ECs exhibited a high-frequency IRSL phenotype when they were replated immediately after irradiation, whereas incubation in plateau-phase conditions reduced the induction of this phenotype and enhanced colony formation. The kinetics of DNA double-strand break repair, which showed a faster time course in confluent ECs than in growing ECs, may contribute to the protective mechanism associated with the IRSL phenotype. These results imply that the IRSL phenotype may be important for determining the angiogenic activity of ECs following irradiation. The present study should contribute to the understanding of the effects of IR on tumor angiogenesis

  14. Emergence of cooperation in phenotypically heterogeneous populations: a replicator dynamics analysis

    International Nuclear Information System (INIS)

    Barreira da Silva Rocha, A; Escobedo, R; Laruelle, A

    2015-01-01

    The emergence of cooperation is analyzed in heterogeneous populations where two kinds of individuals exist according to their phenotypic appearance. Phenotype recognition is assumed for all individuals: individuals are able to identify the type of every other individual, but fail to recognize their own type. Individuals thus behave under partial information conditions. The interactions between individuals are described by the snowdrift game, where individuals can either cooperate or defect. The evolution of such populations is studied in the framework of evolutionary game theory by means of the replicator dynamics. Overlapping generations are considered, so the replicator equations are formulated in discrete-time form. The stability analysis of the dynamical system is carried out and a detailed description of the behavior of trajectories starting from the interior of the state-space is given. We find that the four monomorphic states are unstable and that a polymorphic state exists which is a global attractor for non-degenerate initial states of the population. The result for the discrete-time replicator coincides with the one of the continuous case. (paper)

  15. Gigantol Suppresses Cancer Stem Cell-Like Phenotypes in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Narumol Bhummaphan

    2015-01-01

    Full Text Available As cancer stem cells (CSCs contribute to malignancy, metastasis, and relapse of cancers, potential of compound in inhibition of CSCs has garnered most attention in the cancer research as well as drug development fields recently. Herein, we have demonstrated for the first time that gigantol, a pure compound isolated from Dendrobium draconis, dramatically suppressed stem-like phenotypes of human lung cancer cells. Gigantol at nontoxic concentrations significantly reduced anchorage-independent growth and survival of the cancer cells. Importantly, gigantol significantly reduced the ability of the cancer cells to form tumor spheroids, a critical hallmark of CSCs. Concomitantly, the treatment of the compound was shown to reduce well-known lung CSCs markers, including CD133 and ALDH1A1. Moreover, we revealed that gigantol decreased stemness in the cancer cells by suppressing the activation of protein kinase B (Akt signal which in turn decreased the cellular levels of pluripotency and self-renewal factors Oct4 and Nanog. In conclusion, gigantol possesses CSCs suppressing activity which may facilitate the development of this compound for therapeutic approaches by targeting CSCs.

  16. Matrix rigidity regulates cancer cell growth and cellular phenotype.

    Directory of Open Access Journals (Sweden)

    Robert W Tilghman

    2010-09-01

    Full Text Available The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness of the microenvironment and how this response varies among cancer cell lines.In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: "rigidity dependent" (those which show an increase in cell growth as extracellular rigidity is increased, and "rigidity independent" (those which grow equally on both soft and stiff substrates. Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug.These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

  17. Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

    Science.gov (United States)

    Tilghman, Robert W.; Cowan, Catharine R.; Mih, Justin D.; Koryakina, Yulia; Gioeli, Daniel; Slack-Davis, Jill K.; Blackman, Brett R.; Tschumperlin, Daniel J.; Parsons, J. Thomas

    2010-01-01

    Background The mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines. Methodology/Principal Findings In this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: “rigidity dependent” (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug. Conclusions/Significance These observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models. PMID:20886123

  18. Association of Immunological Cell Profiles with Specific Clinical Phenotypes of Scleroderma Disease

    Science.gov (United States)

    Calzada, David; Mayayo, Teodoro; González-Rodríguez, María Luisa; Rabasco, Antonio María; Lahoz, Carlos

    2014-01-01

    This study aimed to search the correlation among immunological profiles and clinical phenotypes of scleroderma in well-characterized groups of scleroderma patients, comparing forty-nine scleroderma patients stratified according to specific clinical phenotypes with forty-nine healthy controls. Five immunological cell subpopulations (B, CD4+ and CD8+ T-cells, NK, and monocytes) and their respective stages of apoptosis and activation were analyzed by flow cytometry, in samples of peripheral blood mononuclear cells (PBMCs). Analyses of results were stratified according to disease stage, time since the diagnosis, and visceral damage (pulmonary fibrosis, pulmonary hypertension, and cardiac affliction) and by time of treatment with corticosteroids. An increase in the percentages of monocytes and a decrease in the B cells were mainly related to the disease progression. A general apoptosis decrease was found in all phenotypes studied, except in localized scleroderma. An increase of B and NK cells activation was found in patients diagnosed more than 10 years ago. Specific cell populations like monocytes, NK, and B cells were associated with the type of affected organ. This study shows how, in a heterogeneous disease, proper patient's stratification according to clinical phenotypes allows finding specific cellular profiles. Our data may lead to improvements in the knowledge of prognosis factors and to aid in the analysis of future specific therapies. PMID:24818126

  19. Physical constraints in cell fate specification. A case in point: Microgravity and phenotypes differentiation.

    Science.gov (United States)

    Masiello, Maria Grazia; Verna, Roberto; Cucina, Alessandra; Bizzarri, Mariano

    2018-05-01

    Data obtained by studying mammalian cells in absence of gravity strongly support the notion that cell fate specification cannot be understood according to the current molecular model. A paradigmatic case in point is provided by studying cell populations growing in absence of gravity. When the physical constraint (gravity) is 'experimentally removed', cells spontaneously allocate into two morphologically different phenotypes. Such phenomenon is likely enacted by the intrinsic stochasticity, which, in turn, is successively 'canalized' by a specific gene regulatory network. Both phenotypes are thermodynamically and functionally 'compatibles' with the new, modified environment. However, when the two cell subsets are reseeded into the 1g gravity field the two phenotypes collapse into one. Gravity constraints the system in adopting only one phenotype, not by selecting a pre-existing configuration, but more precisely shaping it de-novo through the modification of the cytoskeleton three-dimensional structure. Overall, those findings highlight how macro-scale features are irreducible to lower-scale explanations. The identification of macroscale control parameters - as those depending on the field (gravity, electromagnetic fields) or emerging from the cooperativity among the field's components (tissue stiffness, cell-to-cell connectivity) - are mandatory for assessing boundary conditions for models at lower scales, thus providing a concrete instantiation of top-down effects. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    International Nuclear Information System (INIS)

    Doherty, R.E.; Haywood-Small, S.L.; Sisley, K.; Cross, N.A.

    2011-01-01

    Highlights: ► Isolated ALDH Hi PC3 cells preferentially form primitive holoclone-type colonies. ► Primitive holoclone colonies are predominantly ALDH Lo but contain rare ALDH Hi cells. ► Holoclone-forming cells are not restricted to the ALDH Hi population. ► ALDH phenotypic plasticity occurs in PC3 cells (ALDH Lo to ALDH Hi and vice versa). ► ALDH Hi cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH Lo cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH Hi population, or whether all ALDH Hi cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH Hi cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH Hi cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH Lo population can develop ALDH Hi populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH Hi cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH Hi status enriches for holoclone formation, this activity may be mediated by a minority of ALDH Hi cells.

  1. GenomeRNAi: a database for cell-based RNAi phenotypes.

    Science.gov (United States)

    Horn, Thomas; Arziman, Zeynep; Berger, Juerg; Boutros, Michael

    2007-01-01

    RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at http://rnai.dkfz.de.

  2. A platform for high-throughput bioenergy production phenotype characterization in single cells

    Science.gov (United States)

    Kelbauskas, Laimonas; Glenn, Honor; Anderson, Clifford; Messner, Jacob; Lee, Kristen B.; Song, Ganquan; Houkal, Jeff; Su, Fengyu; Zhang, Liqiang; Tian, Yanqing; Wang, Hong; Bussey, Kimberly; Johnson, Roger H.; Meldrum, Deirdre R.

    2017-01-01

    Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype. The platform comprises two major components: a tandem optical sensor for combined oxygen and pH detection, and a microwell device for isolation and analysis of single and few cells in hermetically sealed sub-nanoliter chambers. Our approach revealed subpopulations of cells with aberrant energy production profiles and enables determination of cellular response variability to electron transfer chain inhibitors and ion uncouplers. PMID:28349963

  3. Biomimetic brain tumor niche regulates glioblastoma cells towards a cancer stem cell phenotype.

    Science.gov (United States)

    Liu, Yung-Chiang; Lee, I-Chi; Chen, Pin-Yuan

    2018-05-01

    Glioblastoma (GBM) is the most malignant primary brain tumor and contains tumorigenic cancer stem cells (CSCs), which support the progression of tumor growth. The selection of CSCs and facilitation of the brain tumor niches may assist the development of novel therapeutics for GBM. Herein, hydrogel materials composed of agarose and hydroxypropyl methyl cellulose (HMC) in different concentrations were established and compared to emulate brain tumor niches and CSC microenvironments within a label-free system. Human GBM cell line, U-87 MG, was cultured on a series of HMC-agarose based culture system. Cell aggregation and spheroids formation were investigated after 4 days of culture, and 2.5% HMC-agarose based culture system demonstrated the largest spheroids number and size. Moreover, CD133 marker expression of GBM cells after 6 days of culture in 2.5% HMC-agarose based culture system was 60%, relatively higher than the control group at only 15%. Additionally, cells on 2.5% HMC-agarose based culture system show the highest chemoresistance, even at the high dose of 500 µM temozolomide for 72 h, the live cell ratio was still > 80%. Furthermore, the results also indicate that the expression of ABCG2 gene was up-regulated after culture in 2.5% HMC-agarose based culture system. Therefore, our results demonstrated that biomimetic brain tumor microenvironment may regulate GBM cells towards the CSC phenotype and expression of CSC characteristics. The microenvironment selection and spheroids formation in HMC-agarose based culture system may provide a label-free CSC selection strategy and drug testing model for future biomedical applications.

  4. Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

    Science.gov (United States)

    Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason; Ng, Cherie T; Krogan, Nevan; Sharan, Roded; Ideker, Trey

    2016-02-24

    Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

  5. Biophysical subsets of embryonic stem cells display distinct phenotypic and morphological signatures.

    Directory of Open Access Journals (Sweden)

    Tom Bongiorno

    Full Text Available The highly proliferative and pluripotent characteristics of embryonic stem cells engender great promise for tissue engineering and regenerative medicine, but the rapid identification and isolation of target cell phenotypes remains challenging. Therefore, the objectives of this study were to characterize cell mechanics as a function of differentiation and to employ differences in cell stiffness to select population subsets with distinct mechanical, morphological, and biological properties. Biomechanical analysis with atomic force microscopy revealed that embryonic stem cells stiffened within one day of differentiation induced by leukemia inhibitory factor removal, with a lagging but pronounced change from spherical to spindle-shaped cell morphology. A microfluidic device was then employed to sort a differentially labeled mixture of pluripotent and differentiating cells based on stiffness, resulting in pluripotent cell enrichment in the soft device outlet. Furthermore, sorting an unlabeled population of partially differentiated cells produced a subset of "soft" cells that was enriched for the pluripotent phenotype, as assessed by post-sort characterization of cell mechanics, morphology, and gene expression. The results of this study indicate that intrinsic cell mechanical properties might serve as a basis for efficient, high-throughput, and label-free isolation of pluripotent stem cells, which will facilitate a greater biological understanding of pluripotency and advance the potential of pluripotent stem cell differentiated progeny as cell sources for tissue engineering and regenerative medicine.

  6. Inflammatory cell phenotypes in AAAs: their role and potential as targets for therapy.

    Science.gov (United States)

    Dale, Matthew A; Ruhlman, Melissa K; Baxter, B Timothy

    2015-08-01

    Abdominal aortic aneurysms (AAAs) are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused ≈15 000 deaths annually in the United States. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4(+) T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Proinflammatory CD4(+) T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to proinflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the proinflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention. © 2015 American Heart Association, Inc.

  7. Inflammatory cell phenotypes in AAAs; their role and potential as targets for therapy

    Science.gov (United States)

    Dale, Matthew A; Ruhlman, Melissa K.; Baxter, B. Timothy

    2015-01-01

    Abdominal aortic aneurysms are characterized by chronic inflammatory cell infiltration. AAA is typically an asymptomatic disease and caused approximately 15,000 deaths annually in the U.S. Previous studies have examined both human and murine aortic tissue for the presence of various inflammatory cell types. Studies show that in both human and experimental AAAs, prominent inflammatory cell infiltration, such as CD4+ T cells and macrophages, occurs in the damaged aortic wall. These cells have the ability to undergo phenotypic modulation based on microenvironmental cues, potentially influencing disease progression. Pro-inflammatory CD4+ T cells and classically activated macrophages dominate the landscape of aortic infiltrates. The skew to pro-inflammatory phenotypes alters disease progression and plays a role in causing chronic inflammation. The local cytokine production and presence of inflammatory mediators, such as extracellular matrix breakdown products, influence the uneven balance of the inflammatory infiltrate phenotypes. Understanding and developing new strategies that target the pro-inflammatory phenotype could provide useful therapeutic targets for a disease with no current pharmacological intervention. PMID:26044582

  8. Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT)-Phenotypic Cells: Are They Cousins or Twins?

    International Nuclear Information System (INIS)

    Kong, Dejuan; Li, Yiwei; Wang, Zhiwei; Sarkar, Fazlul H.

    2011-01-01

    Cancer stem cells (CSCs) are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT), induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors

  9. Cancer Stem Cells and Epithelial-to-Mesenchymal Transition (EMT-Phenotypic Cells: Are They Cousins or Twins?

    Directory of Open Access Journals (Sweden)

    Fazlul H. Sarkar

    2011-02-01

    Full Text Available Cancer stem cells (CSCs are cells within a tumor that possess the capacity to self-renew and maintain tumor-initiating capacity through differentiation into the heterogeneous lineages of cancer cells that comprise the whole tumor. These tumor-initiating cells could provide a resource for cells that cause tumor recurrence after therapy. Although the cell origin of CSCs remains to be fully elucidated, mounting evidence has demonstrated that Epithelial-to-Mesenchymal Transition (EMT, induced by different factors, is associated with tumor aggressiveness and metastasis and these cells share molecular characteristics with CSCs, and thus are often called cancer stem-like cells or tumor-initiating cells. The acquisition of an EMT phenotype is a critical process for switching early stage carcinomas into invasive malignancies, which is often associated with the loss of epithelial differentiation and gain of mesenchymal phenotype. Recent studies have demonstrated that EMT plays a critical role not only in tumor metastasis but also in tumor recurrence and that it is tightly linked with the biology of cancer stem-like cells or cancer-initiating cells. Here we will succinctly summarize the state-of-our-knowledge regarding the molecular similarities between cancer stem-like cells or CSCs and EMT-phenotypic cells that are associated with tumor aggressiveness focusing on solid tumors.

  10. Antiproliferative activity and phenotypic modification induced by selected Peruvian medicinal plants on human hepatocellular carcinoma Hep3B cells.

    Science.gov (United States)

    Carraz, Maëlle; Lavergne, Cédric; Jullian, Valérie; Wright, Michel; Gairin, Jean Edouard; Gonzales de la Cruz, Mercedes; Bourdy, Geneviève

    2015-05-26

    human hepatocytes in vitro. Immunofluorescence experiments on Hep3B cells showed that crude extracts of Schkuhria pinnata and Orthrosanthus chimboracensis could block Hep3B cells in mitosis with an original phenotype. Crude extracts of Perezia coerulescens, Perezia multiflora, Achyrocline alata, Ophryosporus chilca, Otholobium pubescens and Hypericum laricifolium could modify the overall microtubule cytoskeletal dynamics of Hep3B cells in interphase by an original mechanism. Our method allowed us to select 9 extracts which displayed antiproliferative activities associated with original cellular phenotypes on Hep3B cells, regarding known microtubule-targeting drugs. Both chemical and cellular studies are ongoing in order to elucidate natural compounds and cellular mechanisms responsible of the activities described. Copyright © 2015. Published by Elsevier Ireland Ltd.

  11. In vitro analysis of the invasive phenotype of SUM 149, an inflammatory breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Dharmawardhane Suranganie F

    2005-04-01

    Full Text Available Abstract Background Inflammatory breast cancer (IBC is the most lethal form of locally invasive breast cancer known. However, very little information is available on the cellular mechanisms responsible for manifestation of the IBC phenotype. To understand the unique phenotype of IBC, we compared the motile and adhesive interactions of an IBC cell line, SUM 149, to the non-IBC cell line SUM 102. Results Our results demonstrate that both IBC and non-IBC cell lines exhibit similar adhesive properties to basal lamina, but SUM 149 showed a marked increase in adhesion to collagen I. In vitro haptotaxis assays demonstrate that SUM 149 was less invasive, while wound healing assays show a less in vitro migratory phenotype for SUM 149 cells relative to SUM 102 cells. We also demonstrate a role for Rho and E-cadherin in the unique invasive phenotype of IBC. Immunoblotting reveals higher E-cadherin and RhoA expression in the IBC cell line but similar RhoC expression. Rhodamine phalloidin staining demonstrates increased formation of actin stress fibers and larger focal adhesions in SUM 149 relative to the SUM 102 cell line. Conclusion The observed unique actin and cellular architecture as well as the invasive and adhesive responses to the extracellular matrix of SUM 149 IBC cells suggest that the preference of IBC cells for connective tissue, possibly a mediator important for the vasculogenic mimicry via tubulogenesis seen in IBC pathological specimens. Overexpression of E-cadherin and RhoA may contribute to passive dissemination of IBC by promoting cell-cell adhesion and actin cytoskeletal structures that maintain tissue integrity. Therefore, we believe that these findings indicate a passive metastatic mechanism by which IBC cells invade the circulatory system as tumor emboli rather than by active migratory mechanisms.

  12. Dynamic instability of genomic methylation patterns in pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Ooi Steen KT

    2010-09-01

    Full Text Available Abstract Background Genomic methylation patterns are established during gametogenesis, and perpetuated in somatic cells by faithful maintenance methylation. There have been previous indications that genomic methylation patterns may be less stable in embryonic stem (ES cells than in differentiated somatic cells, but it is not known whether different mechanisms of de novo and maintenance methylation operate in pluripotent stem cells compared with differentiating somatic cells. Results In this paper, we show that ablation of the DNA methyltransferase regulator DNMT3L (DNA methyltransferase 3-like in mouse ES cells renders them essentially incapable of de novo methylation of newly integrated retroviral DNA. We also show that ES cells lacking DNMT3L lose DNA methylation over time in culture, suggesting that DNA methylation in ES cells is the result of dynamic loss and gain of DNA methylation. We found that wild-type female ES cells lose DNA methylation at a much faster rate than do male ES cells; this defect could not be attributed to sex-specific differences in expression of DNMT3L or of any DNA methyltransferase. We also found that human ES and induced pluripotent stem cell lines showed marked but variable loss of methylation that could not be attributed to sex chromosome constitution or time in culture. Conclusions These data indicate that DNA methylation in pluripotent stem cells is much more dynamic and error-prone than is maintenance methylation in differentiated cells. DNA methylation requires DNMT3L in stem cells, but DNMT3L is not expressed in differentiating somatic cells. Error-prone maintenance methylation will introduce unpredictable phenotypic variation into clonal populations of pluripotent stem cells, and this variation is likely to be much more pronounced in cultured female cells. This epigenetic variability has obvious negative implications for the clinical applications of stem cells.

  13. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Marie E. Legare, Jamie Bush, Amanda K. Ashley, Taka Kato, William H. Hanneman

    2011-01-01

    Full Text Available Canine and human osteosarcoma (OSA have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

  14. High-Dimensional Phenotyping Identifies Age-Emergent Cells in Human Mammary Epithelia

    Directory of Open Access Journals (Sweden)

    Fanny A. Pelissier Vatter

    2018-04-01

    Full Text Available Summary: Aging is associated with tissue-level changes in cellular composition that are correlated with increased susceptibility to disease. Aging human mammary tissue shows skewed progenitor cell potency, resulting in diminished tumor-suppressive cell types and the accumulation of defective epithelial progenitors. Quantitative characterization of these age-emergent human cell subpopulations is lacking, impeding our understanding of the relationship between age and cancer susceptibility. We conducted single-cell resolution proteomic phenotyping of healthy breast epithelia from 57 women, aged 16–91 years, using mass cytometry. Remarkable heterogeneity was quantified within the two mammary epithelial lineages. Population partitioning identified a subset of aberrant basal-like luminal cells that accumulate with age and originate from age-altered progenitors. Quantification of age-emergent phenotypes enabled robust classification of breast tissues by age in healthy women. This high-resolution mapping highlighted specific epithelial subpopulations that change with age in a manner consistent with increased susceptibility to breast cancer. : Vatter et al. find that single-cell mass cytometry of human mammary epithelial cells from 57 women, from 16 to 91 years old, depicts an in-depth phenotyping of aging mammary epithelia. Subpopulations of altered luminal and progenitor cells that accumulate with age may be at increased risk for oncogenic transformation. Keywords: human mammary epithelia, aging, mass cytometry, single-cell analysis, heterogeneity, breast cancer

  15. Role of Receptor Sialylation in the Ovarian Tumor Cell Phenotype

    Science.gov (United States)

    2014-08-01

    Chemical Society meeting, Baton Rouge, LA, Nov 2012; (3) 19th World Congress on Advances in Oncology conference, Athens, Greece, scheduled for Oct, 2014...dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 2009;69

  16. Condensing Raman spectrum for single-cell phenotype analysis

    KAUST Repository

    Sun, Shiwei

    2015-12-09

    Background In recent years, high throughput and non-invasive Raman spectrometry technique has matured as an effective approach to identification of individual cells by species, even in complex, mixed populations. Raman profiling is an appealing optical microscopic method to achieve this. To fully utilize Raman proling for single-cell analysis, an extensive understanding of Raman spectra is necessary to answer questions such as which filtering methodologies are effective for pre-processing of Raman spectra, what strains can be distinguished by Raman spectra, and what features serve best as Raman-based biomarkers for single-cells, etc. Results In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication.

  17. Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based Heterogeneity in Phenotype and Frequency

    DEFF Research Database (Denmark)

    Simoni, Yannick; Fehlings, Michael; Kloverpris, Henrik N.

    2017-01-01

    Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors...... to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra-epithelial (ie)ILC1-like cells...... that represent a broader category of NK cells in mucosal and non-mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals...

  18. Trading in your spindles for blebs: the amoeboid tumor cell phenotype in prostate cancer

    Directory of Open Access Journals (Sweden)

    Samantha Morley

    2014-08-01

    Full Text Available Prostate cancer (PCa remains a principal cause of mortality in developed countries. Because no clinical interventions overcome resistance to androgen ablation therapy, management of castration resistance and metastatic disease remains largely untreatable. Metastasis is a multistep process in which tumor cells lose cell-cell contacts, egress from the primary tumor, intravasate, survive shear stress within the vasculature and extravasate into tissues to colonize ectopic sites. Tumor cells reestablish migratory behaviors employed during nonneoplastic processes such as embryonic development, leukocyte trafficking and wound healing. While mesenchymal motility is an established paradigm of dissemination, an alternate, 'amoeboid' phenotype is increasingly appreciated as relevant to human cancer. Here we discuss characteristics and pathways underlying the phenotype, and highlight our findings that the cytoskeletal regulator DIAPH3 governs the mesenchymal-amoeboid transition. We also describe our identification of a new class of tumor-derived microvesicles, large oncosomes, produced by amoeboid cells and with potential clinical utility in prostate and other cancers.

  19. PLACENTAL SECRETORY FACTORS INFLUENCE TO THP-1 CELLS PHENOTYPE AND THP-1 CELLS TRANSENDOTHELIAL MIGRATION

    Directory of Open Access Journals (Sweden)

    O. I. Stepanova

    2013-01-01

    Full Text Available Decidual and placental macrophage pools are renewed due to its transendothelial monocyte migration from peripheral blood. Tissue macrophages control placental development and provide fetomaternal immunological tolerance. Preeclamptic pregnancy is accompanied by increased monocyte migration to decidual tissue and local inflammatory events. Regulatory mechanisms of monocyte recruitment to placental and decidual tissues is still unclear. Therefore we investigated the influence soluble placental factors (SPFs during the first- and third-trimester normal pregnancy, as compared to effects of these factors in preeclamptic pregnancy. We studied biological actions of SPF upon transendothelial migration of monocyte-like THP-1 cells and their phenotypic pattern. Transendothelial migration of THP-1 cells was more intensive with firsttrimester SPFs from normal pregnancy, when compared with third-trimester samples, and it was accompanied by decreased CD11a expression. SPFs from pre-eclamptic pregnancy caused an increase in transendothelial migration of THP-1 cells, as compared to SPFs from normal pregnancies, being accompanied by increased CD11b expression. The present study was supported by grants ГК №  02.740.11.0711, НШ-3594.2010.7, МД-150.2011.7 and a grant from St.-Petersburg Goverment for young scientists.

  20. Phenotypic characterization of thymic prelymphoma cells of B10 mice treated with split-dose irradiation

    International Nuclear Information System (INIS)

    Muto, M.; Kubo, E.; Kamisaku, H.; Sado, T.

    1990-01-01

    Using an intrathymic injection assay on B10 Thy-1 congenic mice, it was demonstrated that thymic prelymphoma cells first developed within the thymuses from 4 to 8 days after split-dose irradiation and were detected in more than 63% of the test donor thymuses when examined at 21 and 31 days after irradiation. Moreover, some mice (25%) at 2 mo after split-dose irradiation had already developed thymic lymphomas in their thymuses. To characterize these thymic prelymphoma cells, the thymocytes from B10 Thy-1.1 mice 1 mo after irradiation were stained with anti-CD4 and anti-CD8 mAb and were sorted into four subpopulations. These fractionated cells were injected into the recipient thymuses to examine which subpopulation contained thymic prelymphoma cells. The results indicated that thymic prelymphoma cells existed mainly in CD4- CD8- and CD4- CD8+ thymocyte subpopulations and also in CD4+ CD8+ subpopulation. T cell lymphomas derived from CD4- CD8- prelymphoma cells had mainly CD4- CD8- or CD4- CD8+ phenotypes. T cell lymphomas developed from CD4- CD8+ prelymphoma cells mainly expressed CD4- CD8+ or CD4+ CD8+ phenotype. T cell lymphomas originating from CD4+ CD8+ prelymphoma cells were mainly CD4+ CD8+ but some CD4- CD8+ or CD4+ CD8- cells were also present. These thymic prelymphoma cells were further characterized phenotypically in relation to their expression of the marker defined by the mAb against J11d marker and TL-2 (thymus-leukemia) Ag, which is not expressed on normal thymocytes of B10.Thy-1.2 or B10.Thy-1.1 strain, but appears on the thymocytes of lymphomagenic irradiated mice. The results indicated that the prelymphoma cells existed in J11d+, TL-2+ cells

  1. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-01-01

    Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  2. Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sontag, Ryan L.; Weber, Thomas J.

    2012-05-04

    In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediated toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.

  3. Nonmyeloablative HLA-matched sibling allogeneic hematopoietic stem cell transplantation for severe sickle cell phenotype.

    Science.gov (United States)

    Hsieh, Matthew M; Fitzhugh, Courtney D; Weitzel, R Patrick; Link, Mary E; Coles, Wynona A; Zhao, Xiongce; Rodgers, Griffin P; Powell, Jonathan D; Tisdale, John F

    2014-07-02

    Myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) is curative for children with severe sickle cell disease, but toxicity may be prohibitive for adults. Nonmyeloablative transplantation has been attempted with degrees of preparative regimen intensity, but graft rejection and graft-vs-host disease remain significant. To determine the efficacy, safety, and outcome on end-organ function with this low-intensity regimen for sickle cell phenotype with or without thalassemia. From July 16, 2004, to October 25, 2013, 30 patients aged 16-65 years with severe disease enrolled in this nonmyeloablative transplant study, consisting of alemtuzumab (1 mg/kg in divided doses), total-body irradiation (300 cGy), sirolimus, and infusion of unmanipulated filgrastim mobilized peripheral blood stem cells (5.5-31.7 × 10(6) cells/kg) from human leukocyte antigen-matched siblings. The primary end point was treatment success at 1 year after the transplant, defined as a full donor-type hemoglobin for patients with sickle cell disease and transfusion independence for patients with thalassemia. The secondary end points were the level of donor leukocyte chimerism; incidence of acute and chronic graft-vs-host disease; and sickle cell-thalassemia disease-free survival, immunologic recovery, and changes in organ function, assessed by annual brain imaging, pulmonary function, echocardiographic image, and laboratory testing. Twenty-nine patients survived a median 3.4 years (range, 1-8.6), with no nonrelapse mortality. One patient died from intracranial bleeding after relapse. As of October 25, 2013, 26 patients (87%) had long-term stable donor engraftment without acute or chronic graft-vs-host disease. The mean donor T-cell level was 48% (95% CI, 34%-62%); the myeloid chimerism levels, 86% (95% CI, 70%-100%). Fifteen engrafted patients discontinued immunosuppression medication with continued stable donor chimerism and no graft-vs-host disease. The normalized hemoglobin and

  4. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.

    2010-01-01

    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  5. Step-wise and punctuated genome evolution drive phenotype changes of tumor cells

    International Nuclear Information System (INIS)

    Stepanenko, Aleksei; Andreieva, Svitlana; Korets, Kateryna; Mykytenko, Dmytro; Huleyuk, Nataliya; Vassetzky, Yegor; Kavsan, Vadym

    2015-01-01

    Highlights: • There are the step-wise continuous and punctuated phases of cancer genome evolution. • The system stresses during the different phases may lead to very different responses. • Stable transfection of an empty vector can result in genome and phenotype changes. • Functions of a (trans)gene can be opposite/versatile in cells with different genomes. • Contextually, temozolomide can both promote and suppress tumor cell aggressiveness. - Abstract: The pattern of genome evolution can be divided into two phases: the step-wise continuous phase (step-wise clonal evolution, stable dominant clonal chromosome aberrations (CCAs), and low frequency of non-CCAs, NCCAs) and punctuated phase (marked by elevated NCCAs and transitional CCAs). Depending on the phase, system stresses (the diverse CIN promoting factors) may lead to the very different phenotype responses. To address the contribution of chromosome instability (CIN) to phenotype changes of tumor cells, we characterized CCAs/NCCAs of HeLa and HEK293 cells, and their derivatives after genotoxic stresses (a stable plasmid transfection, ectopic expression of cancer-associated CHI3L1 gene or treatment with temozolomide) by conventional cytogenetics, copy number alterations (CNAs) by array comparative genome hybridization, and phenotype changes by cell viability and soft agar assays. Transfection of either the empty vector pcDNA3.1 or pcDNA3.1-CHI3L1 into 293 cells initiated the punctuated genome changes. In contrast, HeLa-CHI3L1 cells demonstrated the step-wise genome changes. Increased CIN correlated with lower viability of 293-pcDNA3.1 cells but higher colony formation efficiency (CFE). Artificial CHI3L1 production in 293-CHI3L1 cells increased viability and further contributed to CFE. The opposite growth characteristics of 293-CHI3L1 and HeLa-CHI3L1 cells were revealed. The effect and function of a (trans)gene can be opposite and versatile in cells with different genetic network, which is defined by

  6. Step-wise and punctuated genome evolution drive phenotype changes of tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Stepanenko, Aleksei, E-mail: a.a.stepanenko@gmail.com [Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03680 (Ukraine); Andreieva, Svitlana; Korets, Kateryna; Mykytenko, Dmytro [Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03680 (Ukraine); Huleyuk, Nataliya [Institute of Hereditary Pathology, National Academy of Medical Sciences of Ukraine, Lviv 79008 (Ukraine); Vassetzky, Yegor [CNRS UMR8126, Université Paris-Sud 11, Institut de Cancérologie Gustave Roussy, Villejuif 94805 (France); Kavsan, Vadym [Department of Biosynthesis of Nucleic Acids, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03680 (Ukraine)

    2015-01-15

    Highlights: • There are the step-wise continuous and punctuated phases of cancer genome evolution. • The system stresses during the different phases may lead to very different responses. • Stable transfection of an empty vector can result in genome and phenotype changes. • Functions of a (trans)gene can be opposite/versatile in cells with different genomes. • Contextually, temozolomide can both promote and suppress tumor cell aggressiveness. - Abstract: The pattern of genome evolution can be divided into two phases: the step-wise continuous phase (step-wise clonal evolution, stable dominant clonal chromosome aberrations (CCAs), and low frequency of non-CCAs, NCCAs) and punctuated phase (marked by elevated NCCAs and transitional CCAs). Depending on the phase, system stresses (the diverse CIN promoting factors) may lead to the very different phenotype responses. To address the contribution of chromosome instability (CIN) to phenotype changes of tumor cells, we characterized CCAs/NCCAs of HeLa and HEK293 cells, and their derivatives after genotoxic stresses (a stable plasmid transfection, ectopic expression of cancer-associated CHI3L1 gene or treatment with temozolomide) by conventional cytogenetics, copy number alterations (CNAs) by array comparative genome hybridization, and phenotype changes by cell viability and soft agar assays. Transfection of either the empty vector pcDNA3.1 or pcDNA3.1-CHI3L1 into 293 cells initiated the punctuated genome changes. In contrast, HeLa-CHI3L1 cells demonstrated the step-wise genome changes. Increased CIN correlated with lower viability of 293-pcDNA3.1 cells but higher colony formation efficiency (CFE). Artificial CHI3L1 production in 293-CHI3L1 cells increased viability and further contributed to CFE. The opposite growth characteristics of 293-CHI3L1 and HeLa-CHI3L1 cells were revealed. The effect and function of a (trans)gene can be opposite and versatile in cells with different genetic network, which is defined by

  7. Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

    Directory of Open Access Journals (Sweden)

    Katarzyna Wachowicz

    Full Text Available Protein kinase C θ (PKCθ is involved in signaling downstream of the T cell antigen receptor (TCR and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures and in vivo (experimental autoimmune encephalomyelitis model, EAE techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt, accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ-/- CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.

  8. Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment

    Czech Academy of Sciences Publication Activity Database

    Veselý, Pavel; Blase, C.; Matoušková, Eva; Bereiter-Hahn, J.

    2006-01-01

    Roč. 26, - (2006), s. 967-972 ISSN 0250-7005 R&D Projects: GA MZd(CZ) NR8145 Institutional research plan: CEZ:AV0Z50520514 Keywords : podosomes * neoplastic cell morphotype * phenotypic plasticity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.479, year: 2006

  9. IGF-1 has plaque-stabilizing effects in atherosclerosis by altering vascular smooth muscle cell phenotype

    NARCIS (Netherlands)

    von der Thüsen, Jan H.; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J. C.; Biessen, Erik A. L.

    2011-01-01

    Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic

  10. Increased memory phenotypes of CD4+ and CD8+ T cells in ...

    African Journals Online (AJOL)

    Conclusions: Children with SCA in Tanzania show an absolute increase in all leukocyte types, including lymphocytes, with skewing of both CD4+ and CD8+ T cells towards the memory phenotypes. These findings provide insights on the development of adaptive immunity which may have implications on vaccine ...

  11. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes

    DEFF Research Database (Denmark)

    Santos Delgado, Alberto; Wernersson, Rasmus; Jensen, Lars Juhl

    2015-01-01

    3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNAand protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web interface...

  12. Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping.

    Science.gov (United States)

    Juul, Troels; Palm, Fredrik; Nielsen, Per Mose; Bertelsen, Lotte Bonde; Laustsen, Christoffer

    2017-08-01

    It has been demonstrated that hyperpolarized 13 C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13 C MR is suitable for cells isolated from kidney tissue, without prior cell culture. Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13 C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. Ex vivo metabolic phenotyping using hyperpolarized 13 C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457-461, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

  13. Chitosan Feasibility to Retain Retinal Stem Cell Phenotype and Slow Proliferation for Retinal Transplantation

    Directory of Open Access Journals (Sweden)

    Girish K. Srivastava

    2014-01-01

    Full Text Available Retinal stem cells (RSCs are promising in cell replacement strategies for retinal diseases. RSCs can migrate, differentiate, and integrate into retina. However, RSCs transplantation needs an adequate support; chitosan membrane (ChM could be one, which can carry RSCs with high feasibility to support their integration into retina. RSCs were isolated, evaluated for phenotype, and subsequently grown on sterilized ChM and polystyrene surface for 8 hours, 1, 4, and 11 days for analysing cell adhesion, proliferation, viability, and phenotype. Isolated RSCs expressed GFAP, PKC, isolectin, recoverin, RPE65, PAX-6, cytokeratin 8/18, and nestin proteins. They adhered (28 ± 16%, 8 hours and proliferated (40 ± 20 cells/field, day 1 and 244 ± 100 cells/field, day 4 significantly low (P95% and phenotype (cytokeratin 8/18, PAX6, and nestin proteins expression, day 11 on both surfaces (ChM and polystyrene. RSCs did not express alpha-SMA protein on both surfaces. RSCs express proteins belonging to epithelial, glial, and neural cells, confirming that they need further stimulus to reach a final destination of differentiation that could be provided in in vivo condition. ChM does not alternate RSCs behaviour and therefore can be used as a cell carrier so that slow proliferating RSCs can migrate and integrate into retina.

  14. Predicting biomaterial property-dendritic cell phenotype relationships from the multivariate analysis of responses to polymethacrylates

    Science.gov (United States)

    Kou, Peng Meng; Pallassana, Narayanan; Bowden, Rebeca; Cunningham, Barry; Joy, Abraham; Kohn, Joachim; Babensee, Julia E.

    2011-01-01

    Dendritic cells (DCs) play a critical role in orchestrating the host responses to a wide variety of foreign antigens and are essential in maintaining immune tolerance. Distinct biomaterials have been shown to differentially affect the phenotype of DCs, which suggested that biomaterials may be used to modulate immune response towards the biologic component in combination products. The elucidation of biomaterial property-DC phenotype relationships is expected to inform rational design of immuno-modulatory biomaterials. In this study, DC response to a set of 12 polymethacrylates (pMAs) was assessed in terms of surface marker expression and cytokine profile. Principal component analysis (PCA) determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an immature DC phenotype. Partial square linear regression, a multivariate modeling approach, was implemented and successfully predicted biomaterial-induced DC phenotype in terms of surface marker expression from biomaterial properties with R2prediction = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R2prediction = 0.80. These results demonstrated that immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection. PMID:22136715

  15. RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

    Science.gov (United States)

    Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

    2007-01-01

    The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

  16. Gallic acid modulates phenotypic behavior and gene expression in oral squamous cell carcinoma cells by interfering with leptin pathway.

    Science.gov (United States)

    Santos, Eliane Macedo Sobrinho; da Rocha, Rogério Gonçalves; Santos, Hércules Otacílio; Guimarães, Talita Antunes; de Carvalho Fraga, Carlos Alberto; da Silveira, Luiz Henrique; Batista, Paulo Ricardo; de Oliveira, Paulo Sérgio Lopes; Melo, Geraldo Aclécio; Santos, Sérgio Henrique; de Paula, Alfredo Maurício Batista; Guimarães, André Luiz Sena; Farias, Lucyana Conceição

    2018-01-01

    Gallic acid is a polyphenolic compost appointed to interfere with neoplastic cells behavior. Evidence suggests an important role of leptin in carcinogenesis pathways, inducing a proliferative phenotype. We investigated the potential of gallic acid to modulate leptin-induced cell proliferation and migration of oral squamous cell carcinoma cell lines. The gallic acid effect on leptin secretion by oral squamous cell carcinoma cells, as well as the underlying molecular mechanisms, was also assessed. For this, we performed proliferation, migration, immunocytochemical and qPCR assays. The expression levels of cell migration-related genes (MMP2, MMP9, Col1A1, and E-cadherin), angiogenesis (HIF-1α, mir210), leptin signaling (LepR, p44/42 MAPK), apoptosis (casp-3), and secreted leptin levels by oral squamous cell carcinoma cells were also measured. Gallic acid decreased proliferation and migration of leptin-treated oral squamous cell carcinoma cells, and reduced mRNA expression of MMP2, MMP9, Col1A1, mir210, but did not change HIF-1α. Gallic acid decreased levels of leptin secreted by oral squamous cell carcinoma cells, accordingly with downregulation of p44/42 MAPK expression. Thus, gallic acid appears to break down neoplastic phenotype of oral squamous cell carcinoma cells by interfering with leptin pathway. Copyright © 2017 Elsevier GmbH. All rights reserved.

  17. Multiple loci are associated with white blood cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Michael A Nalls

    2011-06-01

    Full Text Available White blood cell (WBC count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2, including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across

  18. Functional and phenotypic heterogeneity of group 3 innate lymphoid cells.

    Science.gov (United States)

    Melo-Gonzalez, Felipe; Hepworth, Matthew R

    2017-03-01

    Group 3 innate lymphoid cells (ILC3), defined by expression of the transcription factor retinoid-related orphan receptor γt, play key roles in the regulation of inflammation and immunity in the gastrointestinal tract and associated lymphoid tissues. ILC3 consist largely of two major subsets, NCR + ILC3 and LTi-like ILC3, but also demonstrate significant plasticity and heterogeneity. Recent advances have begun to dissect the relationship between ILC3 subsets and to define distinct functional states within the intestinal tissue microenvironment. In this review we discuss the ever-expanding roles of ILC3 in the context of intestinal homeostasis, infection and inflammation - with a focus on comparing and contrasting the relative contributions of ILC3 subsets. © 2016 The Authors. Immunology published by John Wiley & Sons Ltd.

  19. Condensing Raman spectrum for single-cell phenotype analysis

    KAUST Repository

    Sun, Shiwei; Wang, Xuetao; Gao, Xin; Ren, Lihui; Su, Xiaoquan; Bu, Dongbo; Ning, Kang

    2015-01-01

    In this work, we have proposed an approach called rDisc to discretize the original Raman spectrum into only a few (usually less than 20) representative peaks (Raman shifts). The approach has advantages in removing noises, and condensing the original spectrum. In particular, effective signal processing procedures were designed to eliminate noise, utilising wavelet transform denoising, baseline correction, and signal normalization. In the discretizing process, representative peaks were selected to signicantly decrease the Raman data size. More importantly, the selected peaks are chosen as suitable to serve as key biological markers to differentiate species and other cellular features. Additionally, the classication performance of discretized spectra was found to be comparable to full spectrum having more than 1000 Raman shifts. Overall, the discretized spectrum needs about 5storage space of a full spectrum and the processing speed is considerably faster. This makes rDisc clearly superior to other methods for single-cell classication.

  20. Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells

    DEFF Research Database (Denmark)

    Vaapil, Marica; Helczynska, Karolina; Villadsen, René

    2012-01-01

    Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less...... is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis....

  1. The phenotype of FancB-mutant mouse embryonic stem cells

    OpenAIRE

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu, Lingchuan; Hasty, Paul

    2011-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslink...

  2. BAG3 promotes the phenotypic transformation of primary rat vascular smooth muscle cells via TRAIL.

    Science.gov (United States)

    Fu, Yao; Chang, Ye; Chen, Shuang; Li, Yuan; Chen, Yintao; Sun, Guozhe; Yu, Shasha; Ye, Ning; Li, Chao; Sun, Yingxian

    2018-05-01

    Under normal physiological condition, the mature vascular smooth muscle cells (VSMCs) show differentiated phenotype. In response to various environmental stimuluses, VSMCs convert from the differentiated phenotype to dedifferentiated phenotype characterized by the increased ability of proliferation/migration and the reduction of contractile ability. The phenotypic transformation of VSMCs played an important role in atherosclerosis. Both Bcl-2-associated athanogene 3 (BAG3) and tumor necrosis factor-related apopt-osis inducing ligand (TRAIL) involved in apoptosis. The relationship between BAG3 and TRAIL and their effects the proliferation and migration in VSMCs are rarely reported. This study investigated the effects of BAG3 on the phenotypic modulation and the potential underlying mechanisms in primary rat VSMCs. Primary rat VSMCs were extracted and cultured in vitro. Cell proliferation was detected by cell counting, real-time cell analyzer (RTCA) and EdU incorporation. Cell migration was detected by wound healing, Transwell and RTCA. BAG3 and TRAIL were detected using real-time PCR and western blotting and the secreted proteins in the cultured media by dot blot. The expression of BAG3 increased with continued passages in cultured primary VSMCs. BAG3 promoted the proliferation and migration of primary rat VSMC in a time-dependent manner. BAG3 significantly increased the expression of TRAIL while had no effects on its receptors. TRAIL knockdown or blocking by neutralizing antibody inhibited the proliferation of VSMCs induced by BAG3. TRAIL knockdown exerted no obvious influence on the migration of VSMCs. Based on this study, we report for the first time that BAG3 was expressed in cultured primary rat VSMCs and the expression of BAG3 increased with continued passages. Furthermore, BAG3 promoted the proliferation of VSMCs via increasing the expression of TRAIL. In addition, we also demonstrated that BAG3 promoted the migration of VSMCs independent of TRAIL

  3. Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype

    Science.gov (United States)

    Gabrusiewicz, Konrad; Rodriguez, Benjamin; Wei, Jun; Hashimoto, Yuuri; Healy, Luke M.; Maiti, Sourindra N.; Wang, Qianghu; Elakkad, Ahmed; Liebelt, Brandon D.; Yaghi, Nasser K.; Ezhilarasan, Ravesanker; Huang, Neal; Weinberg, Jeffrey S.; Prabhu, Sujit S.; Rao, Ganesh; Sawaya, Raymond; Langford, Lauren A.; Bruner, Janet M.; Fuller, Gregory N.; Bar-Or, Amit; Li, Wei; Colen, Rivka R.; Curran, Michael A.; Bhat, Krishna P.; Antel, Jack P.; Cooper, Laurence J.; Sulman, Erik P.; Heimberger, Amy B.

    2016-01-01

    Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages. PMID:26973881

  4. MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

    NARCIS (Netherlands)

    Kosten, I.J.; Spiekstra, S.W.; de Gruijl, T.D.; Gibbs, S.

    2015-01-01

    After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a

  5. A quantitative and dynamic model for plant stem cell regulation.

    Directory of Open Access Journals (Sweden)

    Florian Geier

    Full Text Available Plants maintain pools of totipotent stem cells throughout their entire life. These stem cells are embedded within specialized tissues called meristems, which form the growing points of the organism. The shoot apical meristem of the reference plant Arabidopsis thaliana is subdivided into several distinct domains, which execute diverse biological functions, such as tissue organization, cell-proliferation and differentiation. The number of cells required for growth and organ formation changes over the course of a plants life, while the structure of the meristem remains remarkably constant. Thus, regulatory systems must be in place, which allow for an adaptation of cell proliferation within the shoot apical meristem, while maintaining the organization at the tissue level. To advance our understanding of this dynamic tissue behavior, we measured domain sizes as well as cell division rates of the shoot apical meristem under various environmental conditions, which cause adaptations in meristem size. Based on our results we developed a mathematical model to explain the observed changes by a cell pool size dependent regulation of cell proliferation and differentiation, which is able to correctly predict CLV3 and WUS over-expression phenotypes. While the model shows stem cell homeostasis under constant growth conditions, it predicts a variation in stem cell number under changing conditions. Consistent with our experimental data this behavior is correlated with variations in cell proliferation. Therefore, we investigate different signaling mechanisms, which could stabilize stem cell number despite variations in cell proliferation. Our results shed light onto the dynamic constraints of stem cell pool maintenance in the shoot apical meristem of Arabidopsis in different environmental conditions and developmental states.

  6. IGF-1 Has Plaque-Stabilizing Effects in Atherosclerosis by Altering Vascular Smooth Muscle Cell Phenotype

    Science.gov (United States)

    von der Thüsen, Jan H.; Borensztajn, Keren S.; Moimas, Silvia; van Heiningen, Sandra; Teeling, Peter; van Berkel, Theo J.C.; Biessen, Erik A.L.

    2011-01-01

    Insulin-like growth factor-1 (IGF-1) signaling is important for the maintenance of plaque stability in atherosclerosis due to its effects on vascular smooth muscle cell (vSMC) phenotype. To investigate this hypothesis, we studied the effects of the highly inflammatory milieu of the atherosclerotic plaque on IGF-1 signaling and stability-related phenotypic parameters of murine vSMCs in vitro, and the effects of IGF-1 supplementation on plaque phenotype in an atherosclerotic mouse model. M1-polarized, macrophage-conditioned medium inhibited IGF-1 signaling by ablating IGF-1 and increasing IGF-binding protein 3, increased vSMC apoptosis, and decreased proliferation. Expression of α-actin and col3a1 genes was strongly attenuated by macrophage-conditioned medium, whereas expression of matrix-degrading enzymes was increased. Importantly, all of these effects could be corrected by supplementation with IGF-1. In vivo, treatment with the stable IGF-1 analog Long R3 IGF-1 in apolipoprotein E knockout mice reduced stenosis and core size, and doubled cap/core ratio in early atherosclerosis. In advanced plaques, Long R3 IGF-1 increased the vSMC content of the plaque by more than twofold and significantly reduced the rate of intraplaque hemorrhage. We believe that IGF-1 in atherosclerotic plaques may have a role in preventing plaque instability, not only by modulating smooth muscle cell turnover, but also by altering smooth muscle cell phenotype. PMID:21281823

  7. Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence

    Directory of Open Access Journals (Sweden)

    Natasha S Lewis

    2017-04-01

    Full Text Available Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies.

  8. U-251 revisited: genetic drift and phenotypic consequences of long-term cultures of glioblastoma cells

    International Nuclear Information System (INIS)

    Torsvik, Anja; Stieber, Daniel; Enger, Per Øyvind; Golebiewska, Anna; Molven, Anders; Svendsen, Agnete; Westermark, Bengt; Niclou, Simone P; Olsen, Thale Kristin; Chekenya Enger, Martha; Bjerkvig, Rolf

    2014-01-01

    It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line

  9. Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

    Science.gov (United States)

    Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

    2016-06-02

    After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

  10. Melanoma cells revive an embryonic transcriptional network to dictate phenotypic heterogeneity.

    Science.gov (United States)

    Vandamme, Niels; Berx, Geert

    2014-01-01

    Compared to the overwhelming amount of literature describing how epithelial-to-mesenchymal transition (EMT)-inducing transcription factors orchestrate cellular plasticity in embryogenesis and epithelial cells, the functions of these factors in non-epithelial contexts, such as melanoma, are less clear. Melanoma is an aggressive tumor arising from melanocytes, endowed with unique features of cellular plasticity. The reversible phenotype-switching between differentiated and invasive phenotypes is increasingly appreciated as a mechanism accounting for heterogeneity in melanoma and is driven by oncogenic signaling and environmental cues. This phenotypic switch is coupled with an intriguing and somewhat counterintuitive signaling switch of EMT-inducing transcription factors. In contrast to carcinomas, different EMT-inducing transcription factors have antagonizing effects in melanoma. Balancing between these different EMT transcription factors is likely the key to successful metastatic spread of melanoma.

  11. Induction of expression of two phenotypic markers of pulmonary type II cells in a cultured cell line

    International Nuclear Information System (INIS)

    Henderson, R.F.; Waide, J.J.; Scott, G.G.

    1994-01-01

    The functions of pulmonary type II cells, such as synthesis of pulmonary surfactant and metabolism of inhaled xenobiotics, can be studied in primary isolates of lung cells. However, isolated type II cells, when cultured, quickly lose the phenotypic expressions characteristics of type II cells, including surfactant lipid and protein synthesis and alkaline phosphatase (AP) activity. A cultured cell line that maintained expression of type II cell markers of differentiation would be advantageous for the study of such functions as surfactant synthesis and secretion. Such a cell line would allow generation of a large number of homogeneous cells for study. The purpose of the current study was to induce markers of differentiated type II cells in a cultured cell line to facilitate studies of factors that control surfactant synthesis and secretion

  12. Phenotypic and Functional Alterations in Circulating Memory CD8 T Cells with Time after Primary Infection.

    Directory of Open Access Journals (Sweden)

    Matthew D Martin

    2015-10-01

    Full Text Available Memory CD8 T cells confer increased protection to immune hosts upon secondary viral, bacterial, and parasitic infections. The level of protection provided depends on the numbers, quality (functional ability, and location of memory CD8 T cells present at the time of infection. While primary memory CD8 T cells can be maintained for the life of the host, the full extent of phenotypic and functional changes that occur over time after initial antigen encounter remains poorly characterized. Here we show that critical properties of circulating primary memory CD8 T cells, including location, phenotype, cytokine production, maintenance, secondary proliferation, secondary memory generation potential, and mitochondrial function change with time after infection. Interestingly, phenotypic and functional alterations in the memory population are not due solely to shifts in the ratio of effector (CD62Llo and central memory (CD62Lhi cells, but also occur within defined CD62Lhi memory CD8 T cell subsets. CD62Lhi memory cells retain the ability to efficiently produce cytokines with time after infection. However, while it is was not formally tested whether changes in CD62Lhi memory CD8 T cells over time occur in a cell intrinsic manner or are due to selective death and/or survival, the gene expression profiles of CD62Lhi memory CD8 T cells change, phenotypic heterogeneity decreases, and mitochondrial function and proliferative capacity in either a lymphopenic environment or in response to antigen re-encounter increase with time. Importantly, and in accordance with their enhanced proliferative and metabolic capabilities, protection provided against chronic LCMV clone-13 infection increases over time for both circulating memory CD8 T cell populations and for CD62Lhi memory cells. Taken together, the data in this study reveal that memory CD8 T cells continue to change with time after infection and suggest that the outcome of vaccination strategies designed to elicit

  13. Heat Shock Protein 47: A Novel Biomarker of Phenotypically Altered Collagen-Producing Cells

    International Nuclear Information System (INIS)

    Taguchi, Takashi; Nazneen, Arifa; Al-Shihri, Abdulmonem A.; Turkistani, Khadijah A.; Razzaque, Mohammed S.

    2011-01-01

    Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that helps the molecular maturation of various types of collagens. A close association between increased expression of HSP47 and the excessive accumulation of collagens is found in various human and experimental fibrotic diseases. Increased levels of HSP47 in fibrotic diseases are thought to assist in the increased assembly of procollagen, and thereby contribute to the excessive deposition of collagens in fibrotic areas. Currently, there is not a good universal histological marker to identify collagen-producing cells. Identifying phenotypically altered collagen-producing cells is essential for the development of cell-based therapies to reduce the progression of fibrotic diseases. Since HSP47 has a single substrate, which is collagen, the HSP47 cellular expression provides a novel universal biomarker to identify phenotypically altered collagen-producing cells during wound healing and fibrosis. In this brief article, we explained why HSP47 could be used as a universal marker for identifying phenotypically altered collagen-producing cells

  14. Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Doherty, R.E.; Haywood-Small, S.L. [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom); Sisley, K. [Department of Oncology, Academic Unit of Ophthalmology and Orthopties, University of Sheffield, Sheffield S10 2RX (United Kingdom); Cross, N.A., E-mail: n.cross@shu.ac.uk [Biomedical Research Centre, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Isolated ALDH{sup Hi} PC3 cells preferentially form primitive holoclone-type colonies. Black-Right-Pointing-Pointer Primitive holoclone colonies are predominantly ALDH{sup Lo} but contain rare ALDH{sup Hi} cells. Black-Right-Pointing-Pointer Holoclone-forming cells are not restricted to the ALDH{sup Hi} population. Black-Right-Pointing-Pointer ALDH phenotypic plasticity occurs in PC3 cells (ALDH{sup Lo} to ALDH{sup Hi} and vice versa). Black-Right-Pointing-Pointer ALDH{sup Hi} cells are observed but very rare in PC3 spheroids grown in stem cell medium. -- Abstract: Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH{sup Lo} cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH{sup Hi} population, or whether all ALDH{sup Hi} cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH{sup Hi} cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH{sup Hi} cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH{sup Lo} population can develop ALDH{sup Hi} populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH{sup Hi} cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in

  15. Isolation of stem-like cells from spontaneous feline mammary carcinomas: Phenotypic characterization and tumorigenic potential

    Energy Technology Data Exchange (ETDEWEB)

    Barbieri, Federica; Wurth, Roberto [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy); Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina [Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D' Aosta, National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila, 16129, Genova (Italy); Thellung, Stefano [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy); Daga, Antonio [Laboratory of Translational Oncology, IRCCS Azienda Ospedaliera Universitaria San Martino - IST- Istituto Nazionale Ricerca sul Cancro, L.go R. Benzi, 10, 16132 Genova Italy (Italy); Cilli, Michele [Animal Facility, IRCCS Azienda Ospedaliera Universitaria San Martino - IST- Istituto Nazionale Ricerca sul Cancro, L.go R. Benzi, 10, 16132 Genova Italy (Italy); Ferrari, Angelo [Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D' Aosta, National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Piazza Borgo Pila, 16129, Genova (Italy); Florio, Tullio, E-mail: tullio.florio@unige.it [Section of Pharmacology, Dept. of Internal Medicine Di.M.I., and Center of Excellence for Biomedical Research - University of Genova, Viale Benedetto XV, 2, 16132 Genova (Italy)

    2012-04-15

    Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell-like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-{alpha} and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: Black-Right-Pointing-Pointer Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 Black-Right-Pointing-Pointer These grow as spheres in serum-free medium and self-renew Black-Right-Pointing-Pointer Isolated stem-like cancer cells initiate tumor in immunodeficient mice Black-Right-Pointing-Pointer Xenografted tumors are phenotypically similar to the original tumor Black

  16. Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Phadke, Manali; Krynetskaia, Natalia [Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Mishra, Anurag [Jayne Haines Center for Pharmacogenomics, Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Krynetskiy, Evgeny, E-mail: ekrynets@temple.edu [Temple University School of Pharmacy, Philadelphia, PA 19140 (United States); Jayne Haines Center for Pharmacogenomics, Temple University School of Pharmacy, Philadelphia, PA 19140 (United States)

    2011-07-29

    Highlights: {yields} We examined the effect of glyceraldehyde 3-phosphate (GAPDH) depletion on proliferation of human carcinoma A549 cells. {yields} GAPDH depletion induces accelerated senescence in tumor cells via AMPK network, in the absence of DNA damage. {yields} Metabolic and genetic rescue experiments indicate that GAPDH has regulatory functions linking energy metabolism and cell cycle. {yields} Induction of senescence in LKB1-deficient lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation. -- Abstract: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-{beta}-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of {alpha} subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

  17. In wound repair vimentin mediates the transition of mesenchymal leader cells to a myofibroblast phenotype.

    Science.gov (United States)

    Walker, J L; Bleaken, B M; Romisher, A R; Alnwibit, A A; Menko, A S

    2018-05-02

    Following injury, mesenchymal repair cells are activated to function as leader cells that modulate wound healing. These cells have the potential to differentiate to myofibroblasts, resulting in fibrosis and scarring. The signals underlying these differing pathways are complex and incompletely understood. The ex vivo mock cataract surgery cultures are an attractive model with which to address this question. With this model we study, concurrently, the mechanisms that control mesenchymal leader cell function in injury repair within their native microenvironment, and the signals that induce this same cell population to acquire a myofibroblast phenotype when these cells encounter the environment of the adjacent tissue culture platform. Here, we show that upon injury, the cytoskeletal protein vimentin is released into the extracellular space, binds to the cell surface of the mesenchymal leader cells located at the wound edge in the native matrix environment, and supports wound closure. In pro-fibrotic environments, the extracellular vimentin pool also links specifically to the mesenchymal leader cells, and has an essential role in signaling their fate change to a myofibroblast. These findings suggest a novel role for extracellular, cell-surface-associated vimentin in mediating repair-cell function in wound repair and in transitioning these cells to a myofibroblast phenotype. Movie S1 Movie S1 Collective movement of mesenchymal leader and epithelial follower cells across the tissue culture substrate (ECZ) in response to injury was followed by time-lapse imaging from D0-D3. The mesenchymal cells at the leading edge were easily distinguished morphologically from the lens epithelial follower cells.

  18. Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

    Science.gov (United States)

    Omlor, G W; Nerlich, A G; Tirlapur, U K; Urban, J P; Guehring, T

    2014-12-01

    Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. 3D

  19. Phenotypic and Functional Properties of Tumor-Infiltrating Regulatory T Cells

    Directory of Open Access Journals (Sweden)

    Gap Ryol Lee

    2017-01-01

    Full Text Available Regulatory T (Treg cells maintain immune homeostasis by suppressing excessive immune responses. Treg cells induce tolerance against self- and foreign antigens, thus preventing autoimmunity, allergy, graft rejection, and fetus rejection during pregnancy. However, Treg cells also infiltrate into tumors and inhibit antitumor immune responses, thus inhibiting anticancer therapy. Depleting whole Treg cell populations in the body to enhance anticancer treatments will produce deleterious autoimmune diseases. Therefore, understanding the precise nature of tumor-infiltrating Treg cells is essential for effectively targeting Treg cells in tumors. This review summarizes recent results relating to Treg cells in the tumor microenvironment, with particular emphasis on their accumulation, phenotypic, and functional properties, and targeting to enhance the efficacy of anticancer treatment.

  20. Iso-acoustic focusing of cells for size-insensitive acousto-mechanical phenotyping

    DEFF Research Database (Denmark)

    Augustsson, Per; Karlsen, Jonas Tobias; Su, Hao-Wei

    2016-01-01

    Mechanical phenotyping of single cells is an emerging tool for cell classification, enabling assessment of effective parameters relating to cells' interior molecular content and structure. Here, we present iso-acoustic focusing, an equilibrium method to analyze the effective acoustic impedance...... of single cells in continuous flow. While flowing through a microchannel, cells migrate sideways, influenced by an acoustic field, into streams of increasing acoustic impedance, until reaching their cell-type specific point of zero acoustic contrast. We establish an experimental procedure and provide...... theoretical justifications and models for iso-acoustic focusing. We describe a method for providing a suitable acoustic contrast gradient in a cell-friendly medium, and use acoustic forces to maintain that gradient in the presence of destabilizing forces. Applying this method we demonstrate iso-acoustic...

  1. Phenotypic characterization of the bone marrow stem cells used in regenerative cellular therapy

    International Nuclear Information System (INIS)

    Macias Abraham, Consuelo; Valle Perez, Lazaro O del; Baganet Cobas, Aymara

    2011-01-01

    Regenerative medicine is a novel therapeutic method with broad potential for the treatment of various illnesses, based on the use of bone marrow (BM) stem cells, whose phenotypic characterization is limited. The paper deals with the expression of different cell membrane markers in mononuclear BM cells from 14 patients who underwent autologous cell therapy, obtained by medullary puncture and mobilization to peripheral blood, with the purpose of characterizing the different types of cells present in that heterogeneous cellular population and identifying the adhesion molecules involved in their adhesion. A greater presence was observed of adherent stem cells from the marrow stroma in mononuclear cells obtained directly from the BM; a larger population of CD90 +c ells in mononuclear cells from CD34 -/ CD45 -p eripheral blood with a high expression of molecules CD44 and CD62L, which suggests a greater presence of mesenchymal stem cells (MSC) in mobilized cells from the marrow stroma. The higher levels of CD34 +c ells in peripheral blood stem cells with a low expression of molecules CD117 -a nd DR -s uggests the presence of hematopoietic stem cells, hemangioblasts and progenitor endothelial cells mobilized to peripheral circulation. It was found that mononuclear cells from both the BM and peripheral blood show a high presence of stem cells with expression of adhesion molecule CD44 (MMC marker), probably involved in their migration, settling and differentiation

  2. Isolation of stem-like cells from spontaneous feline mammary carcinomas: Phenotypic characterization and tumorigenic potential

    International Nuclear Information System (INIS)

    Barbieri, Federica; Wurth, Roberto; Ratto, Alessandra; Campanella, Chiara; Vito, Guendalina; Thellung, Stefano; Daga, Antonio; Cilli, Michele; Ferrari, Angelo; Florio, Tullio

    2012-01-01

    Current carcinogenesis theory states that only a small subset of tumor cells, the cancer stem cells or tumor initiating cells (TICs), are responsible for tumor formation and progression. Human breast cancer-initiating cells have been identified as CD44-expressing cells, which retain tumorigenic activity and display stem cell–like properties. Spontaneous feline mammary carcinoma (FMC) is an aggressive cancer, which shows biological similarities to the human tumor counterpart. We report the isolation and phenotypic characterization of FMC-derived stem/progenitor cells, showing in vitro self-renewal, long-lasting proliferation and in vivo tumorigenicity. Twenty-one FMC samples were collected, histologically classified and characterized for the expression of Ki67, EGFR, ER-α and CD44, by immunohistochemistry. By culture in stem cell permissive conditions, we isolated, from 13 FMCs, a CD44-positive subpopulation able to survive and proliferate in vitro as mammospheres of different sizes and morphologies. When injected in NOD/SCID mice, FMC stem-like cells initiate tumors, generating cell heterogeneity and recapitulating the original histotype. In serum-containing medium, spheroid cells showed differentiation properties as shown by morphological changes, the loss of CD44 expression and tumorigenic potential. These data show that stem-defined culture of FMC enriches for TICs and validate the use of these cells as a suitable model for comparative oncology studies of mammary biology and testing therapeutic strategies aimed at eradicating TICs. -- Highlights: ► Feline mammary carcinoma contain a sub-population of stem-like cells expressing CD44 ► These grow as spheres in serum-free medium and self-renew ► Isolated stem-like cancer cells initiate tumor in immunodeficient mice ► Xenografted tumors are phenotypically similar to the original tumor ► Upon differentiation, cells grow as monolayers, loosing the tumorigenic potential

  3. In vitro atrazine exposure affects the phenotypic and functional maturation of dendritic cells

    International Nuclear Information System (INIS)

    Pinchuk, Lesya M.; Lee, Sang-Ryul; Filipov, Nikolay M.

    2007-01-01

    Recent data suggest that some of the immunotoxic effects of the herbicide atrazine, a very widely used pesticide, may be due to perturbations in dendritic cell (DC) function. As consequences of atrazine exposure on the phenotypic and functional maturation of DC have not been studied, our objective was, using the murine DC line, JAWSII, to determine whether atrazine will interfere with DC maturation. First, we characterized the maturation of JAWSII cells in vitro by inducing them to mature in the presence of growth factors and selected maturational stimuli in vitro. Next, we exposed the DC cell line to a concentration range of atrazine and examined its effects on phenotypic and functional maturation of DC. Atrazine exposure interfered with the phenotypic and functional maturation of DC at non-cytotoxic concentrations. Among the phenotypic changes caused by atrazine exposure was a dose-dependent removal of surface MHC-I with a significant decrease being observed at 1 μM concentration. In addition, atrazine exposure decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. When, for comparative purposes, we exposed primary thymic DC to atrazine, MHC-I and CD11c expression was also decreased. Phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation as, albeit at higher concentrations, receptor-mediated antigen uptake was increased by atrazine. Thus, our data suggest that atrazine directly targets DC maturation and that toxicants such as atrazine that efficiently remove MHC-I molecules from the DC surface are likely to contribute to immune evasion

  4. Phenotypic and functional characterization of human mammary stem/progenitor cells in long term culture.

    Directory of Open Access Journals (Sweden)

    Devaveena Dey

    Full Text Available BACKGROUND: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. METHODOLOGY: Single cell suspensions derived from human breast 'organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. PRINCIPAL FINDINGS: We show that primary mammospheres contain a distinct side-population (SP that displays a CD24(low/CD44(low phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high/CD24(low cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1 mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. CONCLUSIONS: Thus, the self-renewal potential of human breast stem cells is

  5. Anti-atherosclerotic plants which modulate the phenotype of vascular smooth muscle cells.

    Science.gov (United States)

    Saleh Al-Shehabi, Tuqa; Iratni, Rabah; Eid, Ali H

    2016-10-15

    Cardiovascular disease (CVD) remains the leading cause of global death, with atherosclerosis being a major contributor to this mortality. Several mechanisms are implicated in the pathogenesis of this disease. A key element in the development and progression of atherosclerotic lesions is the phenotype of vascular smooth muscle cells. Under pathophysiologic conditions such as injury, these cells switch from a contractile to a synthetic phenotype that often possesses high proliferative and migratory capacities. Despite major advances made in the management and treatment of atherosclerosis, mortality associated with this disease remains high. This mandates that other approaches be sought. Herbal medicine, especially for the treatment of CVD, has been gaining more attention in recent years. This is in no small part due to the evidence-based values associated with the consumption of many plants as well as the relatively cheaper prices, easier access and conventional folk medicine "inherited" over generations. Sections: In this review, we provide a brief introduction about the pathogenesis of atherosclerosis then we highlight the role of vascular smooth muscle cells in this disease, especially when a phenotypic switch of these cells arises. We then thoroughly discuss the various plants that show potentially beneficial effects as anti-atherosclerotic, with prime attention given to herbs and plants that inhibit the phenotypic switch of vascular smooth muscle cells. Accumulating evidence provides the justification for the use of botanicals in the treatment or prevention of atherosclerosis. However, further studies, especially clinical ones, are warranted to better define several pharmacological parameters of these herbs, such as toxicity, tolerability, and efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

    Science.gov (United States)

    Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

    2018-03-01

    Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

  7. SAP is required for the development of innate phenotype in H2-M3-restricted CD8+ T cells1

    Science.gov (United States)

    Bediako, Yaw; Bian, Yao; Zhang, Hong; Cho, Hoonsik; Stein, Paul L.; Wang, Chyung-Ru

    2012-01-01

    H2-M3-restricted T cells have a pre-activated surface phenotype, rapidly expand and produce cytokines upon stimulation and as such, are classified as innate T cells. Unlike most innate T cells, M3-restricted T cells also express CD8αβ co-receptors and a diverse TCR repertoire: hallmarks of conventional MHC Ia-restricted CD8+ T cells. Although iNKT cells are also innate lymphocytes, they are selected exclusively on hematopoietic cells (HC), while M3-restricted T cells can be selected on either hematopoietic or thymic epithelial cells (TEC). Moreover, their phenotypes differ depending on what cells mediate their selection. Though there is a clear correlation between selection on HC and development of innate phenotype, the underlying mechanism remains unclear. SAP is required for the development of iNKT cells and mediates signals from SLAM receptors that are exclusively expressed on HC. Based on their dual selection pathway, M3-restricted T cells present a unique model for studying the development of innate T cell phenotype. Using both polyclonal and transgenic mouse models we demonstrate that while M3-restricted T cells are capable of developing in the absence of SAP, SAP is required for HC-mediated selection, development of pre-activated phenotype and heightened effector functions of M3-restricted T cells. These findings are significant because they directly demonstrate the need for SAP in HC-mediated acquisition of innate T cell phenotype and suggest that due to their SAP-dependent HC-mediated selection, M3-restricted T cells develop a pre-activated phenotype and an intrinsic ability to proliferate faster upon stimulation, allowing for an important role in the early response to infection. PMID:23041566

  8. Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

    International Nuclear Information System (INIS)

    Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

    2005-01-01

    Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

  9. Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: implications for immune cell biobanking.

    Directory of Open Access Journals (Sweden)

    Anita Posevitz-Fejfár

    Full Text Available Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum, biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9: at a distal location (shipped overnight and in the central laboratory (processed immediately. PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might

  10. Giant cell lesions with a Noonan-like phenotype: a case report.

    Science.gov (United States)

    Cancino, Claudia Marcela H; Gaião, Léonilson; Sant'Ana Filho, Manoel; Oliveira, Flavio Augusto Marsiaj

    2007-05-01

    The purpose of this article is to describe a case of multiple giant cell lesions of the mandible that occurred in a 14-year-old girl with phenotypic characteristics associated with Noonan Syndrome (NS). NS is a dysmorphic disorder characterized by hypertelorism, short stature, congenital heart defects, short and webbed neck, skeletal anomalies, and bleeding diathesis. A 14-year-old girl with a previous diagnosis of NS (sporadic case) presented with multiple radiolucent lesions in the body and ramus of her mandible. In terms of clinical behavior and the described radiographic characteristics, giant cells lesions with Noonan-like phenotype can be considered a form of cherubism. Therefore, surgical intervention is not necessary, but radiographic follow-up and observation is very important during the control and gradual regression of the lesions.

  11. Phenotypically non-suppressive cells predominate among FoxP3-positive cells in oral lichen planus.

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    Schreurs, Olav; Karatsaidis, Andreas; Schenck, Karl

    2016-11-01

    Oral lichen planus (OLP) is a common T-cell-dominated oral chronic inflammatory disease occurring in periods of remission, quiescence, activity with pronounced inflammation, and acute ulceration. Cell infiltrates in OLP contain varying numbers of CD4 + T cells expressing the transcription factor FoxP3. FoxP3 + CD4 + T cells are, however, a heterogeneous cell population containing suppressive and non-suppressive cells, and their distribution in infiltrates from OLP is unknown. Biopsies were taken from normal oral mucosa (n = 8) and OLP lesions (n = 19), and a set of in situ methods for the determination of the functional phenotype of FoxP3 + CD4 + T cells was applied. Numbers of FoxP3 + CD4 + T cells were highest in the atrophic form of the disease, yet low in the ulcerative form. The main FoxP3 + CD4 + T-cell population observed was FoxP3 + CD45RA - CD25 + CD45RO + and CD15s - , a phenotype delineating a non-suppressive subset. Numbers of cells with an actively suppressing phenotype (FoxP3 + CD45RA - CD25 + CD45RO + and CD15s + ) were, however, about twice as high in reticular lesions as compared with the atrophic form. Many FoxP3 + CD4 + T cells expressed T-bet, the hallmark transcription factor for IFN-γ-producing T cells, indicating that they may enhance immune and inflammatory responses rather than suppress them. The absence of actively suppressing FoxP3 + CD4 + T cells may in part explain why OLP is a remarkably persisting condition, in spite of the presence of substantially high numbers of FoxP3 + CD4 + T cells. The findings emphasize that it is crucial to examine not only numbers but also functional phenotype of FoxP3 + CD4 + T cells in human tissues. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Hypoxia Enhances Differentiation of Adipose Tissue-Derived Stem Cells toward the Smooth Muscle Phenotype

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    Fang Wang

    2018-02-01

    Full Text Available Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p < 0.01. Cells differentiated in 5% oxygen conditions showed greater contraction effect (p < 0.01. Hypoxia influences differentiation of smooth muscle cells from adipose stem cells and 5% oxygen was the optimal condition to generate smooth muscle cells that contract from adipose stem cells.

  13. Association of classical markers and establishment of the dyslipidemic sub-phenotype of sickle cell anemia.

    Science.gov (United States)

    Aleluia, Milena Magalhães; da Guarda, Caroline Conceição; Santiago, Rayra Pereira; Fonseca, Teresa Cristina Cardoso; Neves, Fábia Idalina; de Souza, Regiana Quinto; Farias, Larissa Alves; Pimenta, Felipe Araújo; Fiuza, Luciana Magalhães; Pitanga, Thassila Nogueira; Ferreira, Júnia Raquel Dutra; Adorno, Elisângela Vitória; Cerqueira, Bruno Antônio Veloso; Gonçalves, Marilda de Souza

    2017-04-11

    Sickle cell anemia (SCA) patients exhibit sub-phenotypes associated to hemolysis and vaso-occlusion. The disease has a chronic inflammatory nature that has been also associated to alterations in the lipid profile. This study aims to analyze hematological and biochemical parameters to provide knowledge about the SCA sub-phenotypes previously described and suggest a dyslipidemic sub-phenotype. A cross-sectional study was conducted from 2013 to 2014, and 99 SCA patients in steady state were enrolled. We assessed correlations and associations with hematological and biochemical data and investigated the co-inheritance of -α 3.7Kb -thalassemia (-α 3.7Kb -thal). Correlation analyses were performed using Spearman and Pearson coefficient. The median of quantitative variables between two groups was compared using t-test and Mann-Whitney. P-values <0.05 were considered statistically significant. We found significant association of high lactate dehydrogenase levels with decreased red blood cell count and hematocrit as well as high levels of total and indirect bilirubin. SCA patients with low nitric oxide metabolites had high total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol and reduced very low-density cholesterol, triglycerides, direct bilirubin level and reticulocyte counts. In SCA patients with high-density lipoprotein cholesterol greater than 40 mg/dL, we observed increased red blood cell count, hemoglobin, hematocrit, and fetal hemoglobin and decreased nitric oxide metabolites levels. The presence of -α 3.7Kb -thal was associated with high red blood cell count and low mean corpuscular volume, mean corpuscular hemoglobin, platelet count and total and indirect bilirubin levels. Our results provide additional information about the association between biomarkers and co-inheritance of -α 3.7Kb -thal in SCA, and suggest the role of dyslipidemia and nitric oxide metabolites in the characterization of this sub-phenotype.

  14. 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

  15. Phenotype and Function of CD209+ Bovine Blood Dendritic Cells, Monocyte-Derived-Dendritic Cells and Monocyte-Derived Macrophages.

    Directory of Open Access Journals (Sweden)

    Kun Taek Park

    Full Text Available Phylogenic comparisons of the mononuclear phagocyte system (MPS of humans and mice demonstrate phenotypic divergence of dendritic cell (DC subsets that play similar roles in innate and adaptive immunity. Although differing in phenotype, DC can be classified into four groups according to ontogeny and function: conventional DC (cDC1 and cDC2, plasmacytoid DC (pDC, and monocyte derived DC (MoDC. DC of Artiodactyla (pigs and ruminants can also be sub-classified using this system, allowing direct functional and phenotypic comparison of MoDC and other DC subsets trafficking in blood (bDC. Because of the high volume of blood collections required to study DC, cattle offer the best opportunity to further our understanding of bDC and MoDC function in an outbred large animal species. As reported here, phenotyping DC using a monoclonal antibody (mAb to CD209 revealed CD209 is expressed on the major myeloid population of DC present in blood and MoDC, providing a phenotypic link between these two subsets. Additionally, the present study demonstrates that CD209 is also expressed on monocyte derived macrophages (MoΦ. Functional analysis revealed each of these populations can take up and process antigens (Ags, present them to CD4 and CD8 T cells, and elicit a T-cell recall response. Thus, bDC, MoDC, and MoΦ pulsed with pathogens or candidate vaccine antigens can be used to study factors that modulate DC-driven T-cell priming and differentiation ex vivo.

  16. Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells.

    Science.gov (United States)

    Drawnel, Faye M; Boccardo, Stefano; Prummer, Michael; Delobel, Frédéric; Graff, Alexandra; Weber, Michael; Gérard, Régine; Badi, Laura; Kam-Thong, Tony; Bu, Lei; Jiang, Xin; Hoflack, Jean-Christophe; Kiialainen, Anna; Jeworutzki, Elena; Aoyama, Natsuyo; Carlson, Coby; Burcin, Mark; Gromo, Gianni; Boehringer, Markus; Stahlberg, Henning; Hall, Benjamin J; Magnone, Maria Chiara; Kolaja, Kyle; Chien, Kenneth R; Bailly, Jacques; Iacone, Roberto

    2014-11-06

    Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Faye M. Drawnel

    2014-11-01

    Full Text Available Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

  18. Development of a Functional Schwann Cell Phenotype from Autologous Porcine Bone Marrow Mononuclear Cells for Nerve Repair

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    Michael J. Rutten

    2012-01-01

    Full Text Available Adult bone marrow mononuclear cells (BM-MNCs are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6–8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF expression. Addition of neuregulin (1–25 nM increased p75(NGF levels at 24–48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca2+]i, with nucleotide potency being UTP=ATP>ADP>AMP>adenosine. Suramin blocked the ATP-induced [Ca2+]i but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca2+]i sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

  19. Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models

    International Nuclear Information System (INIS)

    Phelan, J. P.; Reen, F. J.; Dunphy, N.; O'Connor, R.; O'Gara, F.

    2016-01-01

    The role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers. Here we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines. Intracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells. These findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression

  20. Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Gnana-Prakasam, Jaya P.; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Reddy, Sushma K.; Martin, Pamela M.; Thangaraju, Muthusamy; Smith, Sylvia B.; Ganapathy, Vadivel

    2013-01-01

    Purpose. Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated. Methods. Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4. Results. Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells. Conclusions. RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells. PMID:23169885

  1. Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

    Science.gov (United States)

    Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas

    2011-05-01

    The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.

  2. Glycocalyx Degradation Induces a Proinflammatory Phenotype and Increased Leukocyte Adhesion in Cultured Endothelial Cells under Flow.

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    Karli K McDonald

    Full Text Available Leukocyte adhesion to the endothelium is an early step in the pathogenesis of atherosclerosis. Effective adhesion requires the binding of leukocytes to their cognate receptors on the surface of endothelial cells. The glycocalyx covers the surface of endothelial cells and is important in the mechanotransduction of shear stress. This study aimed to identify the molecular mechanisms underlying the role of the glycocalyx in leukocyte adhesion under flow. We performed experiments using 3-D cell culture models, exposing human abdominal aortic endothelial cells to steady laminar shear stress (10 dynes/cm2 for 24 hours. We found that with the enzymatic degradation of the glycocalyx, endothelial cells developed a proinflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion. Our results show an up-regulation of ICAM-1 with degradation compared to non-degraded controls (3-fold increase, p<0.05 and we attribute this effect to a de-regulation in NF-κB activity in response to flow. These results suggest that the glycocalyx is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion. We provide evidence for how the destabilization of this structure may be an early and defining feature in the initiation of atherosclerosis.

  3. Comparative study of the organisation and phenotypes of bladder interstitial cells in human, mouse and rat.

    Science.gov (United States)

    Gevaert, Thomas; Neuhaus, Jochen; Vanstreels, Els; Daelemans, Dirk; Everaerts, Wouter; Der Aa, Frank Van; Timmermans, Jean-Pierre; Roskams, Tania; Steiner, Clara; Pintelon, Isabel; De Ridder, Dirk

    2017-12-01

    With most research on interstitial cells (IC) in the bladder being conducted on animal models, it remains unclear whether all structural and functional data on IC from animal models can be translated to the human context. This prompted us to compare the structural and immunohistochemical properties of IC in bladders from mouse, rat and human. Tissue samples were obtained from the bladder dome and subsequently processed for immunohistochemistry and electron microscopy. The ultrastructural properties of IC were compared by means of electron microscopy and IC were additionally characterized with single/double immunohistochemistry/immunofluorescence. Our results reveal a similar organization of the IC network in the upper lamina propria (ULP), the deep lamina propria (DLP) and the detrusor muscle in human, rat and mouse bladders. Furthermore, despite several similarities in IC phenotypes, we also found several obvious inter-species differences in IC, especially in the ULP. Most remarkably in this respect, ULP IC in human bladder predominantly displayed a myoid phenotype with abundant presence of contractile micro-filaments, while those in rat and mouse bladders showed a fibroblast phenotype. In conclusion, the organization of ULP IC, DLP IC and detrusor IC is comparable in human, rat and mouse bladders, although several obvious inter-species differences in IC phenotypes were found. The present data show that translating research data on IC in laboratory animals to the human setting should be carried out with caution.

  4. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

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    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  5. Functional dynamics of cell surface membrane proteins.

    Science.gov (United States)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

    Science.gov (United States)

    Hu, Kunpeng; Huang, Pinzhu; Luo, Hui; Yao, Zhicheng; Wang, Qingliang; Xiong, Zhiyong; Lin, Jizong; Huang, He; Xu, Shilei; Zhang, Peng; Liu, Bo

    2017-08-01

    Mammalian-enabled (MENA) protein is an actin-regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)-like phenotypes in HCC cells. Real-time-PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA-overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony-forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial-to-mesenchymal transition (EMT) markers, extracellular signal-regulated kinases (ERK) phosphorylation, and the level of β-catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β-catenin signaling pathways.

  7. In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

    International Nuclear Information System (INIS)

    Timraz, Sara B.H.; Farhat, Ilyas A.H.; Alhussein, Ghada; Christoforou, Nicolas; Teo, Jeremy C.M.

    2016-01-01

    In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, and reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.

  8. In-depth evaluation of commercially available human vascular smooth muscle cells phenotype: Implications for vascular tissue engineering

    Energy Technology Data Exchange (ETDEWEB)

    Timraz, Sara B.H., E-mail: sara.timraz@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Farhat, Ilyas A.H., E-mail: ilyas.farhat@outlook.com [Department of Applied Mathematics and Sciences, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Alhussein, Ghada, E-mail: ghada.alhussein@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Christoforou, Nicolas, E-mail: nicolas.christoforou@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates); Department of Biomedical Engineering, Duke University, Durham, NC 27708 (United States); Teo, Jeremy C.M., E-mail: jeremy.teo@kustar.ac.ae [Department of Biomedical Engineering, Khalifa University, PO Box 127788, Abu Dhabi (United Arab Emirates)

    2016-05-01

    In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, and reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.

  9. Electrical Differentiation of Mesenchymal Stem Cells into Schwann-Cell-Like Phenotypes Using Inkjet-Printed Graphene Circuits.

    Science.gov (United States)

    Das, Suprem R; Uz, Metin; Ding, Shaowei; Lentner, Matthew T; Hondred, John A; Cargill, Allison A; Sakaguchi, Donald S; Mallapragada, Surya; Claussen, Jonathan C

    2017-04-01

    Graphene-based materials (GBMs) have displayed tremendous promise for use as neurointerfacial substrates as they enable favorable adhesion, growth, proliferation, spreading, and migration of immobilized cells. This study reports the first case of the differentiation of mesenchymal stem cells (MSCs) into Schwann cell (SC)-like phenotypes through the application of electrical stimuli from a graphene-based electrode. Electrical differentiation of MSCs into SC-like phenotypes is carried out on a flexible, inkjet-printed graphene interdigitated electrode (IDE) circuit that is made highly conductive (sheet resistance electrically stimulated/treated (etMSCs) display significant enhanced cellular differentiation and paracrine activity above conventional chemical treatment strategies [≈85% of the etMSCs differentiated into SC-like phenotypes with ≈80 ng mL -1 of nerve growth factor (NGF) secretion vs. 75% and ≈55 ng mL -1 for chemically treated MSCs (ctMSCs)]. These results help pave the way for in vivo peripheral nerve regeneration where the flexible graphene electrodes could conform to the injury site and provide intimate electrical simulation for nerve cell regrowth. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

    Directory of Open Access Journals (Sweden)

    Kristin Dawson

    Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

  11. Regulatory T cells expanded from HIV-1-infected individuals maintain phenotype, TCR repertoire and suppressive capacity.

    Directory of Open Access Journals (Sweden)

    Mathieu Angin

    Full Text Available While modulation of regulatory T cell (Treg function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4(+ Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region, characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

  12. Dynamical Adaptation in Terrorist Cells/Networks

    DEFF Research Database (Denmark)

    Hussain, Dil Muhammad Akbar; Ahmed, Zaki

    2010-01-01

    Typical terrorist cells/networks have dynamical structure as they evolve or adapt to changes which may occur due to capturing or killing of a member of the cell/network. Analytical measures in graph theory like degree centrality, betweenness and closeness centralities are very common and have long...

  13. Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome.

    Science.gov (United States)

    Crosas-Molist, Eva; Meirelles, Thayna; López-Luque, Judit; Serra-Peinado, Carla; Selva, Javier; Caja, Laia; Gorbenko Del Blanco, Darya; Uriarte, Juan José; Bertran, Esther; Mendizábal, Yolanda; Hernández, Vanessa; García-Calero, Carolina; Busnadiego, Oscar; Condom, Enric; Toral, David; Castellà, Manel; Forteza, Alberto; Navajas, Daniel; Sarri, Elisabet; Rodríguez-Pascual, Fernando; Dietz, Harry C; Fabregat, Isabel; Egea, Gustavo

    2015-04-01

    Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level. Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls. In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation. © 2015 American Heart Association, Inc.

  14. Methylator phenotype of malignant germ cell tumours in children identifies strong candidates for chemotherapy resistance.

    Science.gov (United States)

    Jeyapalan, J N; Noor, D A Mohamed; Lee, S-H; Tan, C L; Appleby, V A; Kilday, J P; Palmer, R D; Schwalbe, E C; Clifford, S C; Walker, D A; Murray, M J; Coleman, N; Nicholson, J C; Scotting, P J

    2011-08-09

    Yolk sac tumours (YSTs) and germinomas are the two major pure histological subtypes of germ cell tumours. To date, the role of DNA methylation in the aetiology of this class of tumour has only been analysed in adult testicular forms and with respect to only a few genes. A bank of paediatric tumours was analysed for global methylation of LINE-1 repeat elements and global methylation of regulatory elements using GoldenGate methylation arrays. Both germinomas and YSTs exhibited significant global hypomethylation of LINE-1 elements. However, in germinomas, methylation of gene regulatory regions differed little from control samples, whereas YSTs exhibited increased methylation at a large proportion of the loci tested, showing a 'methylator' phenotype, including silencing of genes associated with Caspase-8-dependent apoptosis. Furthermore, we found that the methylator phenotype of YSTs was coincident with higher levels of expression of the DNA methyltransferase, DNA (cytosine-5)-methyltransferase 3B, suggesting a mechanism underlying the phenotype. Epigenetic silencing of a large number of potential tumour suppressor genes in YSTs might explain why they exhibit a more aggressive natural history than germinomas and silencing of genes associated with Caspase-8-dependent cell death might explain the relative resistance of YSTs to conventional therapy.

  15. Adult hippocampus derived soluble factors induce a neuronal-like phenotype in mesenchymal stem cells.

    Science.gov (United States)

    Rivera, Francisco J; Sierralta, Walter D; Minguell, Jose J; Aigner, Ludwig

    2006-10-02

    Bone marrow-derived mesenchymal stem cells (MSCs) are not restricted in their differentiation fate to cells of the mesenchymal lineage. They acquire a neural phenotype in vitro and in vivo after transplantation in the central nervous system. Here we investigated whether soluble factors derived from different brain regions are sufficient to induce a neuronal phenotype in MSCs. We incubated bone marrow-derived MSCs in conditioned medium (CM) derived from adult hippocampus (HCM), cortex (CoCM) or cerebellum (CeCM) and analyzed the cellular morphology and the expression of neuronal and glial markers. In contrast to muscle derived conditioned medium, which served as control, conditioned medium derived from the different brain regions induced a neuronal morphology and the expression of the neuronal markers GAP-43 and neurofilaments in MSCs. Hippocampus derived conditioned medium had the strongest activity. It was independent of NGF or BDNF; and it was restricted to the neuronal differentiation fate, since no induction of the astroglial marker GFAP was observed. The work indicates that soluble factors present in the brain are sufficient to induce a neuronal phenotype in MSCs.

  16. Phenotype Analysis and Quantification of Proliferating Cells in the Cortical Gray Matter of the Adult Rat

    International Nuclear Information System (INIS)

    Mori, Tetsuji; Wakabayashi, Taketoshi; Takamori, Yasuharu; Kitaya, Kotaro; Yamada, Hisao

    2009-01-01

    In intact adult mammalian brains, there are two neurogenic regions: the subependymal zone and the subgranular layer of the hippocampus. Even outside these regions, small numbers of proliferating precursors do exist. Many studies suggest that the majority of these are oligodendrocyte precursors that express NG2, a chondroitin sulfate proteoglycan, and most of the residual proliferating cells seem to be endothelial cells. However, it is still unclear whether NG2-immunonegative proliferating precursors are present, because previous studies have neglected their possible existence. In this study, we systematically analyzed the phenotypes of the proliferating cells in the intact adult rat cortical gray matter. We improved our techniques and carefully characterized the proliferating cells, because there were several problems with identifying and quantifying the proliferating cells: the detection of NG2-expressing cells was dependent on the fixation condition; there were residual proliferating leukocytes in the blood vessels; and two anti-NG2 antibodies gave rise to different staining patterns. Moreover, we used two methods, BrdU and Ki67 immunostaining, to quantify the proliferating cells. Our results strongly suggest that in the intact adult cerebral cortical gray matter, there were only two types of proliferating cells: the majority were NG2-expressing cells, including pericytes, and the rest were endothelial cells

  17. Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector.

    Science.gov (United States)

    Chakkaramakkil Verghese, Santhosh; Goloviznina, Natalya A; Kurre, Peter

    2016-11-19

    Fanconi anemia (FA) is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC)-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV) particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

  18. Phenotypic correction of Fanconi anemia cells in the murine bone marrow after carrier cell mediated delivery of lentiviral vector

    Directory of Open Access Journals (Sweden)

    Santhosh Chakkaramakkil Verghese

    2016-11-01

    Full Text Available Abstract Fanconi anemia (FA is an autosomal-recessive disorder associated with hematopoietic failure and it is a candidate for hematopoietic stem cell (HSC-directed gene therapy. However, the characteristically reduced HSC numbers found in FA patients, their ineffective mobilization from the marrow, and re-oxygenation damage during ex vivo manipulation have precluded clinical success using conventional in vitro approaches. We previously demonstrated that lentiviral vector (LV particles reversibly attach to the cell surface where they gain protection from serum complement neutralization. We reasoned that cellular delivery of LV to the bone marrow niche could avoid detrimental losses during FA HSC mobilization and in vitro modification. Here, we demonstrate that a VSV-G pseudotyped lentivector, carrying the FANCC transgene, can be transmitted from carrier to bystander cells. In cell culture and transplantation models of FA, we further demonstrate that LV carrier cells migrate along SDF-1α gradients and transfer vector particles that stably integrate and phenotypically correct the characteristic DNA alkylator sensitivity in murine and human FA-deficient target bystander cells. Altogether, we demonstrate that cellular homing mechanisms can be harnessed for the functional phenotype correction in murine FA hematopoietic cells.

  19. 27-hydroxycholesterol induces the transition of MCF7 cells into a mesenchymal phenotype.

    Science.gov (United States)

    Torres, Cristian G; Ramírez, María E; Cruz, Pamela; Epuñan, María J; Valladares, Luis E; Sierralta, Walter D

    2011-08-01

    A decrease in the expression of E-cadherin and β-catenin, paralleling the loss of adherens junction complex, was observed in MCF7 cells exposed for longer than 48 h to 2 µM 27-hydroxycholesterol (27OHC), indicating an epithelial-mesenchymal transition (EMT). Upon removal of 27OHC from the culture medium, the cells released by the exposure of 72 h to the oxysterol grew as loosely packed cell groups. In these cells, accumulation of E-cadherin and β-catenin in the cytoplasm and the prolonged expression of epidermal growth factor receptor 2 (EGFR2/neu) in the plasma membrane were observed, suggesting that the acquired phenotype was related to the expression of this tyrosine kinase-growth factor receptor. The results presented here are discussed on the basis of the claimed relationship between 27OHC, hypercholesterolemia, macrophage infiltration and therapy-resistant ERα+ breast cancer incidence.

  20. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes.

    Science.gov (United States)

    Han, Yo-Han; Kee, Ji-Ye; Kim, Dae-Seung; Mun, Jeong-Geon; Jeong, Mi-Young; Park, Sang-Hyun; Choi, Byung-Min; Park, Sung-Joo; Kim, Hyun-Jung; Um, Jae-Young; Hong, Seung-Heon

    2016-08-27

    Arctigenin (ARC) has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC). In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT) through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2) and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis.

  1. Arctigenin Inhibits Lung Metastasis of Colorectal Cancer by Regulating Cell Viability and Metastatic Phenotypes

    Directory of Open Access Journals (Sweden)

    Yo-Han Han

    2016-08-01

    Full Text Available Arctigenin (ARC has been shown to have an anti-cancer effect in various cell types and tissues. However, there have been no studies concerning metastatic colorectal cancer (CRC. In this study, we investigated the anti-metastatic properties of ARC on colorectal metastasis and present a potential candidate drug. ARC induced cell cycle arrest and apoptosis in CT26 cells through the intrinsic apoptotic pathway via MAPKs signaling. In several metastatic phenotypes, ARC controlled epithelial-mesenchymal transition (EMT through increasing the expression of epithelial marker E-cadherin and decreasing the expressions of mesenchymal markers; N-cadherin, vimentin, β-catenin, and Snail. Moreover, ARC inhibited migration and invasion through reducing of matrix metalloproteinase-2 (MMP-2 and MMP-9 expressions. In an experimental metastasis model, ARC significantly inhibited lung metastasis of CT26 cells. Taken together, our study demonstrates the inhibitory effects of ARC on colorectal metastasis.

  2. NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Jennifer Ann Foltz

    2016-11-01

    Full Text Available Canines spontaneously develop many cancers similar to humans - including osteosarcoma, leukemia, and lymphoma - offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46, the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3-/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3-/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3-/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and GM-CSF as measured by Luminex. Like human NK cells, CD3-/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median= 20,283-fold in 21 days. Further, we identify a minor Null population (CD3-/CD21-/CD14-/NKp46- with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3-/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells, and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46- subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy.

  3. High-resolution phenotypic profiling of natural products-induced effects on the single-cell level

    KAUST Repository

    Kremb, Stephan Georg; Voolstra, Christian R.

    2017-01-01

    Natural products (NPs) are highly evolved molecules making them a valuable resource for new therapeutics. Here we demonstrate the usefulness of broad-spectrum phenotypic profiling of NP-induced perturbations on single cells with imaging-based High

  4. Regulatory T cell frequencies and phenotypes following anti-viral vaccination.

    Directory of Open Access Journals (Sweden)

    A Charlotte M T de Wolf

    Full Text Available Regulatory T cells (Treg function in the prevention of excessive inflammation and maintenance of immunological homeostasis. However, these cells may also interfere with resolution of infections or with immune reactions following vaccination. Effects of Treg on vaccine responses are nowadays investigated, but the impact of vaccination on Treg homeostasis is still largely unknown. This may be a relevant safety aspect, since loss of tolerance through reduced Treg may trigger autoimmunity. In exploratory clinical trials, healthy adults were vaccinated with an influenza subunit vaccine plus or minus the adjuvant MF59®, an adjuvanted hepatitis B subunit vaccine or a live attenuated yellow fever vaccine. Frequencies and phenotypes of resting (rTreg and activated (aTreg subpopulations of circulating CD4+ Treg were determined and compared to placebo immunization. Vaccination with influenza vaccines did not result in significant changes in Treg frequencies and phenotypes. Vaccination with the hepatitis B vaccine led to slightly increased frequencies of both rTreg and aTreg subpopulations and a decrease in expression of functionality marker CD39 on aTreg. The live attenuated vaccine resulted in a decrease in rTreg frequency, and an increase in expression of activation marker CD25 on both subpopulations, possibly indicating a conversion from resting to migratory aTreg due to vaccine virus replication. To study the more local effects of vaccination on Treg in lymphoid organs, we immunized mice and analyzed the CD4+ Treg frequency and phenotype in draining lymph nodes and spleen. Vaccination resulted in a transient local decrease in Treg frequency in lymph nodes, followed by a systemic Treg increase in the spleen. Taken together, we showed that vaccination with vaccines with an already established safe profile have only minimal impact on frequencies and characteristics of Treg over time. These findings may serve as a bench-mark of inter-individual variation

  5. Phenotypic characterisation of cell populations in the brains of horses experimentally infected with West Nile virus.

    Science.gov (United States)

    Delcambre, G H; Liu, J; Streit, W J; Shaw, G P J; Vallario, K; Herrington, J; Wenzlow, N; Barr, K L; Long, M T

    2017-11-01

    West Nile virus (WNV), a mosquito borne member of the Flaviviridae, is one of the most commonly diagnosed agents of viral encephalitis in horses and people worldwide. A cassette of markers for formalin-fixed paraffin-embedded tissue and an archive of tissues from experimental infections in the horse were used to investigate the equine neuroimmune response to WNV meningoencephalomyelitis to phenotype the early response to WNV infection in the horse. Quantitative analysis using archived tissue from experimentally infected horses. The thalamus and hindbrain from 2 groups of 6 horses were compared and consisted of a culture positive tissues from WNV experimentally horses, in the other, normal horses. Formalin-fixed paraffin-embedded tissue from the thalamus and hindbrain were immunolabeled for microglia, astrocytes, B cells, macrophages/neutrophils, CD3 + T cells. Fresh frozen tissues were immunolabeled for CD4 + and CD8 + T lymphocyte cell markers. Cell counts were obtained using a computer software program. Differences, after meeting assumptions of abnormality, were computed using a general linear model with a Tukey test (Phorses, Iba-1 + microglia, CD3 + T lymphocyte and MAC387 + macrophage staining were significantly increased. The T cell response for the WNV-challenged horses was mixed, composed of CD4 + and CD8 + T lymphocytes. A limited astrocyte response was also observed in WNV-challenged horses, and MAC387 + and B cells were the least abundant cell populations. The results of this study were limited by a single collection time post-infection. Furthermore, a comprehensive analysis of cellular phenotypes is needed for naturally infected horses. Unfortunately, in clinical horses, there is high variability of sampling in terms of days post-infection and tissue handling. The data show that WNV-challenged horses recruit a mixed T cell population at the onset of neurologic disease. © 2017 EVJ Ltd.

  6. Population dynamics in vasopressin cells.

    Science.gov (United States)

    Leng, Gareth; Brown, Colin; Sabatier, Nancy; Scott, Victoria

    2008-01-01

    Most neurons sense and code change, and when presented with a constant stimulus they adapt, so as to be able to detect a fresh change. However, for some things it is important to know their absolute level; to encode such information, neurons must sustain their response to an unchanging stimulus while remaining able to respond to a change in that stimulus. One system that encodes the absolute level of a stimulus is the vasopressin system, which generates a hormonal signal that is proportional to plasma osmolality. Vasopressin cells sense plasma osmolality and secrete appropriate levels of vasopressin from the neurohypophysis as needed to control water excretion; this requires sustained secretion under basal conditions and the ability to increase (or decrease) secretion should plasma osmolality change. Here we explore the mechanisms that enable vasopressin cells to fulfill this function, and consider how coordination between the cells might distribute the secretory load across the population of vasopressin cells. 2008 S. Karger AG, Basel.

  7. Fractionated irradiation-induced EMT-like phenotype conferred radioresistance in esophageal squamous cell carcinoma

    Science.gov (United States)

    Zhang, Hongfang; Luo, Honglei; Jiang, Zhenzhen; Yue, Jing; Hou, Qiang; Xie, Ruifei; Wu, Shixiu

    2016-01-01

    The efficacy of radiotherapy, one major treatment modality for esophageal squamous cell carcinoma (ESCC) is severely attenuated by radioresistance. Epithelial-to-mesenchymal transition (EMT) is a cellular process that determines therapy response and tumor progression. However, whether EMT is induced by ionizing radiation and involved in tumor radioresistance has been less studied in ESCC. Using multiple fractionated irradiation, the radioresistant esophageal squamous cancer cell line KYSE-150R had been established from its parental cell line KYSE-150. We found KYSE-150R displayed a significant EMT phenotype with an elongated spindle shape and down-regulated epithelial marker E-cadherin and up-regulated mesenchymal marker N-cadherin in comparison with KYSE-150. Furthermore, KYSE-150R also possessed some stemness-like properties characterized by density-dependent growth promotion and strong capability for sphere formation and tumorigenesis in NOD-SCID mice. Mechanical studies have revealed that WISP1, a secreted matricellular protein, is highly expressed in KYSE-150R and mediates EMT-associated radioresistance both in ESCC cells and in xenograft tumor models. Moreover, WISP1 has been demonstrated to be closely associated with the EMT phenotype observed in ESCC patients and to be an independent prognosis factor of ESCC patients treated with radiotherapy. Our study highlighted WISP1 as an attractive target to reverse EMT-associated radioresistance in ESCC and can be used as an independent prognostic factor of patients treated with radiotherapy. PMID:27125498

  8. Characterization of DNA repair phenotypes of Xeroderma pigmentosum cell lines by a paralleled in vitro test

    International Nuclear Information System (INIS)

    Raffin, A.L.

    2009-06-01

    DNA is constantly damaged modifying the genetic information for which it encodes. Several cellular mechanisms as the Base Excision Repair (BER) and the Nucleotide Excision Repair (NER) allow recovering the right DNA sequence. The Xeroderma pigmentosum is a disease characterised by a deficiency in the NER pathway. The aim of this study was to propose an efficient and fast test for the diagnosis of this disease as an alternative to the currently available UDS test. DNA repair activities of XP cell lines were quantified using in vitro miniaturized and paralleled tests in order to establish DNA repair phenotypes of XPA and XPC deficient cells. The main advantage of the tests used in this study is the simultaneous measurement of excision or excision synthesis (ES) of several lesions by only one cellular extract. We showed on one hand that the relative ES of the different lesions depend strongly on the protein concentration of the nuclear extract tested. Working at high protein concentration allowed discriminating the XP phenotype versus the control one, whereas it was impossible under a certain concentration's threshold. On the other hand, while the UVB irradiation of control cells stimulated their repair activities, this effect was not observed in XP cells. This study brings new information on the XPA and XPC protein roles during BER and NER and underlines the complexity of the regulations of DNA repair processes. (author)

  9. Fractionated irradiation-induced EMT-like phenotype conferred radioresistance in esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Zhang, Hongfang; Luo, Honglei; Jiang, Zhenzhen; Yue, Jing; Hou, Qiang; Xie, Ruifei; Wu, Shixiu

    2016-01-01

    The efficacy of radiotherapy, one major treatment modality for esophageal squamous cell carcinoma (ESCC) is severely attenuated by radioresistance. Epithelial-to-mesenchymal transition (EMT) is a cellular process that determines therapy response and tumor progression. However, whether EMT is induced by ionizing radiation and involved in tumor radioresistance has been less studied in ESCC. Using multiple fractionated irradiation, the radioresistant esophageal squamous cancer cell line KYSE-150R had been established from its parental cell line KYSE-150. We found KYSE-150R displayed a significant EMT phenotype with an elongated spindle shape and down-regulated epithelial marker E-cadherin and up-regulated mesenchymal marker N-cadherin in comparison with KYSE-150. Furthermore, KYSE-150R also possessed some stemness-like properties characterized by density-dependent growth promotion and strong capability for sphere formation and tumorigenesis in NOD-SCID mice. Mechanical studies have revealed that WISP1, a secreted matricellular protein, is highly expressed in KYSE-150R and mediates EMT-associated radioresistance both in ESCC cells and in xenograft tumor models. Moreover, WISP1 has been demonstrated to be closely associated with the EMT phenotype observed in ESCC patients and to be an independent prognosis factor of ESCC patients treated with radiotherapy. Our study highlighted WISP1 as an attractive target to reverse EMT-associated radioresistance in ESCC and can be used as an independent prognostic factor of patients treated with radiotherapy

  10. Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury.

    Science.gov (United States)

    Thyberg, J

    1998-07-01

    Smooth muscle cells build up the media of mammalian arteries and constitute one of the principal cell types in atherosclerotic and restenotic lesions. Accordingly, they show a high degree of plasticity and are able to shift from a differentiated, contractile phenotype to a less differentiated, synthetic phenotype, and then back again. This modulation occurs as a response to vascular injury and includes a prominent structural reorganization with loss of myofilaments and formation of an extensive endoplasmic reticulum and a large Golgi complex. At the same time, the expression of cytoskeletal proteins and other gene products is altered. As a result, the cells lose their contractility and become able to migrate from the media to the intima, proliferate, and secrete extracellular matrix components, thereby contributing to the formation of intimal thickenings. The mechanisms behind this change in morphology and function of the smooth muscle cells are still incompletely understood. A crucial role has been ascribed to basement membrane proteins such as laminin and collagen type IV and adhesive proteins such as fibronectin. A significant role is also played by mitogenic proteins such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). An improved knowledge of the regulation of smooth muscle differentiated properties represents an important part in the search for new methods of prevention and treatment of vascular disease.

  11. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    2011-02-01

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  12. Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

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    Tamara Murmann

    Full Text Available Small cell lung carcinomas (SCLCs represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

  13. Phenotypic plasticity and epithelial-to-mesenchymal transition in the behaviour and therapeutic response of oral squamous cell carcinoma.

    Science.gov (United States)

    Vig, Navin; Mackenzie, Ian C; Biddle, Adrian

    2015-10-01

    It is increasingly recognised that phenotypic plasticity, apparently driven by epigenetic mechanisms, plays a key role in tumour behaviour and markedly influences the important processes of therapeutic survival and metastasis. An important source of plasticity in malignancy is epithelial-to-mesenchymal transition (EMT), a common epigenetically controlled event that results in transition of malignant cells between different phenotypic states that confer motility and enhance survival. In this review, we discuss the importance of phenotypic plasticity and its contribution to cellular heterogeneity in oral squamous cell carcinoma with emphasis on aspects of drug resistance and EMT. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells

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    T Vinardell

    2012-02-01

    Full Text Available Hydrostatic pressure (HP is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-β3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs, synovial membrane derived stem cells (SDSCs and infrapatellar fat pad derived stem cells (FPSCs were subjected to 10 MPa of cyclic HP (4 h/day and different concentrations of TGF-β3 (0, 1 and 10 ng/mL for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-β3 stimulation. HP in the absence of TGF-β3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-β3 (1 ng/mL, HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-β3 (10 ng/mL. Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.

  15. Glucocorticoids promote a glioma stem cell-like phenotype and resistance to chemotherapy in human glioblastoma primary cells

    DEFF Research Database (Denmark)

    Kostopoulou, Ourania N; Mohammad, Abdul-Aleem; Bartek, Jiri

    2018-01-01

    Glioma stem cells (GSCs) are glioblastoma (GBM) cells that are resistant to therapy and can give rise to recurrent tumors. The identification of patient-related factors that support GSCs is thus necessary to design effective therapies for GBM patients. Glucocorticoids (GCs) are used to treat GBM......-associated edema. However, glucocorticoids participate in the physiological response to psychosocial stress, which has been linked to poor cancer prognosis. This raises concern that glucocorticoids affect the tumor and GSCs. Here, we treated primary human GBM cells with dexamethasone and evaluated GC......-driven changes in cell morphology, proliferation, migration, gene expression, secretory activity and growth as neurospheres. Dexamethasone treatment of GBM cells appeared to promote the development of a GSC-like phenotype and conferred resistance to physiological stress and chemotherapy. We also analyzed...

  16. Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines

    International Nuclear Information System (INIS)

    Martowicz, Agnieszka; Spizzo, Gilbert; Gastl, Guenther; Untergasser, Gerold

    2012-01-01

    The epithelial cell adhesion molecule (EpCAM) has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype. For this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAM high breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3). Moreover, EpCAM low breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t) and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth. In comparison to non-specific silencing controls (n/s-crtl) knockdown of EpCAM (E#2) in EpCAM high cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAM low cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes. The role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer

  17. Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells

    International Nuclear Information System (INIS)

    Dozmorov, Mikhail G; Kyker, Kimberly D; Saban, Ricardo; Knowlton, Nicholas; Dozmorov, Igor; Centola, Michael B; Hurst, Robert E

    2006-01-01

    The extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy. Five bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified. Approximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions

  18. Phenotypic and functional analysis of CD1a+ dendritic cells from cats chronically infected with feline immunodeficiency virus.

    Science.gov (United States)

    Zhang, Lin; Reckling, Stacie; Dean, Gregg A

    2015-10-01

    Numerous studies suggest dendritic cell (DC) dysfunction is central to the dysregulated immune response during HIV infection; however, in vivo studies are lacking. In the present study we used feline immunodeficiency virus (FIV) infection of cats as a model for HIV-1 infection to assess the maturation and function of dendritic cells, in vivo and in vitro. We compared CD1a+ DC migration, surface phenotype, endocytosis, mixed leukocyte reaction (MLR) and regulatory T cell (Treg) phenotype induction by CD1a+ cells isolated from lymph nodes of FIV-infected and control cats. Results showed that resident CD1a+ DC in lymph nodes of chronically FIV-infected cats are phenotypically mature, can stimulate normal primary T cell proliferation, override Treg suppression and do not skew toward Treg induction. In contrast, FIV infection had deleterious effects on antigen presentation and migratory capacity of CD1a+ cells in tissues. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Oxygen-Related Differences in Cellular and Vesicular Phenotypes Observed for Ovarian Cell Cancer Lines

    DEFF Research Database (Denmark)

    Søndergaard, Evo K. Lindersson; Pugholm, Lotte Hatting; Bæk, Rikke

    2016-01-01

    Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients c...... produced more EVs.The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.......Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients...

  20. The phenotype of FancB-mutant mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan; Hasty, Paul

    2011-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  1. A monolithic glass chip for active single-cell sorting based on mechanical phenotyping.

    Science.gov (United States)

    Faigle, Christoph; Lautenschläger, Franziska; Whyte, Graeme; Homewood, Philip; Martín-Badosa, Estela; Guck, Jochen

    2015-03-07

    The mechanical properties of biological cells have long been considered as inherent markers of biological function and disease. However, the screening and active sorting of heterogeneous populations based on serial single-cell mechanical measurements has not been demonstrated. Here we present a novel monolithic glass chip for combined fluorescence detection and mechanical phenotyping using an optical stretcher. A new design and manufacturing process, involving the bonding of two asymmetrically etched glass plates, combines exact optical fiber alignment, low laser damage threshold and high imaging quality with the possibility of several microfluidic inlet and outlet channels. We show the utility of such a custom-built optical stretcher glass chip by measuring and sorting single cells in a heterogeneous population based on their different mechanical properties and verify sorting accuracy by simultaneous fluorescence detection. This offers new possibilities of exact characterization and sorting of small populations based on rheological properties for biological and biomedical applications.

  2. The phenotype of FancB-mutant mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States)

    2011-07-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  3. A matter of identity — Phenotype and differentiation potential of human somatic stem cells

    Directory of Open Access Journals (Sweden)

    S.E.P. New

    2015-07-01

    Full Text Available Human somatic stem cells with neural differentiation potential can be valuable for developing cell-based therapies, including treatment of birth-related defects, while avoiding issues associated with cell reprogramming. Precisely defining the “identity” and differentiation potential of somatic stem cells from different sources, has proven difficult, given differences in sets of specific markers, protocols used and lack of side-by-side characterization of these cells in different studies. Therefore, we set to compare expression of mesenchymal and neural markers in human umbilical cord-derived mesenchymal stem cells (UC-MSCs, pediatric adipose-derived stem cells (p-ADSCs in parallel with human neural stem cells (NSCs. We show that UC-MSCs at a basal level express mesenchymal and so-called “neural” markers, similar to that we previously reported for the p-ADSCs. All somatic stem cell populations studied, independently from tissue and patient of origin, displayed a remarkably similar expression of surface markers, with the main difference being the restricted expression of CD133 and CD34 to NSCs. Expression of certain surface and neural markers was affected by the expansion medium used. As predicted, UC-MSCs and p-ADSCs demonstrated tri-mesenchymal lineage differentiation potential, though p-ADSCs display superior chondrogenic differentiation capability. UC-MSCs and p-ADSCs responded also to neurogenic induction by up-regulating neuronal markers, but crucially they appeared morphologically immature when compared with differentiated NSCs. This highlights the need for further investigation into the use of these cells for neural therapies. Crucially, this study demonstrates the lack of simple means to distinguish between different cell types and the effect of culture conditions on their phenotype, and indicates that a more extensive set of markers should be used for somatic stem cell characterization, especially when developing therapeutic

  4. Phenotypic, ultra-structural, and functional characterization of bovine peripheral blood dendritic cell subsets.

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    Janet J Sei

    Full Text Available Dendritic cells (DC are multi-functional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets directly ex vivo, without further in vitro manipulation. Multi-color flow cytometric analysis revealed that three DC subsets could be identified. Bovine plasmacytoid DC were phenotypically identified by a unique pattern of cell surface protein expression including CD4, exhibited an extensive endoplasmic reticulum and Golgi apparatus, efficiently internalized and degraded exogenous antigen, and were the only peripheral blood cells specialized in the production of type I IFN following activation with Toll-like receptor (TLR agonists. Conventional DC were identified by expression of a different pattern of cell surface proteins including CD11c, MHC class II, and CD80, among others, the display of extensive dendritic protrusions on their plasma membrane, expression of very high levels of MHC class II and co-stimulatory molecules, efficient internalization and degradation of exogenous antigen, and ready production of detectable levels of TNF-alpha in response to TLR activation. Our investigations also revealed a third novel DC subset that may be a precursor of conventional DC that were MHC class II+ and CD11c-. These cells exhibited a smooth plasma membrane with a rounded nucleus, produced TNF-alpha in response to TLR-activation (albeit lower than CD11c+ DC, and were the least efficient in internalization/degradation of exogenous antigen. These studies define three bovine blood DC subsets with distinct phenotypic and functional characteristics which can be analyzed during immune responses to pathogens and vaccinations of cattle.

  5. MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Hisataka Ogawa

    Full Text Available Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors.

  6. Cell kinetics and genetic instabilities in differentiated type early gastric cancers with different mucin phenotype.

    Science.gov (United States)

    Shibata, Naomi; Watari, Jiro; Fujiya, Mikihiro; Tanno, Satoshi; Saitoh, Yusuke; Kohgo, Yutaka

    2003-01-01

    To clarify the biological impact and molecular pathogenesis of cellular phenotype in differentiated-type gastric cancers (DGCs), we investigated cell kinetics and genetic instabilities in early stage of DGCs. A total of 43 early gastric cancers (EGCs) were studied. EGCs were divided into 3 phenotypic categories: gastric (G type, n = 11), ordinary (O type, n = 20), and complete intestinal (CI type, n = 12) based on the combination of HGM, ConA, MUC2, and CD10. Proliferative index (PI), apoptotic index (AI), and p53 overexpression were investigated by immunohistochemical staining with anti-Ki-67, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method, and p53 antibody, respectively. Using a high-resolution fluorescent microsatellite analysis system, microsatellite instability (MSI) and loss of heterozygosity (LOH) were examined. Frameshift mutation analysis of transforming growth factor-beta type II receptor (TGF-betaRII) and bcl-2-associated X (BAX) in cancers with MSI was also performed. The mean AI/PI ratio values were 0.04 for G-type, 0.10 for O-type, and 0.13 for CI-type cancers--significantly lower in G type than in O and CI types (P = 0.02 and P = 0.001, respectively). No difference in the incidence of MSI and LOH was seen among the 3 cellular phenotypes. However, the major pattern of MSI, which showed drastic and widely dispersed changes and is related to an increased risk for cancer, was significantly higher in G and O types than in CI type (P cancers. These results indicate that G-type cancers are likely to show more aggressive behaviors than CI-type cancers, and that O-type cancers show the intermediate characteristics of both types. However, the molecular pathogenesis of each phenotypic cancer is not associated with microsatellite alterations. Copyright 2003, Elsevier Science (USA). All rights reserved.

  7. Interleukin-6 from subchondral bone mesenchymal stem cells contributes to the pathological phenotypes of experimental osteoarthritis

    Science.gov (United States)

    Wu, Xiaofeng; Cao, Lei; Li, Fan; Ma, Chao; Liu, Guangwang; Wang, Qiugen

    2018-01-01

    As a main cause of morbidity in the aged population, osteoarthritis (OA) is characterized by cartilage destruction, synovium inflammation, osteophytes, and subchondral bone sclerosis. To date its etiology remains elusive. Recent data highlight an important role of subchondral bone in the onset and progression of OA. Therefore, elucidating the mechanisms underlying abnormal subchondral bone could be of importance in the treatment of OA. Interleukin-6 is a proinflammatory cytokine involved in many physiological and pathological processes. Although in vitro and in vivo studies have indicated that IL-6 is an important cytokine in the physiopathogenesis of OA, its effects on subchondral bone have not been studied in OA animal models. In this study, we aimed to i) investigate the role of IL-6 in the pathological phenotypes of OA subchondral bone MSCs including increase in cell numbers, mineralization disorder and abnormal type I collagen production; ii) explore whether the systemic blockade of IL-6 signaling could alleviate the pathological phenotypes of experimental OA. We found that IL-6 was over-secreted by OA subchondral bone MSCs compared with normal MSCs and IL-6/STAT3 signaling was over-activated in subchondral bone MSCs, which contributed to the pathological phenotypes of OA subchondral bone MSCs. More importantly, systemic inhibition of IL-6/STAT3 signaling with IL-6 antibody or STAT3 inhibitor AG490 decreased the severity of pathological phenotypes of OA subchondral bone MSCs and cartilage lesions in OA. Our findings provide strong evidence for a pivotal role for IL-6 signaling in OA and open up new therapeutic perspectives. PMID:29736207

  8. β-MSCs: successful fusion of MSCs with β-cells results in a β-cell like phenotype.

    Science.gov (United States)

    Azizi, Zahra; Lange, Claudia; Paroni, Federico; Ardestani, Amin; Meyer, Anke; Wu, Yonghua; Zander, Axel R; Westenfelder, Christof; Maedler, Kathrin

    2016-08-02

    Bone marrow mesenchymal stromal cells (MSC) have anti-inflammatory, anti-apoptotic and immunosuppressive properties and are a potent source for cell therapy. Cell fusion has been proposed for rapid generation of functional new reprogrammed cells. In this study, we aimed to establish a fusion protocol of bone marrow-derived human MSCs with the rat beta-cell line (INS-1E) as well as human isolated pancreatic islets in order to generate insulin producing beta-MSCs as a cell-based treatment for diabetes.Human eGFP+ puromycin+ MSCs were co-cultured with either stably mCherry-expressing rat INS-1E cells or human dispersed islet cells and treated with phytohemagglutinin (PHA-P) and polyethylene glycol (PEG) to induce fusion. MSCs and fused cells were selected by puromycin treatment.With an improved fusion protocol, 29.8 ± 2.9% of all MSCs were β-MSC heterokaryons based on double positivity for mCherry and eGFP.After fusion and puromycin selection, human NKX6.1 and insulin as well as rat Neurod1, Nkx2.2, MafA, Pdx1 and Ins1 mRNA were highly elevated in fused human MSC/INS-1E cells, compared to the mixed control population. Such induction of beta-cell markers was confirmed in fused human MSC/human dispersed islet cells, which showed elevated NEUROD1, NKX2.2, MAFA, PDX1 and insulin mRNA compared to the mixed control. Fused cells had higher insulin content and improved insulin secretion compared to the mixed control and insulin positive beta-MSCs also expressed nuclear PDX1. We established a protocol for fusion of human MSCs and beta cells, which resulted in a beta cell like phenotype. This could be a novel tool for cell-based therapies of diabetes.

  9. Dysregulation of gene expression in the artificial human trisomy cells of chromosome 8 associated with transformed cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Hisakatsu Nawata

    Full Text Available A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression.

  10. Ethanol inhibits B16-BL6 melanoma metastasis and cell phenotypes associated with metastasis.

    Science.gov (United States)

    Kushiro, Kyoko; Núñez, Nomelí P

    2012-01-01

    Every year, approximately 68,000 new cases of malignant melanoma are diagnosed in the US. Ethanol consumption inhibits metastasis of melanoma in mice, but the mechanism is not well understood. C57BL/6J ob/+ mice, given either water or 20% ethanol, were injected intravenously with B16-BL6 melanoma cells to determine pulmonary metastasis. The effects of ethanol on cell phenotypes and markers of the epithelial-to-mesenchymal transition were determined in cell culture. In mice, ethanol consumption inhibited experimental pulmonary metastasis. This inhibition was associated with decreased body weight, and levels of systemic leptin, and insulin. In cell culture, ethanol inhibited B16-BL6 cell motility, invasion, and anchorage-independent growth. Additionally, ethanol reduced Snai1 expression and increased E-cadherin expression. Lastly, ethanol increased the expression of Kiss1 metastasis-suppressor and the metastasis suppressor Nm23/nucleoside diphosphate kinase. In both animal and in cell culture conditions, ethanol inhibited the metastatic ability of B16-BL6 melanoma cells.

  11. Phenotypic plasticity and effects of selection on cell division symmetry in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Uttara N Lele

    Full Text Available Aging has been demonstrated in unicellular organisms and is presumably due to asymmetric distribution of damaged proteins and other components during cell division. Whether the asymmetry-induced aging is inevitable or an adaptive and adaptable response is debated. Although asymmetric division leads to aging and death of some cells, it increases the effective growth rate of the population as shown by theoretical and empirical studies. Mathematical models predict on the other hand, that if the cells divide symmetrically, cellular aging may be delayed or absent, growth rate will be reduced but growth yield will increase at optimum repair rates. Therefore in nutritionally dilute (oligotrophic environments, where growth yield may be more critical for survival, symmetric division may get selected. These predictions have not been empirically tested so far. We report here that Escherichia coli grown in oligotrophic environments had greater morphological and functional symmetry in cell division. Both phenotypic plasticity and genetic selection appeared to shape cell division time asymmetry but plasticity was lost on prolonged selection. Lineages selected on high nutrient concentration showed greater frequency of presumably old or dead cells. Further, there was a negative correlation between cell division time asymmetry and growth yield but there was no significant correlation between asymmetry and growth rate. The results suggest that cellular aging driven by asymmetric division may not be hardwired but shows substantial plasticity as well as evolvability in response to the nutritional environment.

  12. Alteration of Lymphocyte Phenotype and Function in Sickle Cell Anemia: Implications for Vaccine Responses

    Science.gov (United States)

    Balandya, Emmanuel; Reynolds, Teri; Obaro, Stephen; Makani, Julie

    2016-01-01

    Individuals with sickle cell anemia (SCA) have increased susceptibility to infections, secondary to impairment of immune function. Besides the described dysfunction in innate immunity, including impaired opsonization and phagocytosis of bacteria, evidence of dysfunction of T and B lymphocytes in SCA has also been reported. This includes reduction in the proportion of circulating CD4+ and CD8+ T cells, reduction of CD4+ helper : CD8+ suppressor T cell ratio, aberrant activation and dysfunction of regulatory T cells (Treg), skewing of CD4+ T cells towards Th2 response and loss of IgM-secreting CD27+IgMhighIgDlow memory B cells. These changes occur on the background of immune activation characterized by predominance of memory CD4+ T cell phenotypes, increased Th17 signaling and elevated levels of C-reactive protein and pro-inflammatory cytokines IL-6 and TNF-α, which may affect the immunogenicity and protective efficacy of vaccines available to prevent infections in SCA. Thus, in order to optimize the use of vaccines in SCA, a thorough understanding of T and B lymphocyte functions and vaccine reactivity among individuals with SCA is needed. Studies should be encouraged of different SCA populations, including sub-Saharan Africa where the burden of SCA is highest. This article summarizes our current understanding of lymphocyte biology in SCA, and highlights areas that warrant future research. PMID:27237467

  13. Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

    Directory of Open Access Journals (Sweden)

    Aditi Gupta

    2013-01-01

    Full Text Available Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics.

  14. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

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    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  15. Phenotypic and gene expression changes between low (glucose-responsive) and High (glucose non-responsive) MIN-6 beta cells

    DEFF Research Database (Denmark)

    O´Driscoll, L.; Gammell, p.; McKierman, E.

    2006-01-01

    The long-term potential to routinely use replacement beta cells/islets as cell therapy for type 1 diabetes relies on our ability to culture such cells/islets, in vitro, while maintaining their functional status. Previous beta cell studies, by ourselves and other researchers, have indicated...... that the glucose-stimulated insulin secretion (GSIS) phenotype is relatively unstable, in long-term culture. This study aimed to investigate phenotypic and gene expression changes associated with this loss of GSIS, using the MIN-6 cell line as model. Phenotypic differences between MIN-6(L, low passage) and MIN-6(H......, high passage) were determined by ELISA (assessing GSIS and cellular (pro)insulin content), proliferation assays, phase contrast light microscopy and analysis of alkaline phosphatase expression. Differential mRNA expression was investigated using microarray, bioinformatics and real-time PCR technologies...

  16. uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells.

    Science.gov (United States)

    Laurenzana, Anna; Chillà, Anastasia; Luciani, Cristina; Peppicelli, Silvia; Biagioni, Alessio; Bianchini, Francesca; Tenedini, Elena; Torre, Eugenio; Mocali, Alessandra; Calorini, Lido; Margheri, Francesca; Fibbi, Gabriella; Del Rosso, Mario

    2017-09-15

    In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma. © 2017 UICC.

  17. Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.

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    Yoshihiro Sowa

    Full Text Available Recent studies have shown that adipose-derived stromal/stem cells (ASCs contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs. This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2 and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta. NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.

  18. The fossil record of phenotypic integration and modularity: A deep-time perspective on developmental and evolutionary dynamics.

    Science.gov (United States)

    Goswami, Anjali; Binder, Wendy J; Meachen, Julie; O'Keefe, F Robin

    2015-04-21

    Variation is the raw material for natural selection, but the factors shaping variation are still poorly understood. Genetic and developmental interactions can direct variation, but there has been little synthesis of these effects with the extrinsic factors that can shape biodiversity over large scales. The study of phenotypic integration and modularity has the capacity to unify these aspects of evolutionary study by estimating genetic and developmental interactions through the quantitative analysis of morphology, allowing for combined assessment of intrinsic and extrinsic effects. Data from the fossil record in particular are central to our understanding of phenotypic integration and modularity because they provide the only information on deep-time developmental and evolutionary dynamics, including trends in trait relationships and their role in shaping organismal diversity. Here, we demonstrate the important perspective on phenotypic integration provided by the fossil record with a study of Smilodon fatalis (saber-toothed cats) and Canis dirus (dire wolves). We quantified temporal trends in size, variance, phenotypic integration, and direct developmental integration (fluctuating asymmetry) through 27,000 y of Late Pleistocene climate change. Both S. fatalis and C. dirus showed a gradual decrease in magnitude of phenotypic integration and an increase in variance and the correlation between fluctuating asymmetry and overall integration through time, suggesting that developmental integration mediated morphological response to environmental change in the later populations of these species. These results are consistent with experimental studies and represent, to our knowledge, the first deep-time validation of the importance of developmental integration in stabilizing morphological evolution through periods of environmental change.

  19. T cell clones which share T cell receptor epitopes differ in phenotype, function and specificity

    NARCIS (Netherlands)

    Yssel, H.; Blanchard, D.; Boylston, A.; de Vries, J. E.; Spits, H.

    1986-01-01

    Recently, we described a monoclonal antibody (3D6) that reacts with the T cell receptor (Ti) of the T leukemic cell line HPB-ALL and that cross-reacts with 2-10% of the T cells of normal healthy individuals. In this study we report the establishment of T cell clones that are 3D6+ but that differ in

  20. Notch Signaling Is Associated With ALDH Activity And An Aggressive Metastatic Phenotype In Murine Osteosarcoma Cells

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    Xiaodong eMu

    2013-06-01

    Full Text Available Osteosarcoma (OS is the most common primary malignancy of bone, and pulmonary metastatic disease accounts for nearly all mortality. However, little is known about the biochemical signaling alterations that drive the progression of metastatic disease. Two murine OS cell populations, K7M2 and K12, are clonally related but differ significantly in their metastatic phenotypes and therefore represent excellent tools for studying metastatic OS molecular biology. K7M2 cells are highly metastatic, whereas K12 cells display limited metastatic potential. Here we report that the expression of Notch genes (Notch1, 2, 4 are up-regulated, including downstream targets Hes1 and Stat3, in the highly metastatic K7M2 cells compared to the less metastatic K12 cells, indicating that the Notch signaling pathway is more active in K7M2 cells. We have previously described that K7M2 cells exhibit higher levels of aldehyde dehydrogenase (ALDH activity. Here we report that K7M2 cell ALDH activity is reduced with Notch inhibition, suggesting that ALDH activity may be regulated in part by the Notch pathway. Notch signaling is also associated with increased resistance to oxidative stress, migration, invasion, and VEGF expression in vitro. However, Notch inhibition did not significantly alter K7M2 cell proliferation. In conclusion, we provide evidence that Notch signaling is associated with ALDH activity and increased metastatic behavior in OS cells. Both Notch and ALDH are putative molecular targets for the treatment and prevention of OS metastasis.

  1. Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes.

    Science.gov (United States)

    Reed-Geaghan, Erin G; Wright, Margaret C; See, Lauren A; Adelman, Peter C; Lee, Kuan Hsien; Koerber, H Richard; Maricich, Stephen M

    2016-04-13

    The extent to which the skin instructs peripheral somatosensory neuron maturation is unknown. We studied this question in Merkel cell-neurite complexes, where slowly adapting type I (SAI) neurons innervate skin-derived Merkel cells. Transgenic mice lacking Merkel cells had normal dorsal root ganglion (DRG) neuron numbers, but fewer DRG neurons expressed the SAI markers TrkB, TrkC, and Ret. Merkel cell ablation also decreased downstream TrkB signaling in DRGs, and altered the expression of genes associated with SAI development and function. Skin- and Merkel cell-specific deletion of Bdnf during embryogenesis, but not postnatal Bdnf deletion or Ntf3 deletion, reproduced these results. Furthermore, prototypical SAI electrophysiological signatures were absent from skin regions where Bdnf was deleted in embryonic Merkel cells. We conclude that BDNF produced by Merkel cells during a precise embryonic period guides SAI neuron development, providing the first direct evidence that the skin instructs sensory neuron molecular and functional maturation. Peripheral sensory neurons show incredible phenotypic and functional diversity that is initiated early by cell-autonomous and local environmental factors found within the DRG. However, the contribution of target tissues to subsequent sensory neuron development remains unknown. We show that Merkel cells are required for the molecular and functional maturation of the SAI neurons that innervate them. We also show that this process is controlled by BDNF signaling. These findings provide new insights into the regulation of somatosensory neuron development and reveal a novel way in which Merkel cells participate in mechanosensation. Copyright © 2016 the authors 0270-6474/16/364362-15$15.00/0.

  2. A Developed NK-92MI Cell Line with Siglec-7neg Phenotype Exhibits High and Sustainable Cytotoxicity against Leukemia Cells

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    Chin-Han Huang

    2018-04-01

    Full Text Available Altered sialic acid processing that leads to upregulation of cell surface sialylation is recognized as a key change in malignant tissue glycosylation. This cancer-associated hypersialylation directly impacts the signaling interactions between tumor cells and their surrounding microenvironment, especially the interactions mediated by immune cell surface sialic acid-binding immunoglobulin-like lectins (Siglecs to relay inhibitory signals for cytotoxicity. First, we obtained a Siglec-7neg NK-92MI cell line, NK-92MI-S7N, by separating a group of Siglec-7neg cell population from an eight-month-long-term NK-92MI in vitro culture by fluorescence-activated cell sorting (FACS. The effect of Siglec-7 loss on NK-92MI-S7N cells was characterized by the cell morphology, proliferation, and cytotoxic activity via FACS, MTS assay, cytotoxic assay, and natural killer (NK degranulation assay. We found the expression levels of Siglec-7 in NK-92MI were negatively correlated with NK cytotoxicity against leukemia cells. This NK-92MI-S7N cell not only shared very similar phenotypes with its parental cells but also possessed a high and sustainable killing activity. Furthermore, this Siglec-7neg NK line was unexpectedly capable of eliminating a NK-92MI-resistant leukemia cell, THP-1, through enhancing the effector-target interaction. In this study, a NK cell line with high and sustainable cytotoxicity was established and this cell may provide a potential application in NK-based treatment for leukemia patients.

  3. Cyclebase 3.0: a multi-organism database on cell-cycle regulation and phenotypes.

    Science.gov (United States)

    Santos, Alberto; Wernersson, Rasmus; Jensen, Lars Juhl

    2015-01-01

    The eukaryotic cell division cycle is a highly regulated process that consists of a complex series of events and involves thousands of proteins. Researchers have studied the regulation of the cell cycle in several organisms, employing a wide range of high-throughput technologies, such as microarray-based mRNA expression profiling and quantitative proteomics. Due to its complexity, the cell cycle can also fail or otherwise change in many different ways if important genes are knocked out, which has been studied in several microscopy-based knockdown screens. The data from these many large-scale efforts are not easily accessed, analyzed and combined due to their inherent heterogeneity. To address this, we have created Cyclebase--available at http://www.cyclebase.org--an online database that allows users to easily visualize and download results from genome-wide cell-cycle-related experiments. In Cyclebase version 3.0, we have updated the content of the database to reflect changes to genome annotation, added new mRNA and protein expression data, and integrated cell-cycle phenotype information from high-content screens and model-organism databases. The new version of Cyclebase also features a new web interface, designed around an overview figure that summarizes all the cell-cycle-related data for a gene. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. The phenotypes of podocytes and parietal epithelial cells may overlap in diabetic nephropathy.

    Science.gov (United States)

    Andeen, Nicole K; Nguyen, Tri Q; Steegh, Floor; Hudkins, Kelly L; Najafian, Behzad; Alpers, Charles E

    2015-11-01

    Reversal of diabetic nephropathy (DN) has been achieved in humans and mice, but only rarely and under special circumstances. As progression of DN is related to podocyte loss, reversal of DN requires restoration of podocytes. Here, we identified and quantified potential glomerular progenitor cells that could be a source for restored podocytes. DN was identified in 31 human renal biopsy cases and separated into morphologically early or advanced lesions. Markers of podocytes (WT-1, p57), parietal epithelial cells (PECs) (claudin-1), and cell proliferation (Ki-67) were identified by immunohistochemistry. Podocyte density was progressively reduced with DN. Cells marking as podocytes (p57) were present infrequently on Bowman's capsule in controls, but significantly increased in histologically early DN. Ki-67-expressing cells were identified on the glomerular tuft and Bowman's capsule in DN, but rarely in controls. Cells marking as PECs were present on the glomerular tuft, particularly in morphologically advanced DN. These findings show evidence of phenotypic plasticity in podocyte and PEC populations and are consistent with studies in the BTBR ob/ob murine model in which reversibility of DN occurs with podocytes potentially regenerating from PEC precursors. Thus, our findings support, but do not prove, that podocytes may regenerate from PEC progenitors in human DN. If so, progression of DN may represent a modifiable net balance between podocyte loss and regeneration.

  5. Interpreting phenotypic antibiotic tolerance and persister cells as evolution via epigenetic inheritance.

    Science.gov (United States)

    Day, Troy

    2016-04-01

    Epigenetic inheritance is the transmission of nongenetic material such as gene expression levels, RNA and other biomolecules from parents to offspring. There is a growing realization that such forms of inheritance can play an important role in evolution. Bacteria represent a prime example of epigenetic inheritance because a large array of cellular components is transmitted to offspring, in addition to genetic material. Interestingly, there is an extensive and growing empirical literature showing that many bacteria can form 'persister' cells that are phenotypically resistant or tolerant to antibiotics, but most of these results are not interpreted within the context of epigenetic inheritance. Instead, persister cells are usually viewed as a genetically encoded bet-hedging strategy that has evolved in response to a fluctuating environment. Here I show, using a relatively simple model, that many of these empirical findings can be more simply understood as arising from a combination of epigenetic inheritance and cellular noise. I therefore suggest that phenotypic drug tolerance in bacteria might represent one of the best-studied examples of evolution under epigenetic inheritance. © 2016 John Wiley & Sons Ltd.

  6. The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures

    International Nuclear Information System (INIS)

    Krause, Silva; Maffini, Maricel V; Soto, Ana M; Sonnenschein, Carlos

    2010-01-01

    Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human

  7. [Construction of 2-dimensional tumor microvascular architecture phenotype in non-small cell lung cancer].

    Science.gov (United States)

    Liu, Jin-kang; Wang, Xiao-yi; Xiong, Zeng; Zhou, Hui; Zhou, Jian-hua; Fu, Chun-yan; Li, Bo

    2008-08-01

    To construct a technological platform of 2-dimensional tumor microvascular architecture phenotype (2D-TAMP) expression. Thirty samples of non-small cell lung cancer (NSCLC) were collected after surgery. The corresponding sections of tumor tissue specimens to the slice of CT perfusion imaging were selected. Immunohistochemical staining,Gomori methenamine silver stain, and electron microscope observation were performed to build a technological platform of 2D-TMAP expression by detecting the morphology and the integrity of basement membrane of microvasculature, microvascular density, various microvascular subtype, the degree of the maturity and lumenization of microvasculature, and the characteristics of immunogenetics of microvasculature. The technological platform of 2D-TMAP expression was constructed successfully. There was heterogeneity in 2D-TMAP expression of non-small cell lung cancer. The microvascular of NSCLC had certain characteristics. 2D-TMAP is a key technology that can be used to observe the overall state of micro-environment in tumor growth.

  8. Phenotypic analysis of perennial airborne allergen-specific CD4+ T cells in atopic and non-atopic individuals.

    Science.gov (United States)

    Crack, L R; Chan, H W; McPherson, T; Ogg, G S

    2011-11-01

    Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. © 2011 Blackwell Publishing Ltd.

  9. Effects of icotinib on advanced non-small cell lung cancer with different EGFR phenotypes.

    Science.gov (United States)

    Pan, Huiyun; Liu, Rong; Li, Shengjie; Fang, Hui; Wang, Ziwei; Huang, Sheng; Zhou, Jianying

    2014-09-01

    Icotinib is the first oral epidermal growth factor receptor (EGFR) tyrosine kinase receptor inhibitor, which has been proven to exert significant inhibitory effects on non-small cell lung cancer in vitro. Clinical evidence has showed that the efficacy of Icotinib on retreating advanced non-small cell lung cancer is comparable to Gefitinib. However, different phenotypes of EGFR can affect the therapeutic outcomes of EGFR tyrosine kinase receptor inhibitor. Therefore, our study focused on efficacy and safety of Icotinib in patients with advanced non-small cell lung cancer of different EGPR phenotypes. Clinical data of patients with advanced non-small cell lung cancer who received Icotinib treatment from August, 2011 to May, 2013 were retrospectively analyzed. Kaplan-Meier analysis was used for survival analysis and comparison. 18 wild-type EGFR and 51 mutant type were found in a total of 69 patients. Objective response rate of patients with mutant type EGFR was 54.9 % and disease control rate was 86.3 %. Objective response rate of wild-type patients was 11.1 % (P = 0.0013 vs mutant type), disease control rate was 50.0 % (P = 0.0017). Median progression-free survival (PFS) of mutant type and wild-type patients were 9.7 and 2.6 months, respectively (P Icotinib included rash, diarrhea, itching skin with occurrence rates of 24.6 % (17/69), 13.0 % (9/69), and 11.6 % (8/69), respectively. Most adverse reactions were grade I-II. Icotinib has great efficacy in EGFR mutated patients, making it an optimal regimen to treat EGFR mutated patients. Furthermore, most of adverse reactions associated with Icotinib treatment were tolerable.

  10. Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Yener Bülent

    2007-10-01

    Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

  11. Nuclear import mechanism for myocardin family members and their correlation with vascular smooth muscle cell phenotype.

    Science.gov (United States)

    Nakamura, Seiji; Hayashi, Ken'ichiro; Iwasaki, Kazuhiro; Fujioka, Tomoaki; Egusa, Hiroshi; Yatani, Hirofumi; Sobue, Kenji

    2010-11-26

    Myocardin (Mycd), which is essential for the differentiation of the smooth muscle cell lineage, is constitutively located in the nucleus, although its family members, myocardin-related transcription factors A and B (MRTF-A/B), mostly reside in the cytoplasm and translocate to the nucleus in response to Rho signaling. The mechanism for their nuclear import is unclear. Here we investigated the mechanism for the nuclear import of Mycd family members and demonstrated any correlation between such mechanism and the phenotype of vascular smooth muscle cells (VSMCs). In cultured VSMCs, the knockdown of importin β1 inhibited the nuclear import of Mycd and MRTF-A/B. Their NH(2)-terminal basic domain was identified as a binding site for importin α/β1 by in vitro analyses. However, Mycd had a higher affinity for importin α/β1 than did MRTF-A/B, even in the absence of G-actin, and Mycd affinity for importin α1/β1 was stronger than for any other importin α/β1 heterodimers. The binding of Mycd to importin α/β1 was insensitive to G-actin, whereas that of MRTF-A/B was differently inhibited by G-actin. In dedifferentiated VSMCs, the levels of importins α1 and β1 were reduced concomitant with down-regulation of Mycd, serum response factor, and smooth muscle cell markers. By contrast, in differentiated VSMCs, their expressions were up-regulated. Thus, the nuclear import of Mycd family members in VSMCs depends on importin α/β1, and their relative affinities for importin α/β1 heterodimers determine Mycd nuclear import. The expression of Mycd nuclear import machineries is related to the expression levels of VSMC phenotype-dependent smooth muscle cell markers.

  12. Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry.

    Directory of Open Access Journals (Sweden)

    Giovanna Clavarino

    Full Text Available A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45 and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.

  13. Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection

    Science.gov (United States)

    Kamaladasa, A.; Wickramasinghe, N.; Adikari, T. N.; Gomes, L.; Shyamali, N. L. A.; Salio, M.; Cerundolo, V.; Ogg, G. S.

    2016-01-01

    Summary Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)‐γ and interleukin (IL)−4 ex‐vivo enzyme‐linked immunospot (ELISPOT) assays following stimulation with alpha‐galactosyl‐ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4+ subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus‐specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl‐6 (P = 0·0003) and both Bcl‐6 and inducible T cell co‐stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4+ iNKT cells, with reduced expression of CD161 markers. PMID:26874822

  14. Cranberry and Grape Seed Extracts Inhibit the Proliferative Phenotype of Oral Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Kourt Chatelain

    2011-01-01

    Full Text Available Proanthocyanidins, compounds highly concentrated in dietary fruits, such as cranberries and grapes, demonstrate significant cancer prevention potential against many types of cancer. The objective of this study was to evaluate cranberry and grape seed extracts to quantitate and compare their anti-proliferative effects on the most common type of oral cancer, oral squamous cell carcinoma. Using two well-characterized oral squamous cell carcinoma cell lines, CAL27 and SCC25, assays were performed to evaluate the effects of cranberry and grape seed extract on phenotypic behaviors of these oral cancers. The proliferation of both oral cancer cell lines was significantly inhibited by the administration of cranberry and grape seed extracts, in a dose-dependent manner. In addition, key regulators of apoptosis, caspase-2 and caspase-8, were concomitantly up-regulated by these treatments. However, cranberry and grape seed extracts elicited differential effects on cell adhesion, cell morphology, and cell cycle regulatory pathways. This study represents one of the first comparative investigations of cranberry and grape seed extracts and their anti-proliferative effects on oral cancers. Previous findings using purified proanthocyanidin from grape seed extract demonstrated more prominent growth inhibition, as well as apoptosis-inducing, properties on CAL27 cells. These observations provide evidence that cranberry and grape seed extracts not only inhibit oral cancer proliferation but also that the mechanism of this inhibition may function by triggering key apoptotic regulators in these cell lines. This information will be of benefit to researchers interested in elucidating which dietary components are central to mechanisms involved in the mediation of oral carcinogenesis and progression.

  15. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...

  16. Lifespan Extension and Sustained Expression of Stem Cell Phenotype of Human Breast Epithelial Stem Cells in a Medium with Antioxidants

    Directory of Open Access Journals (Sweden)

    Kai-Hung Wang

    2016-01-01

    Full Text Available We have previously reported the isolation and culture of a human breast epithelial cell type with stem cell characteristics (Type I HBEC from reduction mammoplasty using the MSU-1 medium. Subsequently, we have developed several different normal human adult stem cell types from different tissues using the K-NAC medium. In this study, we determined whether this low calcium K-NAC medium with antioxidants (N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate is a better medium to grow human breast epithelial cells. The results clearly show that the K-NAC medium is a superior medium for prolonged growth (cumulative population doubling levels ranged from 30 to 40 of normal breast epithelial cells that expressed stem cell phenotypes. The characteristics of these mammary stem cells include deficiency in gap junctional intercellular communication, expression of Oct-4, and the ability to differentiate into basal epithelial cells and to form organoid showing mammary ductal and terminal end bud-like structures. Thus, this new method of growing Type I HBECs will be very useful in future studies of mammary development, breast carcinogenesis, chemoprevention, and cancer therapy.

  17. Characterization of T cell clones from chagasic patients: predominance of CD8 surface phenotype in clones from patients with pathology

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    Washington R. Cuna

    1995-08-01

    Full Text Available Human Chagas' disease, caused by the protozoan Trypanosoma cruzi, is associated with pathological processes whose mechanisms are not known. To address this question, T cell lines were developed from chronic chagasic patients peripheral blood mononuclear cells (PBMC and cloned. These T cell clones (TCC were analyzed phenotypically with monoclonal antibodies by the use of a fluorescence microscope. The surface phenotype of the TCC from the asymptomatic patient were predominantly CD4 positive (86%. On the contrary, the surface phenotype CD8 was predominant in the TCC from the patients suffering from cardiomegaly with right bundle branch block (83%, bradycardia with megacolon (75 % and bradycardia (75%. Future studies will be developed in order to identify the antigens eliciting these T cell subpopulations.

  18. Phenotypic characterization of aberrant stem and progenitor cell populations in myelodysplastic syndromes.

    Science.gov (United States)

    Ostendorf, Benjamin N; Flenner, Eva; Flörcken, Anne; Westermann, Jörg

    2018-01-01

    Recent reports have revealed myelodysplastic syndromes (MDS) to arise from cancer stem cells phenotypically similar to physiological hematopoietic stem cells. Myelodysplastic hematopoiesis maintains a hierarchical organization, but the proportion of several hematopoietic compartments is skewed and multiple surface markers are aberrantly expressed. These aberrant antigen expression patterns hold diagnostic and therapeutic promise. However, eradication of MDS requires targeting of early myelodysplasia propagating stem cells. This warrants an exact assessment of the differentiation stage at which aberrant expression occurs in transformed hematopoiesis. Here, we report results on the prospective and extensive dissection of the hematopoietic hierarchy in 20 patients with either low-risk MDS or MDS with excess blasts and compare it to hematopoiesis in patients with non-malignancy-associated cytopenia or B cell lymphoma without bone marrow infiltration. We found patients with MDS with excess blasts to exhibit characteristic expansions of specific immature progenitor compartments. We also identified the aberrant expression of several markers including ALDH, CLL-1, CD44, and CD47 to be specific features of hematopoiesis in MDS with excess blasts. We show that amongst these, aberrant CLL-1 expression manifested at the early uncommitted hematopoietic stem cell level, suggesting a potential role as a therapeutic target.

  19. Phenotypic characterization of aberrant stem and progenitor cell populations in myelodysplastic syndromes.

    Directory of Open Access Journals (Sweden)

    Benjamin N Ostendorf

    Full Text Available Recent reports have revealed myelodysplastic syndromes (MDS to arise from cancer stem cells phenotypically similar to physiological hematopoietic stem cells. Myelodysplastic hematopoiesis maintains a hierarchical organization, but the proportion of several hematopoietic compartments is skewed and multiple surface markers are aberrantly expressed. These aberrant antigen expression patterns hold diagnostic and therapeutic promise. However, eradication of MDS requires targeting of early myelodysplasia propagating stem cells. This warrants an exact assessment of the differentiation stage at which aberrant expression occurs in transformed hematopoiesis. Here, we report results on the prospective and extensive dissection of the hematopoietic hierarchy in 20 patients with either low-risk MDS or MDS with excess blasts and compare it to hematopoiesis in patients with non-malignancy-associated cytopenia or B cell lymphoma without bone marrow infiltration. We found patients with MDS with excess blasts to exhibit characteristic expansions of specific immature progenitor compartments. We also identified the aberrant expression of several markers including ALDH, CLL-1, CD44, and CD47 to be specific features of hematopoiesis in MDS with excess blasts. We show that amongst these, aberrant CLL-1 expression manifested at the early uncommitted hematopoietic stem cell level, suggesting a potential role as a therapeutic target.

  20. Non-Cell Autonomous Effects of the Senescence-Associated Secretory Phenotype in Cancer Therapy

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    Tareq Saleh

    2018-05-01

    Full Text Available In addition to promoting various forms of cell death, most conventional anti-tumor therapies also promote senescence. There is now extensive evidence that therapy-induced senescence (TIS might be transient, raising the concern that TIS could represent an undesirable outcome of therapy by providing a mechanism for tumor dormancy and eventual disease recurrence. The senescence-associated secretory phenotype (SASP is a hallmark of TIS and may contribute to aberrant effects of cancer therapy. Here, we propose that the SASP may also serve as a major driver of escape from senescence and the re-emergence of proliferating tumor cells, wherein factors secreted from the senescent cells contribute to the restoration of tumor growth in a non-cell autonomous fashion. Accordingly, anti-SASP therapies might serve to mitigate the deleterious outcomes of TIS. In addition to providing an overview of the putative actions of the SASP, we discuss recent efforts to identify and eliminate senescent tumor cells.

  1. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

    Directory of Open Access Journals (Sweden)

    Emilio Satoshi Hara

    Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

  2. The dynamic landscape of the cell nucleus.

    Science.gov (United States)

    Austin, Christopher M; Bellini, Michel

    2010-01-01

    While the cell nucleus was described for the first time almost two centuries ago, our modern view of the nuclear architecture is primarily based on studies from the last two decades. This surprising late start coincides with the development of new, powerful strategies to probe for the spatial organization of nuclear activities in both fixed and live cells. As a result, three major principles have emerged: first, the nucleus is not just a bag filled with nucleic acids and proteins. Rather, many distinct functional domains, including the chromosomes, resides within the confines of the nuclear envelope. Second, all these nuclear domains are highly dynamic, with molecules exchanging rapidly between them and the surrounding nucleoplasm. Finally, the motion of molecules within the nucleoplasm appears to be mostly driven by random diffusion. Here, the emerging roles of several subnuclear domains are discussed in the context of the dynamic functions of the cell nucleus.

  3. Increased osmolarity and cell clustering preserve canine notochordal cell phenotype in culture

    NARCIS (Netherlands)

    Spillekom, S.; Smolders, L.A.; Grinwis, G.C.M.; Arkesteijn, I.T.M.; Ito, K.; Meij, B.P.; Tryfonidou, M.A.

    2014-01-01

    Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive

  4. Maternal Adaptive Immune Cells in Decidua Parietalis Display a More Activated and Coinhibitory Phenotype Compared to Decidua Basalis

    Directory of Open Access Journals (Sweden)

    Martin Solders

    2017-01-01

    Full Text Available The maternal part of the placenta, the decidua, consists of maternal immune cells, decidual stromal cells, and extravillous fetal trophoblasts. In a successful pregnancy, these cell compartments interact to provide an intricate balance between fetal tolerance and antimicrobial defense. These processes are still poorly characterized in the two anatomically different decidual tissues, basalis and parietalis. We examined immune cells from decidua basalis and parietalis from term placentas (n=15 with flow cytometry. By using multivariate discriminant analysis, we found a clear separation between the two decidual compartments based on the 81 investigated parameters. Decidua parietalis lymphocytes displayed a more activated phenotype with a higher expression of coinhibitory markers than those isolated from basalis and contained higher frequencies of T regulatory cells. Decidua basalis contained higher proportions of monocytes, B cells, and mucosal-associated invariant T (MAIT cells. The basalis B cells were more immature, and parietalis MAIT cells showed a more activated phenotype. Conventional T cells, NK cells, and MAIT cells from both compartments potently responded with the production of interferon-γ and/or cytotoxic molecules in response to stimulation. To conclude, leukocytes in decidua basalis and parietalis displayed remarkable phenotypic disparities, indicating that the corresponding stromal microenvironments provide different immunoregulatory signals.

  5. [Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

    Science.gov (United States)

    Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

    2012-07-01

    To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

  6. Mechanical phenotyping of cells and extracellular matrix as grade and stage markers of lung tumor tissues.

    Science.gov (United States)

    Panzetta, Valeria; Musella, Ida; Rapa, Ida; Volante, Marco; Netti, Paolo A; Fusco, Sabato

    2017-07-15

    The mechanical cross-talk between cells and the extra-cellular matrix (ECM) regulates the properties, functions and healthiness of the tissues. When this is disturbed it changes the mechanical state of the tissue components, singularly or together, and cancer, along with other diseases, may start and progress. However, the bi-univocal mechanical interplay between cells and the ECM is still not properly understood. In this study we show how a microrheology technique gives us the opportunity to evaluate the mechanics of cells and the ECM at the same time. The mechanical phenotyping was performed on the surgically removed tissues of 10 patients affected by adenocarcinoma of the lung. A correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Our findings suggest a sort of asymmetric modification of the mechanical properties of the cells and the extra-cellular matrix in the tumor, being the more compliant cell even though it resides in a stiffer matrix. Overall, the simultaneous mechanical characterization of the tissues constituents (cells and ECM) provided new support for diagnosis and offered alternative points of analysis for cancer mechanobiology. When the integrity of the mechanical cross-talk between cells and the extra-cellular matrix is disturbed cancer, along with other diseases, may initiate and progress. Here, we show how a new technique gives the opportunity to evaluate the mechanics of cells and the ECM at the same time. It was applied on surgically removed tissues of 10 patients affected by adenocarcinoma of the lung and a correlation between the mechanics and the grade and stage of the tumor was reported and compared to the mechanical characteristics of the healthy tissue. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  7. Interstitial flows promote an amoeboid cell phenotype and motility of breast cancer cells

    Science.gov (United States)

    Tung, Chih-Kuan; Huang, Yu Ling; Zheng, Angela; Wu, Mingming

    2015-03-01

    Lymph nodes, the drainage systems for interstitial flows, are clinically known to be the first metastatic sites of many cancer types including breast and prostate cancers. Here, we demonstrate that breast cancer cell morphology and motility is modulated by interstitial flows in a cell-ECM adhesion dependent manner. The average aspect ratios of the cells are significantly lower (or are more amoeboid like) in the presence of the flow in comparison to the case when the flow is absent. The addition of exogenous adhesion molecules within the extracellular matrix (type I collagen) enhances the overall aspect ratio (or are more mesenchymal like) of the cell population. Using measured cell trajectories, we find that the persistence of the amoeboid cells (aspect ratio less than 2.0) is shorter than that of mesenchymal cells. However, the maximum speed of the amoeboid cells is larger than that of mesenchymal cells. Together these findings provide the novel insight that interstitial flows promote amoeboid cell morphology and motility and highlight the plasticity of tumor cell motility in response to its biophysical environment. Supported by NIH Grant R21CA138366.

  8. Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype.

    Science.gov (United States)

    Zhang, Yuanyuan; He, Yujiang; Bharadwaj, Shantaram; Hammam, Nevin; Carnagey, Kristen; Myers, Regina; Atala, Anthony; Van Dyke, Mark

    2009-08-01

    Recent studies have shown that extracellular matrix (ECM) substitutes can have a dramatic impact on cell growth, differentiation and function. However, these ECMs are often applied generically and have yet to be developed for specific cell types. In this study, we developed tissue-specific ECM-based coating substrates for skin, skeletal muscle and liver cell cultures. Cellular components were removed from adult skin, skeletal muscle, and liver tissues, and the resulting acellular matrices were homogenized and dissolved. The ECM solutions were used to coat culture dishes. Tissue matched and non-tissue matched cell types were grown on these coatings to assess adhesion, proliferation, maintenance of phenotype and cell function at several time points. Each cell type showed better proliferation and differentiation in cultures containing ECM from their tissue of origin. Although subtle compositional differences in the three ECM types were not investigated in this study, these results suggest that tissue-specific ECMs provide a culture microenvironment that is similar to the in vivo environment when used as coating substrates, and this new culture technique has the potential for use in drug development and the development of cell-based therapies.

  9. Tumor Cells Express FcγRl Which Contributes to Tumor Cell Growth and a Metastatic Phenotype

    Directory of Open Access Journals (Sweden)

    M. Bud Nelson

    2001-01-01

    Full Text Available High levels of circulating immune complexes containing tumor-associated antigens are associated with a poor prognosis for individuals with cancer. The ability of B cells, previously exposed to tumor-associated antigens, to promote both in vitro and in vivo tumor growth formed the rationale to evaluate the mechanism by which immune complexes may promote tumor growth. In elucidating this mechanism, FcγRl expression by tumor cells was characterized by flow cytometry, polymerase chain reaction, and sequence analysis. Immune complexes containing shed tumor antigen and anti-shed tumor antigen Ab cross-linked FcγRl-expressing tumor cells, which resulted in an induction of tumor cell proliferation and of shed tumor antigen production. Use of selective tyrosine kinase inhibitors demonstrated that tumor cell proliferation induced by immune complex cross-linking of FcγRl is dependent on the tyrosine kinase signal transduction pathway. A selective inhibitor of phosphatidylinositol-3 kinase also inhibited this induction of tumor cell proliferation. These findings support a role for immune complexes and FcγRl expression by tumor cells in augmentation of tumor growth and a metastatic phenotype.

  10. Tumor cell phenotype is sustained by selective MAPK oxidation in mitochondria.

    Directory of Open Access Journals (Sweden)

    Soledad Galli

    2008-06-01

    Full Text Available Mitochondria are major cellular sources of hydrogen peroxide (H(2O(2, the production of which is modulated by oxygen availability and the mitochondrial energy state. An increase of steady-state cell H(2O(2 concentration is able to control the transition from proliferating to quiescent phenotypes and to signal the end of proliferation; in tumor cells thereby, low H(2O(2 due to defective mitochondrial metabolism can contribute to sustain proliferation. Mitogen-activated protein kinases (MAPKs orchestrate signal transduction and recent data indicate that are present in mitochondria and regulated by the redox state. On these bases, we investigated the mechanistic connection of tumor mitochondrial dysfunction, H(2O(2 yield, and activation of MAPKs in LP07 murine tumor cells with confocal microscopy, in vivo imaging and directed mutagenesis. Two redox conditions were examined: low 1 microM H(2O(2 increased cell proliferation in ERK1/2-dependent manner whereas high 50 microM H(2O(2 arrested cell cycle by p38 and JNK1/2 activation. Regarding the experimental conditions as a three-compartment model (mitochondria, cytosol, and nuclei, the different responses depended on MAPKs preferential traffic to mitochondria, where a selective activation of either ERK1/2 or p38-JNK1/2 by co-localized upstream kinases (MAPKKs facilitated their further passage to nuclei. As assessed by mass spectra, MAPKs activation and efficient binding to cognate MAPKKs resulted from oxidation of conserved ERK1/2 or p38-JNK1/2 cysteine domains to sulfinic and sulfonic acids at a definite H(2O(2 level. Like this, high H(2O(2 or directed mutation of redox-sensitive ERK2 Cys(214 impeded binding to MEK1/2, caused ERK2 retention in mitochondria and restricted shuttle to nuclei. It is surmised that selective cysteine oxidations adjust the electrostatic forces that participate in a particular MAPK-MAPKK interaction. Considering that tumor mitochondria are dysfunctional, their inability to

  11. Single-cell analysis reveals early manifestation of cancerous phenotype in pre-malignant esophageal cells.

    Directory of Open Access Journals (Sweden)

    Jiangxin Wang

    Full Text Available Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett's esophagus (BE as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

  12. In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

    Science.gov (United States)

    Reyes-Aguirre, Luis I; Quintero, Heberto; Estrada-Leyva, Brenda; Lamas, Mónica

    2018-01-01

    microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

  13. Demonstration of a novel HIV-1 restriction phenotype from a human T cell line.

    Directory of Open Access Journals (Sweden)

    Yanxing Han

    2008-07-01

    Full Text Available Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s. Further characterization of this novel gene product(s will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1.

  14. Human haemodynamic frequency harmonics regulate the inflammatory phenotype of vascular endothelial cells.

    Science.gov (United States)

    Feaver, Ryan E; Gelfand, Bradley D; Blackman, Brett R

    2013-01-01

    Haemodynamic variations are inherent to blood vessel geometries (such as bifurcations) and correlate with regional development of inflammation and atherosclerosis. However, the complex frequency spectrum characteristics from these haemodynamics have never been exploited to test whether frequency variations are critical determinants of endothelial inflammatory phenotype. Here we utilize an experimental Fourier transform analysis to systematically manipulate individual frequency harmonics from human carotid shear stress waveforms applied in vitro to human endothelial cells. The frequency spectrum, specifically the 0 th and 1st harmonics, is a significant regulator of inflammation, including NF-κB activity and downstream inflammatory phenotype. Further, a harmonic-based regression-model predicts eccentric NF-κB activity observed in the human internal carotid artery. Finally, short interfering RNA-knockdown of the mechanosensor PECAM-1 reverses frequency-dependent regulation of NF-κB activity. Thus, PECAM-1 may have a critical role in the endothelium's exquisite sensitivity to complex shear stress frequency harmonics and provide a mechanism for the focal development of vascular inflammation.

  15. Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers

    International Nuclear Information System (INIS)

    Karaçalı, Bilge; Vamvakidou, Alexandra P; Tözeren, Aydın

    2007-01-01

    Three-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images. Histologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system. Accuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images. Proposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development

  16. Acquiring Chondrocyte Phenotype from Human Mesenchymal Stem Cells under Inflammatory Conditions

    Directory of Open Access Journals (Sweden)

    Masahiro Kondo

    2014-11-01

    Full Text Available An inflammatory milieu breaks down the cartilage matrix and induces chondrocyte apoptosis, resulting in cartilage destruction in patients with cartilage degenerative diseases, such as rheumatoid arthritis or osteoarthritis. Because of the limited regenerative ability of chondrocytes, defects in cartilage are irreversible and difficult to repair. Mesenchymal stem cells (MSCs are expected to be a new tool for cartilage repair because they are present in the cartilage and are able to differentiate into multiple lineages of cells, including chondrocytes. Although clinical trials using MSCs for patients with cartilage defects have already begun, its efficacy and repair mechanisms remain unknown. A PubMed search conducted in October 2014 using the following medical subject headings (MeSH terms: mesenchymal stromal cells, chondrogenesis, and cytokines resulted in 204 articles. The titles and abstracts were screened and nine articles relevant to “inflammatory” cytokines and “human” MSCs were identified. Herein, we review the cell biology and mechanisms of chondrocyte phenotype acquisition from human MSCs in an inflammatory milieu and discuss the clinical potential of MSCs for cartilage repair.

  17. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues.

    Science.gov (United States)

    Cifola, Ingrid; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A

    2011-06-13

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  18. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues

    International Nuclear Information System (INIS)

    Cifola, Ingrid; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A

    2011-01-01

    Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

  19. Direct Cell-Cell Contact between Mesenchymal Stem Cells and Endothelial Progenitor Cells Induces a Pericyte-Like Phenotype In Vitro

    Directory of Open Access Journals (Sweden)

    Markus Loibl

    2014-01-01

    Full Text Available Tissue engineering techniques for the regeneration of large bone defects require sufficient vascularisation of the applied constructs to ensure a sufficient supply of oxygen and nutrients. In our previous work, prevascularised 3D scaffolds have been successfully established by coculture of bone marrow derived stem cells (MSCs and endothelial progenitor cells (EPCs. We identified stabilising pericytes (PCs as part of newly formed capillary-like structures. In the present study, we report preliminary data on the interactions between MSCs and EPCs, leading to the differentiation of pericyte-like cells. MSCs and EPCs were seeded in transwell cultures, direct cocultures, and single cultures. Cells were cultured for 10 days in IMDM 10% FCS or IMDM 5% FCS 5% platelet lysate medium. Gene expression of PC markers, CD146, NG2, αSMA, and PDGFR-β, was analysed using RT-PCR at days 0, 3, 7, and 10. The upregulation of CD146, NG2, and αSMA in MSCs in direct coculture with EPCs advocates the MSCs’ differentiation towards a pericyte-like phenotype in vitro. These results suggest that pericyte-like cells derive from MSCs and that cell-cell contact with EPCs is an important factor for this differentiation process. These findings emphasise the concept of coculture strategies to promote angiogenesis for cell-based tissue engineered bone grafts.

  20. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer’s Disease

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    Philip W. Brownjohn

    2017-04-01

    Full Text Available Summary: Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP processing and the accumulation of APP-derived amyloid β (Aβ peptides are key processes in Alzheimer's disease (AD. We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation. : In this article, Livesey and colleagues perform a phenotypic drug screen in a human stem cell model of Alzheimer's disease. The anthelminthic avermectins are identified as a family of compounds that increase the production of short Aβ peptides over longer more toxic Aβ forms. The effect is analogous to existing γ-secretase modulators, but is independent of the core γ-secretase complex. Keywords: neural stem cells, Alzheimer's disease, phenotypic screening, iPSCs, human neurons, dementia, Down syndrome, amyloid beta, ivermectin, selamectin

  1. Curtailed T-cell activation curbs effector differentiation and generates CD8+ T cells with a naturally-occurring memory stem cell phenotype.

    Science.gov (United States)

    Zanon, Veronica; Pilipow, Karolina; Scamardella, Eloise; De Paoli, Federica; De Simone, Gabriele; Price, David A; Martinez Usatorre, Amaia; Romero, Pedro; Mavilio, Domenico; Roberto, Alessandra; Lugli, Enrico

    2017-09-01

    Human T memory stem (T SCM ) cells with superior persistence capacity and effector functions are emerging as important players in the maintenance of long-lived T-cell memory and are thus considered an attractive population to be used in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their generation remain poorly defined. Here we show that curtailed T-cell receptor stimulation curbs human effector CD8 + T-cell differentiation and allows the generation of CD45RO - CD45RA + CCR7 + CD27 + CD95 + -phenotype cells from highly purified naïve T-cell precursors, resembling naturally-occurring human T SCM . These cells proliferate extensively in vitro and in vivo, express low amounts of effector-associated genes and transcription factors and undergo considerable self-renewal in response to IL-15 while retaining effector differentiation potential. Such a phenotype is associated with a lower number of mitochondria compared to highly-activated effector T cells committed to terminal differentiation. These results shed light on the molecular signals that are required to generate long-lived memory T cells with potential application in adoptive cell transfer immunotherapy. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co.KGaA, Weinheim.

  2. RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

    Science.gov (United States)

    De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini

    2017-01-10

    The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by

  3. NCR1+ cells in dogs show phenotypic characteristics of natural killer cells.

    Science.gov (United States)

    Grøndahl-Rosado, Christine; Bønsdorff, Tina B; Brun-Hansen, Hege C; Storset, Anne K

    2015-03-01

    No specific markers for natural killer (NK) cells in dogs have currently been described. NCR1 (NKp46, CD355) has been considered a pan species NK cell marker and is expressed on most or all NK cells in all species investigated except for the pig which has both a NCR1(+) and a NCR1(-) population. In this study peripheral blood mononuclear cells (PBMC) from 14 healthy dogs, 37 dogs with a clinical diagnosis, including a dog diagnosed with LGL leukemia, and tissue samples from 8 dogs were evaluated for NCR1(+) expression by a cross reacting anti bovine NCR1 antibody. CD3(-)NCR1(+) cells were found in the blood of 93 % of healthy dogs and comprised up to 2.5 % of lymphocytes in PBMC. In a selection of healthy dogs, sampling and immunophenotyping were repeated throughout a period of 1 year revealing a substantial variation in the percentage of CD3(-)NCR1(+) over time. Dogs allocated to 8 disease groups had comparable amounts of CD3(-)NCR1(+) cells in PBMC to the healthy individuals. All organs examined including liver, spleen and lymph nodes contained CD3(-)NCR1(+) cells. Circulating CD3(-)NCR1(+) cells were further characterized as CD56(-)GranzymeB(+)CD8(-). A CD3(+)NCR1(+) population was observed in PBMC in 79 % of the healthy dogs examined representing at the most 4.8 % of the lymphocyte population. In canine samples examined for CD56 expression, CD56(+) cells were all CD3(+) and NCR1(-). To our knowledge, this is the first examination of NCR1 expression in the dog. The study shows that this NK cell associated receptor is expressed both on populations of CD3(+) and CD3(-) blood lymphocytes in dogs and the receptor is found on a CD3(+) GranzymeB(+) CD8(+) leukemia. Our results support that CD56 is expressed only on CD3(+) cells in dogs and shows that NCR1 defines a different CD3(+) lymphocyte population than CD56(+)CD3(+) cells in this species. CD3(-)NCR1(+) cells may represent canine NK cells.

  4. SMAD4 Loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells

    International Nuclear Information System (INIS)

    Chen, Yu-Wen; Hsiao, Pi-Jung; Weng, Ching-Chieh; Kuo, Kung-Kai; Kuo, Tzu-Lei; Wu, Deng-Chyang; Hung, Wen-Chun; Cheng, Kuang-Hung

    2014-01-01

    SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized. The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down. Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment. This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to

  5. SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

    Science.gov (United States)

    Chen, Yu-Wen; Hsiao, Pi-Jung; Weng, Ching-Chieh; Kuo, Kung-Kai; Kuo, Tzu-Lei; Wu, Deng-Chyang; Hung, Wen-Chun; Cheng, Kuang-Hung

    2014-03-14

    SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized. The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down. Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment. This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to

  6. Human Stromal (Mesenchymal) Stem Cells from Bone Marrow, Adipose Tissue and Skin Exhibit Differences in Molecular Phenotype and Differentiation Potential

    DEFF Research Database (Denmark)

    Al-Nbaheen, May; Vishnubalaji, Radhakrishnan; Ali, Dalia

    2013-01-01

    Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow......, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin......, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols....

  7. Cytokine and Phenotypic Cell Profiles of Leishmania infantum Infection in the Dog

    Directory of Open Access Journals (Sweden)

    Carla Maia

    2012-01-01

    Full Text Available Leishmaniasis has reemerged in recent years showing a wider geographic distribution and increased global incidence of human and canine disease than previously known. Dogs are the main domestic/peridomestic reservoir hosts of zoonotic visceral leishmaniasis caused by Leishmania infantum. Since the evolution of leishmaniasis and clinical appearance is a consequence of complex interactions between the parasite and host immune response, a profound knowledge about the immune profile developed in dog's infection is crucial for vaccine and immunomodulatory therapy design. The main goal of this paper is to compile the recent advances made on cytokine and phenotypic cell profiles in different tissues and organs of dogs infected with L. infantum. This paper also stressed that the knowledge of the immune responses developed, namely, in liver, lymph node, and spleen is very limited. All data emphasizes that more research on canine leishmaniasis is necessary for the development of new and efficacious tools to control zoonotic leishmaniasis.

  8. Induction of adipocyte-like phenotype in human mesenchymal stem cells by hypoxia

    DEFF Research Database (Denmark)

    Fink, Trine; Abildtrup, Lisbeth Ann; Fogd, Kirsten

    2004-01-01

    Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most...... commonly encountered laboratory condition. The study reported here was designed to determine whether oxygen has a role in the differentiation of hMSCs into adipocytes. Indeed, when exposed to atmosphere containing only 1% of oxygen, the formation of adipocyte-like phenotype with cytoplasmic lipid....... High level of induction, however, was observed with the PPAR-gamma-induced angiopoietin-related gene, PGAR. The lack of an adipocyte-specific transcription pattern thus indicates that despite accumulation of the lipid, true adipogenic differentiation did not take place. In conclusion, hypoxia appears...

  9. Phenotypic alteration of CD8+ T cells in chronic lymphocytic leukemia is associated with epigenetic reprogramming.

    Science.gov (United States)

    Wu, Jiazhu; Xu, Xiaojing; Lee, Eun-Joon; Shull, Austin Y; Pei, Lirong; Awan, Farrukh; Wang, Xiaoling; Choi, Jeong-Hyeon; Deng, Libin; Xin, Hong-Bo; Zhong, Wenxun; Liang, Jinhua; Miao, Yi; Wu, Yujie; Fan, Lei; Li, Jianyong; Xu, Wei; Shi, Huidong

    2016-06-28

    Immunosuppression is a prevalent clinical feature in chronic lymphocytic leukemia (CLL) patients, with many patients demonstrating increased susceptibility to infections as well as increased failure of an antitumor immune response. However, much is currently not understood regarding the precise mechanisms that attribute to this immunosuppressive phenotype in CLL. To provide further clarity to this particular phenomenon, we analyzed the T-cell profile of CLL patient samples within a large cohort and observed that patients with an inverted CD4/CD8 ratio had a shorter time to first treatment as well as overall survival. These observations coincided with higher expression of the immune checkpoint receptor PD-1 in CLL patient CD8+ T cells when compared to age-matched healthy donors. Interestingly, we discovered that increased PD-1 expression in CD8+ T cells corresponds with decreased DNA methylation levels in a distal upstream locus of the PD-1 gene PDCD1. Further analysis using luciferase reporter assays suggests that the identified PDCD1 distal upstream region acts as an enhancer for PDCD1 transcription and this region becomes demethylated during activation of naïve CD8+ T cells by anti-CD3/anti-CD28 antibodies and IL2. Finally, we conducted a genome-wide DNA methylation analysis comparing CD8+ T cells from CLL patients against healthy donors and identified additional differentially methylated genes with known immune regulatory functions including CCR6 and KLRG1. Taken together, our findings reveal the occurrence of epigenetic reprogramming taking place within CLL patient CD8+ T cells and highlight the potential mechanism of how immunosuppression is accomplished in CLL.

  10. Retroviral-mediated gene therapy for the differentiation of primary cells into a mineralizing osteoblastic phenotype.

    Science.gov (United States)

    Phillips, Jennifer E; García, Andrés J

    2008-01-01

    Bone tissue engineering has emerged as a promising strategy for the repair of critical-sized skeletal fractures. However, the clinical application of this approach has been limited by the availability of a robust mineralizing cell source. Non-osteogenic cells, such as skin fibroblasts, are an attractive cell-source alternative because they are easy to harvest from autologous donor skin biopsies and display a high capacity for in vitro expansion. We have recently demonstrated that retroviral gene delivery of the osteoblastic transcription factor Runx2/Cbfa1 promotes osteogenic differentiation in primary dermal fibroblasts cultured in monolayer. Notably, sustained expression of Runx2 was not sufficient to promote functional osteogenesis in these cells, and co-treatment with the steroid hormone dexamethasone was required to induce deposition of biologically-equivalent matrix mineralization. On the basis of these results, we then investigated the osteogenic capacity of these genetically engineered fibroblasts when seeded on polymeric scaffolds in vitro and in vivo. These experiments demonstrated that Runx2-expressing fibroblasts seeded on collagen scaffolds produce significant levels of matrix mineralization after 28 days in vivo implantation in a subcutaneous, heterotopic site. Overall, these results offer evidence that transcription factor-based gene therapy may be a powerful strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. This concept may also be applied to control functional differentiation in a broad range of cell types and tissue engineering applications. The chapter below outlines detailed methods for the isolation and ex vivo genetic modification of primary dermal fibroblasts using retroviral-mediated delivery of the Runx2 transgene in both monolayer culture and three-dimensional scaffolds.

  11. Induction of the GABA cell phenotype: an in vitro model for studying neurodevelopmental disorders.

    Directory of Open Access Journals (Sweden)

    Sivan Subburaju

    Full Text Available Recent studies of the hippocampus have suggested that a network of genes is associated with the regulation of the GAD₆₇ (GAD1 expression and may play a role in γ-amino butyric acid (GABA dysfunction in schizophrenia (SZ and bipolar disorder (BD. To obtain a more detailed understanding of how GAD₆₇ regulation may result in GABAergic dysfunction, we have developed an in vitro model in which GABA cells are differentiated from the hippocampal precursor cell line, HiB5. Growth factors, such as PDGF, and BDNF, regulate the GABA phenotype by inducing the expression of GAD₆₇ and stimulating the growth of cellular processes, many with growth cones that form appositions with the cell bodies and processes of other GAD₆₇-positive cells. These changes are associated with increased expression of acetylated tubulin, microtubule-associated protein 2 (MAP2 and the post-synaptic density protein 95 (PSD95. The addition of BDNF, together with PDGF, increases the levels of mRNA and protein for GAD₆₇, as well as the high affinity GABA uptake protein, GAT1. These changes are associated with increased concentrations of GABA in the cytoplasm of "differentiated" HiB5 neurons. In the presence of Ca²⁺ and K⁺, newly synthesized GABA is released extracellularly. When the HiB5 cells appear to be fully differentiated, they also express GAD₆₅, parvalbumin and calbindin, and GluR subtypes as well as HDAC1, DAXX, PAX5, Runx2, associated with GAD₆₇ regulation. Overall, these results suggest that the HiB5 cells can differentiate into functionally mature GABA neurons in the presence of gene products that are associated with GAD₆₇ regulation in the adult hippocampus.

  12. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei.

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    Patcharaporn Tippayawat

    Full Text Available Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined.Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry.B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+ (and CD8(+ T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum.Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

  13. 3D collagen fibrillar microstructure guides pancreatic cancer cell phenotype and serves as a critical design parameter for phenotypic models of EMT.

    Directory of Open Access Journals (Sweden)

    T J Puls

    Full Text Available Pancreatic cancer, one of the deadliest cancers, is characterized by high rates of metastasis and intense desmoplasia, both of which are associated with changes in fibrillar type I collagen composition and microstructure. Epithelial to mesenchymal transition (EMT, a critical step of metastasis, also involves a change in extracellular matrix (ECM context as cells detach from basement membrane (BM and engage interstitial matrix (IM. The objective of this work was to develop and apply an in-vitro three-dimensional (3D tumor-ECM model to define how ECM composition and biophysical properties modulate pancreatic cancer EMT. Three established pancreatic ductal adenocarcinoma (PDAC lines were embedded within 3D matrices prepared with type I collagen Oligomer (IM at various fibril densities to control matrix stiffness or Oligomer and Matrigel combined at various ratios while maintaining constant matrix stiffness. Evaluation of cell morphology and protein expression at both the cellular- and population-levels revealed a spectrum of matrix-driven EMT phenotypes that were dependent on ECM composition and architecture as well as initial PDAC phenotype. In general, exposure to fibrillar IM was sufficient to drive EMT, with cells displaying spindle-shaped morphology and mesenchymal markers, and non-fibrillar BM promoted more epithelial behavior. When cultured within low density Oligomer, only a subpopulation of epithelial BxPC-3 cells displayed EMT while mesenchymal MiaPaCa-2 cells displayed more uniform spindle-shaped morphologies and mesenchymal marker expression. Interestingly, as IM fibril density increased, associated changes in spatial constraints and matrix stiffness resulted in all PDAC lines growing as tight clusters; however mesenchymal marker expression was maintained. Collectively, the comparison of these results to other in-vitro tumor models highlights the role of IM fibril microstructure in guiding EMT heterogeneity and showcases the potential

  14. Turbulent Dynamics of Epithelial Cell Cultures

    Science.gov (United States)

    Blanch-Mercader, C.; Yashunsky, V.; Garcia, S.; Duclos, G.; Giomi, L.; Silberzan, P.

    2018-05-01

    We investigate the large length and long time scales collective flows and structural rearrangements within in vitro human bronchial epithelial cell (HBEC) cultures. Activity-driven collective flows result in ensembles of vortices randomly positioned in space. By analyzing a large population of vortices, we show that their area follows an exponential law with a constant mean value and their rotational frequency is size independent, both being characteristic features of the chaotic dynamics of active nematic suspensions. Indeed, we find that HBECs self-organize in nematic domains of several cell lengths. Nematic defects are found at the interface between domains with a total number that remains constant due to the dynamical balance of nucleation and annihilation events. The mean velocity fields in the vicinity of defects are well described by a hydrodynamic theory of extensile active nematics.

  15. Cell fate determination dynamics in bacteria

    Science.gov (United States)

    Kuchina, Anna; Espinar, Lorena; Cagatay, Tolga; Garcia-Ojalvo, Jordi; Suel, Gurol

    2010-03-01

    The fitness of an organism depends on many processes that serve the purpose to adapt to changing environment in a robust and coordinated fashion. One example of such process is cellular fate determination. In the presence of a variety of alternative responses each cell adopting a particular fate represents a ``choice'' that must be tightly regulated to ensure the best survival strategy for the population taking into account the broad range of possible environmental challenges. We investigated this problem in the model organism B.Subtilis which under stress conditions differentiates terminally into highly resistant spores or initiates an alternative transient state of competence. The dynamics underlying cell fate choice remains largely unknown. We utilize quantitative fluorescent microscopy to track the activities of genes involved in these responses on a single-cell level. We explored the importance of temporal interactions between competing cell fates by re- engineering the differentiation programs. I will discuss how the precise dynamics of cellular ``decision-making'' governed by the corresponding biological circuits may enable cells to adjust to diverse environments and determine survival.

  16. Dynamic phenotypic plasticity in photosynthesis and biomass patterns in Douglas-fir seedlings

    Science.gov (United States)

    A. C. Koehn; G. I. McDonald; D. L. Turner; D. L. Adams

    2010-01-01

    As climate changes, understanding the mechanisms long-lived conifers use to adapt becomes more important. Light gradients within a forest stand vary constantly with the changes in climate, and the minimum light required for survival plays a major role in plant community dynamics. This study focuses on the dynamic plasticity of Douglas-fir (Pseudotsuga menziesii var....

  17. Linking dynamic phenotyping with metabolite analysis to study natural variation in drought responses of Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Lorraine H.C. Fisher

    2016-11-01

    Full Text Available Drought is an important environmental stress limiting the productivity of major crops worldwide. Understanding drought tolerance and possible mechanisms for improving drought resistance is therefore a prerequisite to develop drought-tolerant crops that produce significant yields with reduced amounts of water. Brachypodium distachyon (Brachypodium is a key model species for cereals, forage grasses and energy grasses. In this study, initial screening of a Brachypodium germplasm collection consisting of 138 different ecotypes exposed to progressive drought, highlighted the natural variation in morphology, biomass accumulation and responses to drought stress. A core set of ten ecotypes, classified as being either tolerant, susceptible or intermediate, in response to drought stress, were exposed to mild or severe (respectively 15% and 0% soil water content drought stress and phenomic parameters linked to growth and colour changes were assessed. When exposed to severe drought stress, phenotypic data and metabolite profiling combined with multivariate analysis revealed a remarkable consistency in separating the selected ecotypes into their different pre-defined drought tolerance groups. Increases in several metabolites, including for the phytohormones jasmonic acid and salicylic acid, and TCA-cycle intermediates, were positively correlated with biomass yield and with reduced yellow pixel counts; suggestive of delayed senescence, both key target traits for crop improvement to drought stress. While metabolite analysis also separated ecotypes into the distinct tolerance groupings after exposure to mild drought stress, similar analysis of the phenotypic data failed to do so, confirming the value of metabolomics to investigate early responses to drought stress. The results highlight the potential of combining the analyses of phenotypic and metabolic responses to identify key mechanisms and markers associated with drought tolerance in both the Brachypodium

  18. Distinct migration and contact dynamics of resting and IL-2-activated human natural killer cells

    Directory of Open Access Journals (Sweden)

    Per Erik Olofsson

    2014-03-01

    Full Text Available Natural killer (NK cells serve as one of the first lines of defense against viral infections and transformed cells. NK cell cytotoxicity is not dependent on antigen presentation by target cells, but is dependent on integration of activating and inhibitory signals triggered by receptor–ligand interactions formed at a tight intercellular contact between the NK and target cell, i.e. the immune synapse. We have studied the single-cell migration behavior and target-cell contact dynamics of resting and IL-2-activated human peripheral blood NK cells. Small populations of NK cells and target cells were confined in microwells and imaged by fluorescence microscopy for >8 h. Only the IL-2-activated population of NK cells showed efficient cytotoxicity against the human embryonic kidney (HEK 293T target cells. We found that although the average migration speeds were comparable, activated NK cells showed significantly more dynamic migration behavior, with more frequent transitions between periods of low and high motility. Resting NK cells formed fewer and weaker contacts with target cells, which manifested as shorter conjugation times and in many cases a complete lack of post-conjugation attachment to target cells. Activated NK cells were approximately twice as big as the resting cells, displayed a more migratory phenotype, and were more likely to employ motile scanning of the target cell surface during conjugation. Taken together, our experiments quantify, at the single-cell level, how activation by IL-2 leads to altered NK cell cytotoxicity, migration behavior and contact dynamics.

  19. When aging reaches CD4+ T-cells: phenotypic and functional changes

    Directory of Open Access Journals (Sweden)

    Marco Antonio Moro-García

    2013-05-01

    Full Text Available Beyond midlife, the immune system shows aging features and its defensive capability becomes impaired, by a process known as immunosenescence that involves many changes in the innate and adaptive responses. Innate immunity seems to be better preserved globally, while the adaptive immune response exhibits profound age-dependent modifications. Elderly people display a decline in numbers of naïve T-cells in peripheral blood and lymphoid tissues, while, in contrast, their proportion of highly differentiated effector and memory T-cells, such as the CD28null T-cells, increases markedly. Naïve and memory CD4+ T-cells constitute a highly dynamic system with constant homeostatic and antigen-driven proliferation, influx, and loss of T-cells. Thymic activity dwindles with age and essentially ceases in the later decades of life, severely constraining the generation of new T-cells. Homeostatic control mechanisms are very effective at maintaining a large and diverse subset of naïve CD4+ T-cells throughout life, but although later than in CD8+T-cell compartment, these mechanisms ultimately fail with age.

  20. Development and Characterization of a Brain Endothelial Cell Phenotype using Human Induced Pluripotent Stem Cells

    DEFF Research Database (Denmark)

    Goldeman, Charlotte; Saaby, Lasse; Holst, Bjørn

    for experiments the following day. The model was monitored by measuring the trans-endothelial electrical resistance (TEER). RA had an inductive effect on the model, shown by an elevation in barrier tightness which correlated with the presence of tight junction proteins, shown by confocal microscopy images which...... be used to investigate drug transport in vitro, and screen candidates for permeation properties. One recent approach is to develop in vitro models of the BBB using human induced pluripotent stem cells (hIPSCs) as described by Stebbins et al. (2015).The aim of the present study was to investigate whether...... the published protocols were generically applicable and thus to develop and characterize in vitro models of the BBB using hIPSCs from different sources. Two stem cell lines, Bioni010-C and WTSli024-A, were seeded and maintained on Matrigel in mTesR1 media. Cells were then seeded as single cells at different...

  1. Interlaminar differences in the pyramidal cell phenotype in parietal cortex of an Indian bat, cynopterus sphinx.

    Science.gov (United States)

    Srivastava, U C; Pathak, S V

    2010-10-30

    To study interlaminar phenotypic variations in the pyramidal neurons of parietal isocortex in bat (Cynopterus sphinx), Golgi and Nissl methods have been employed. The parietal isocortex is relatively thin in the bat as compared to prototheria with layer III, V and VI accounting for more than two—thirds of total cortical thickness. Thick cell free layer I and thinnest accentuated layer II are quite in connotation with other chiropterids. Poor demarcation of layer III/IV in the present study is also in connotation with primitive eutherian mammal (i.e. prototherian) and other chiropterids. Most of the pyramidal cells in the different layers of the parietal isocortex are of typical type as seen in other eutherians but differ significantly in terms of soma shape and size, extent of dendritic arbor, diameter of dendrites and spine density. Percentage of pyramidal neurons, diameter of apical dendrite and spine density on apical dendrite appear to follow an increasing trend from primitive to advanced mammals; but extent of dendrites are probably governed by the specific life patterns of these mammals. It is thus concluded that 'typical' pyramidal neurons in parietal isocortex are similar in therians but different from those in prototherians. It is possible that these cells might have arisen among early eutherians after divergence from prototherian stock.

  2. LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

    Science.gov (United States)

    Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

    2017-11-28

    Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

  3. The Janus-faced role of ezrin in "linking" cells to either normal or metastatic phenotype.

    Science.gov (United States)

    Brambilla, Daria; Fais, Stefano

    2009-11-15

    In the majority of eukaryotic cells, the ezrin, radixin and moesin (ERM) proteins are involved in many physiologic functions including regulation of actin cytoskeleton, control of cell shape, adhesion, motility and modulation of signal transduction pathways. In a previous study, we used a dominant negative ezrin-mutant to address ezrin involvement in remodeling of actin cytoskeleton and subsequently we depicted ezrin key role in melanoma cell migration and progression. Herein, we highlight recent advances on ezrin involvement in the metastatic phenomenon, including also some more neglected ezrin-related functions. Novel molecular processes driven by ezrin activation include: phagocytosis, acquisition of resistance to chemotherapeutics and triggering of programmed cell death signals. Recent data support an integrated role of ezrin also in development of tumor malignancy. On one hand, ezrin may be responsible of deranged execution of specific known functions such as adhesion and motility and on the other, it may also participate to unique metastatic determinants, through the establishment of aberrant linkages with tumor-related proteins. For instance, ezrin misslocalization, absence or deranged activity has started to be correlated with tumor progression in many tumors of different species, including humans. Concomitantly, ezrin may act simultaneously as a regulatory or deregulatory chaperon in both normal and tumor cells. It is still to be established whether this Janus-faced feature of ezrin is due to some unknown transforming Zelig-like property or to the fact that a tumor-associated molecule preferentially links to ezrin thus distracting it from its normal connections. However, the contribution of ezrin functional deregulation to the acquisition of the metastatic phenotype appears clear and ezrin or ezrin aberrant associations may represent good candidates for future anti-tumor therapies.

  4. Clinical Significance of Immuno phenotypic Markers in Pediatric T-cell Acute Lymphoblastic Leukemia

    International Nuclear Information System (INIS)

    SIDHOM, I.; SHAABAN, Kh.; SOLIMAN, S.; HAMDY, N.; YASSIN, D.; SALEM, Sh.; HASSANEIN, H.; MANSOUR, M.T.; EZZAT, S.; EL-ANWAR, W.

    2008-01-01

    Background: Cell-marker profiling has led to conflicting conclusions about its prognostic significance in T-ALL. Aim: To investigate the prevalence of the expression of CD34, CD10 and myeloid associated antigens (CD13/ CD33) in childhood T-ALL and to relate their presence to initial clinical and biologic features and early response to therapy. Patients and Methods: This study included 67 consecutive patients with newly diagnosed T-ALL recruited from the Children's Cancer Hospital in Egypt during the time period from July 2007 to June 2008. Immuno phenotypic markers and minimal residual disease (MRD) were studied by five-color flow cytometry. Results: The frequency of CD34 was 34.9%, CD10 33.3%, while CD13/CD33 was 18.8%. No significant association was encountered between CD34, CD10 or myeloid antigen positivity and the presenting clinical features as age, sex, TLC and CNS leukemia. Only CD10+ expression had significant association with initial CNS involvement (p=0.039). CD34 and CD13/CD33 expression was significantly associated with T-cell maturation stages (p<0.05). No relationship was observed for age, TLC, gender, NCI risk or CNS involvement with early response to therapy illustrated by BM as well as MRD day 15 and day 42. CD34+, CD13/CD33+ and early T-cell stage had high MRD levels on day 15 that was statistically highly significant (p<0.01), but CD10+ had statistically significant lower MRD level on day 15 (p=0.049). However, only CD34 retained its significance at an MRD cut-off level of 0.01%. Conclusion: CD34, CD10, CD13/CD33 expression, as well as T-cell maturation stages, may have prognostic significance in pediatric T-ALL as they have a significant impact on early clearance of leukemic cells detected by MRD day 15.

  5. Genome-wide recombination dynamics are associated with phenotypic variation in maize.

    Science.gov (United States)

    Pan, Qingchun; Li, Lin; Yang, Xiaohong; Tong, Hao; Xu, Shutu; Li, Zhigang; Li, Weiya; Muehlbauer, Gary J; Li, Jiansheng; Yan, Jianbing

    2016-05-01

    Meiotic recombination is a major driver of genetic diversity, species evolution, and agricultural improvement. Thus, an understanding of the genetic recombination landscape across the maize (Zea mays) genome will provide insight and tools for further study of maize evolution and improvement. Here, we used c. 50 000 single nucleotide polymorphisms to precisely map recombination events in 12 artificial maize segregating populations. We observed substantial variation in the recombination frequency and distribution along the ten maize chromosomes among the 12 populations and identified 143 recombination hot regions. Recombination breakpoints were partitioned into intragenic and intergenic events. Interestingly, an increase in the number of genes containing recombination events was accompanied by a decrease in the number of recombination events per gene. This kept the overall number of intragenic recombination events nearly invariable in a given population, suggesting that the recombination variation observed among populations was largely attributed to intergenic recombination. However, significant associations between intragenic recombination events and variation in gene expression and agronomic traits were observed, suggesting potential roles for intragenic recombination in plant phenotypic diversity. Our results provide a comprehensive view of the maize recombination landscape, and show an association between recombination, gene expression and phenotypic variation, which may enhance crop genetic improvement. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  6. Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics.

    Directory of Open Access Journals (Sweden)

    Yu-Fen Chang

    Full Text Available Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact. This combination can be applied to single rodent primary adult cardiomyocytes, and human stem cell derived cardiomyocytes, enabling contactless small molecule evaluation for inhibitors of sodium, potassium and calcium channels suggesting it may be useful for early toxicity work. In pancreatic beta-cells it reveals the effects of glucose and the KATP inhibitor gliclazide.

  7. Mesothelioma tumor cells modulate dendritic cell lipid content, phenotype and function.

    Directory of Open Access Journals (Sweden)

    Joanne K Gardner

    Full Text Available Dendritic cells (DCs play an important role in the generation of anti-cancer immune responses, however there is evidence that DCs in cancer patients are dysfunctional. Lipid accumulation driven by tumor-derived factors has recently been shown to contribute to DC dysfunction in several human cancers, but has not yet been examined in mesothelioma. This study investigated if mesothelioma tumor cells and/or their secreted factors promote increases in DC lipid content and modulate DC function. Human monocyte-derived DCs (MoDCs were exposed to human mesothelioma tumor cells and tumor-derived factors in the presence or absence of lipoproteins. The data showed that immature MoDCs exposed to mesothelioma cells or factors contained increased lipid levels relative to control DCs. Lipid accumulation was associated with reduced antigen processing ability (measured using a DQ OVA assay, upregulation of the co-stimulatory molecule, CD86, and production of the tolerogenic cytokine, IL-10. Increases in DC lipid content were further enhanced by co-exposure to mesothelioma-derived factors and triglyceride-rich lipoproteins, but not low-density lipoproteins. In vivo studies using a murine mesothelioma model showed that the lipid content of tumor-infiltrating CD4+ CD8α- DCs, CD4- CD8α- DCs DCs and plasmacytoid DCs increased with tumor progression. Moreover, increasing tumor burden was associated with reduced proliferation of tumor-antigen-specific CD8+ T cells in tumor-draining lymph nodes. This study shows that mesothelioma promotes DC lipid acquisition, which is associated with altered activation status and reduced capacity to process and present antigens, which may impair the ability of DCs to generate effective anti mesothelioma T cell responses.

  8. Phenotypic modulation of auto-reactive cells by insertion of tolerogenic molecules via MSC-derived exosomes.

    Science.gov (United States)

    Mokarizadeh, Aram; Delirezh, Nowruz; Morshedi, Ahhmad; Mosayebi, Ghasem; Farshid, Amir-Abbas; Dalir-Naghadeh, Bahram

    2012-01-01

    Auto-reactive cells-mediated immune responses are responsible for the current tissue damages during autoimmunity. Accordingly, functional modulation of auto-reactive cells has been a pivotal aim in many of recent studies. In the current study, we investigated the possibility for insertion of regulatory molecules onto auto-reactive cells through exosomal nano-shuttles as a novel approach for phenotype modification of auto-reactive cells. The exosomes were isolated from supernatant of mesenchymal stem cells culture. Resultant exosomes co-cultured with lymphocytes were harvested from established EAE mice in the presence of antigenic MOG35-55 peptide. After 24 hr, insertion of exosomal tolerogenic molecules (PD-L1, TGF-β, galectin-1) onto auto-reactive cells were explored through flow cytometry. The potency of exosomal inserted membrane molecules to modulate phenotype of auto-reactive lymphocytes was assessed upon ELISA test for their-derived cytokines IFN-γ and IL-17. Incorporation of exosomal molecules into lymohocytes' membrane was confirmed by flow cytometric analyses for surface levels of mentioned molecules. Additionally, the decreased secretion of IFN-γ and IL-17 were detected in exosome pre-treated lymphocytes upon stimulation with MOG peptide. Mesenchymal stem cells -derived exosomes showed to be efficient organelles for insertion of bioactive tolerogenic molecules onto auto-reactive cells and modulation of their phenotypes.

  9. β-Cell Ca(2+) dynamics and function are compromised in aging.

    Science.gov (United States)

    Barker, Christopher J; Li, Luosheng; Köhler, Martin; Berggren, Per-Olof

    2015-01-01

    Defects in pancreatic β-cell function and survival are key components in type 2 diabetes (T2D). An age-dependent deterioration in β-cell function has also been observed, but little is known about the molecular mechanisms behind this phenomenon. Our previous studies indicate that the regulation of cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) may be critical and that this is dependent on the proper function of the mitochondria. The [Ca(2+)]i dynamics of the pancreatic β-cell are driven by an interplay between glucose-induced influx of extracellular Ca(2+) via voltage-dependent Ca(2+) channels and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-mediated liberation of Ca(2+) from intracellular stores. Our previous work has indicated a direct relationship between disruption of Ins(1,4,5)P3-mediated Ca(2+) regulation and loss of β-cell function, including disturbed [Ca(2+)]i dynamics and compromised insulin secretion. To investigate these processes in aging we used three mouse models, a premature aging mitochondrial mutator mouse, a mature aging phenotype (C57BL/6) and an aging-resistant phenotype (129). Our data suggest that age-dependent impairment in mitochondrial function leads to modest changes in [Ca(2+)]i dynamics in mouse β-cells, particularly in the pattern of [Ca(2+)]i oscillations. These changes are driven by modifications in both PLC/Ins(1,4,5)P3-mediated Ca(2+) mobilization from intracellular stores and decreased β-cell Ca(2+) influx over the plasma membrane. Our findings underscore an important concept, namely that even relatively small, time-dependent changes in β-cell signal-transduction result in compromised insulin release and in a diabetic phenotype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. JNK-associated scattered growth of YD-10B oral squamous carcinoma cells while maintaining the epithelial phenotype

    International Nuclear Information System (INIS)

    Lee, Gayoung; Kim, Hyun-Man

    2017-01-01

    Cell scattering of epithelial carcinoma cancer cells is one of the critical event in tumorigenesis. Cells losing epithelial cohesion detach from aggregated epithelial cell masses and may migrate to fatal organs through metastasis. The present study investigated the molecular mechanism by which squamous cell carcinoma cells grow scattered at the early phase of transformation while maintaining the epithelial phenotype. We studied YD-10B cells, which are established from human oral squamous cell carcinoma, because the cells grow scattered without the development of E-cadherin junctions (ECJs) under routine culture conditions despite the high expression of functional E-cadherin. The functionality of their E-cadherin was demonstrated in that YD-10B cells developed ECJs, transiently or persistently, when they were cultured on substrates coated with a low amount of fibronectin or to confluence. The phosphorylation of JNK was up-regulated in YD-10B cells compared with that in human normal oral keratinocyte cells or human squamous cell carcinoma cells, which grew aggregated along with well-organized ECJs. The suppression of JNK activity induced the aggregated growth of YD-10B cells concomitant with the development of ECJs. These results indicate for the first time that inherently up-regulated JNK activity induces the scattered growth of the oral squamous cell carcinoma cells through down-regulating the development of ECJ despite the expression of functional E-cadherin, a hallmark of the epithelial phenotype. - Highlights: • JNK dissociates YD-10B oral squamous cell carcinoma cells. • JNK suppresses the development of E-cadherin junctions of oral carcinoma cells. • Suppression of JNK activity reverses the scattered growth of oral carcinoma cells.

  11. Tissue-Resident Memory CD8+ T Cells: From Phenotype to Function

    Directory of Open Access Journals (Sweden)

    David J. Topham

    2018-03-01

    Full Text Available Tissue-resident memory CD8+ T cells are an important first line of defense from infection in peripheral non-lymphoid tissues, such as the mucosal tissues of the respiratory, digestive, and urogenital tracts. This memory T cell subset is established late during resolution of primary infection of those tissues, has a distinct genetic signature, and is often defined by the cell surface expression of CD69, CD103, CD49a, and CD44 in both mouse and human studies. The stimuli that program or imprint the unique gene expression and cell surface phenotypes on TRM are beginning to be defined, but much work remains to be done. It is not clear, for example, when and where the TRM precursors receive these signals, and there is evidence that supports imprinting in both the lymph node and the peripheral tissue sites. In most studies, expression of CD49a, CD103, and CD69 on T cells in the tissues appears relatively late in the response, suggesting there are precise environmental cues that are not present at the height of the acute response. CD49a and CD103 are not merely biomarkers of TRM, they confer substrate specificities for cell adhesion to collagen and E-cadherin, respectively. Yet, little attention has been paid to how expression affects the positioning of TRM in the peripheral tissues. CD103 and CD49a are not mutually exclusive, and not always co-expressed, although whether they can compensate for one another is unknown. In fact, they may define different subsets of TRM in certain tissues. For instance, while CD49a+CD8+ memory T cells can be found in almost all peripheral tissues, CD103 appears to be more restricted. In this review, we discuss the evidence for how these hallmarks of TRM affect positioning of T cells in peripheral sites, how CD49a and CD103 differ in expression and function, and why they are important for immune protection conferred by TRM in mucosal tissues such as the respiratory tract.

  12. Gamma c-signaling cytokines induce a regulatory T cell phenotype in malignant CD4+ T lymphocytes

    DEFF Research Database (Denmark)

    Kasprzycka, Monika; Zhang, Qian; Witkiewicz, Agnieszka

    2008-01-01

    In this study, we demonstrate that malignant mature CD4(+) T lymphocytes derived from cutaneous T cell lymphomas (CTCL) variably display some aspects of the T regulatory phenotype. Whereas seven cell lines representing a spectrum of primary cutaneous T cell lymphoproliferative disorders expressed...... that FOXP3-expressing cells were common among the CD7-negative enlarged atypical and small lymphocytes at the early skin patch and plaque stages. Their frequency was profoundly diminished at the tumor stage and in the CTCL lymph node lesions with or without large cell transformation. These results indicate...

  13. 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

  14. Cell-Permeable Parkin Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals

    Science.gov (United States)

    Duong, Tam; Kim, Jaetaek; Ruley, H. Earl; Jo, Daewoong

    2014-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder of complex etiology characterized by the selective loss of dopaminergic neurons, particularly in the substantia nigra. Parkin, a tightly regulated E3 ubiquitin ligase, promotes the survival of dopaminergic neurons in both PD and Parkinsonian syndromes induced by acute exposures to neurotoxic agents. The present study assessed the potential of cell-permeable parkin (CP-Parkin) as a neuroprotective agent. Cellular uptake and tissue penetration of recombinant, enzymatically active parkin was markedly enhanced by the addition of a hydrophobic macromolecule transduction domain (MTD). The resulting CP-Parkin proteins (HPM13 and PM10) suppressed dopaminergic neuronal toxicity in cells and mice exposed to 6-hydroxydopamine (6-OHDH) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These included enhanced survival and dopamine expression in cultured CATH.a and SH-SY5Y neuronal cells; and protection against MPTP-induced damage in mice, notably preservation of tyrosine hydroxylase-positive cells with enhanced dopamine expression in the striatum and midbrain, and preservation of gross motor function. These results demonstrate that CP-Parkin proteins can compensate for intrinsic limitations in the parkin response and provide a therapeutic strategy to augment parkin activity in vivo. PMID:25019626

  15. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

    Directory of Open Access Journals (Sweden)

    Rust Steven

    2013-02-01

    Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

  16. Vascular smooth muscle cell differentiation to an osteogenic phenotype involves matrix metalloproteinase-2 modulation by homocysteine.

    Science.gov (United States)

    Liu, Tingjiao; Lin, Jinghan; Ju, Ting; Chu, Lei; Zhang, Liming

    2015-08-01

    Arterial calcification is common in vascular diseases and involves conversion of vascular smooth muscle cells (VSMCs) to an osteoblast phenotype. Clinical studies suggest that the development of atherosclerosis can be promoted by homocysteine (HCY), but the mechanisms remain unclear. Here, we determined whether increases in HCY levels lead to an increase in VSMC calcification and differentiation, and examined the role of an extracellular matrix remodeler, matrix metalloproteinase-2 (MMP-2). Rat VSMCs were exposed to calcification medium in the absence or presence of HCY (10, 100 or 200 μmol/L) or an MMP-2 inhibitor (10(-6) or 10(-5) mol/L). MTT assays were performed to determine the cytotoxicity of the MMP-2 inhibitor in calcification medium containing 200 μmol/L HCY. Calcification was assessed by measurements of calcium deposition and alkaline phosphatase (ALP) activity as well as von Kossa staining. Expression of osteocalcin, bone morphogenetic protein (BMP)-2, and osteopontin, and MMP-2 was determined by immunoblotting. Calcification medium induced osteogenic differentiation of VSMCs. HCY promoted calcification, increased osteocalcin and BMP-2 expression, and decreased expression of osteopontin. MMP-2 expression was increased by HCY in a dose-dependent manner in VSMCs exposed to both control and calcification medium. The MMP-2 inhibitor decreased the calcium content and ALP activity, and attenuated the osteoblastic phenotype of VSMCs. Vascular calcification and osteogenic differentiation of VSMCs were positively regulated by HCY through increased/restored MMP-2 expression, increased expression of calcification proteins, and decreased anti-calcification protein levels. In summary, MMP-2 inhibition may be a protective strategy against VSMC calcification.

  17. Examination of MARCO activity on dendritic cell phenotype and function using a gene knockout mouse.

    Directory of Open Access Journals (Sweden)

    Hiroshi Komine

    Full Text Available We have reported the upregulation of MARCO, a member of the class A scavenger receptor family, on the surface of murine and human dendritic cells (DCs pulsed with tumor lysates. Exposure of murine tumor lysate-pulsed DCs to an anti-MARCO antibody led to loss of dendritic-like processes and enhanced migratory capacity. In this study, we have further examined the biological and therapeutic implications of MARCO expression by DCs. DCs generated from the bone marrow (bm of MARCO knockout (MARCO⁻/⁻ mice were phenotypically similar to DCs generated from the bm of wild-type mice and produced normal levels of IL-12 and TNF-α when exposed to LPS. MARCO⁻/⁻ DCs demonstrated enhanced migratory capacity in response to CCL-21 in vitro. After subcutaneous injection into mice, MARCO⁻/⁻ TP-DCs migrated more efficiently to the draining lymph node leading to enhanced generation of tumor-specific IFN-γ producing T cells and improved tumor regression and survival in B16 melanoma-bearing mice. These results support targeting MARCO on the surface of DCs to improve trafficking and induction of anti-tumor immunity.

  18. Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

    Directory of Open Access Journals (Sweden)

    Patrick Kaiser

    2014-02-01

    Full Text Available In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN, the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

  19. CD133+CD24lo defines a 5-Fluorouracil-resistant colon cancer stem cell-like phenotype

    Science.gov (United States)

    Paschall, Amy V.; Yang, Dafeng; Lu, Chunwan; Redd, Priscilla S.; Choi, Jeong-Hyeon; Heaton, Christopher M.; Lee, Jeffrey R.; Nayak-Kapoor, Asha; Liu, Kebin

    2016-01-01

    The chemotherapeutic agent 5-Fluorouracil (5-FU) is the most commonly used drug for patients with advanced colon cancer. However, development of resistance to 5-FU is inevitable in almost all patients. The mechanism by which colon cancer develops 5-FU resistance is still unclear. One recently proposed theory is that cancer stem-like cells underlie colon cancer 5-FU resistance, but the phenotypes of 5-FU-resistant colon cancer stem cells are still controversial. We report here that 5-FU treatment selectively enriches a subset of CD133+ colon cancer cells in vitro. 5-FU chemotherapy also increases CD133+ tumor cells in human colon cancer patients. However, sorted CD133+ colon cancer cells exhibit no increased resistance to 5-FU, and CD133 levels exhibit no correlation with colon cancer patient survival or cancer recurrence. Genome-wide analysis of gene expression between sorted CD133+ colon cancer cells and 5-FU-selected colon cancer cells identifies 207 differentially expressed genes. CD24 is one of the genes whose expression level is lower in the CD133+ and 5-FU-resistant colon cancer cells as compared to CD133+ and 5-FU-sensitive colon cancer cells. Consequently, CD133+CD24lo cells exhibit decreased sensitivity to 5-FU. Therefore, we determine that CD133+CD24lo phenotype defines 5-FU-resistant human colon cancer stem cell-like cells. PMID:27659530

  20. Red blood cell dynamics: from cell deformation to ATP release.

    Science.gov (United States)

    Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

    2011-10-01

    The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release. This journal is © The Royal Society of Chemistry 2011

  1. Intestinal Stem Cell Dynamics: A Story of Mice and Humans.

    Science.gov (United States)

    Hodder, Michael C; Flanagan, Dustin J; Sansom, Owen J

    2018-06-01

    Stem cell dynamics define the probability of accumulating mutations within the intestinal epithelium. In this issue of Cell Stem Cell, Nicholson et al. (2018) report that human intestinal stem cell dynamics differ significantly from those of mice and establish that oncogenic mutations are more likely to expand; therefore, "normal" epithelium may carry multiple mutations. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Galectin-3 impairment of MYCN-dependent apoptosis-sensitive phenotype is antagonized by nutlin-3 in neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Veronica Veschi

    Full Text Available MYCN amplification occurs in about 20-25% of human neuroblastomas and characterizes the majority of the high-risk cases, which display less than 50% prolonged survival rate despite intense multimodal treatment. Somehow paradoxically, MYCN also sensitizes neuroblastoma cells to apoptosis, understanding the molecular mechanisms of which might be relevant for the therapy of MYCN amplified neuroblastoma. We recently reported that the apoptosis-sensitive phenotype induced by MYCN is linked to stabilization of p53 and its proapoptotic kinase HIPK2. In MYCN primed neuroblastoma cells, further activation of both HIPK2 and p53 by Nutlin-3 leads to massive apoptosis in vitro and to tumor shrinkage and impairment of metastasis in xenograft models. Here we report that Galectin-3 impairs MYCN-primed and HIPK2-p53-dependent apoptosis in neuroblastoma cells. Galectin-3 is broadly expressed in human neuroblastoma cell lines and tumors and is repressed by MYCN to induce the apoptosis-sensitive phenotype. Despite its reduced levels, Galectin-3 can still exert residual antiapoptotic effects in MYCN amplified neuroblastoma cells, possibly due to its specific subcellular localization. Importantly, Nutlin-3 represses Galectin-3 expression, and this is required for its potent cell killing effect on MYCN amplified cell lines. Our data further characterize the apoptosis-sensitive phenotype induced by MYCN, expand our understanding of the activity of MDM2-p53 antagonists and highlight Galectin-3 as a potential biomarker for the tailored p53 reactivation therapy in patients with high-risk neuroblastomas.

  3. Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Lauren H. Mangum

    2017-01-01

    Full Text Available Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS, which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP or from adipose associated with debrided burned skin (BH. Most (95–99% cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p<0.05. Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes.

  4. Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

    Science.gov (United States)

    Mangum, Lauren H.; Stone, Randolph; Wrice, Nicole L.; Larson, David A.; Florell, Kyle F.; Christy, Barbara A.; Herzig, Maryanne C.; Cap, Andrew P.

    2017-01-01

    Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs) for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS), which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL) is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP) or from adipose associated with debrided burned skin (BH). Most (95–99%) cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p < 0.05). Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes. PMID:29138638

  5. Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype.

    Science.gov (United States)

    Huang, Chen; Zhang, Jing; Ao, Mingxin; Li, Ying; Zhang, Chun; Xu, Yonggen; Li, Xuemin; Wang, Wei

    2012-02-01

    Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction. Copyright © 2011 Wiley Periodicals, Inc.

  6. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype

    DEFF Research Database (Denmark)

    Martinat, Cecile; Bacci, Jean-Jacques; Leete, Thomas

    2006-01-01

    's disease. We sought to identify genes that can potentiate maturation of ES cell cultures to the midbrain DA neuron phenotype. A number of transcription factors have been implicated in the development of midbrain DA neurons by expression analyses and loss-of-function knockout mouse studies, including Nurr1......Midbrain dopamine (DA) neurons play a central role in the regulation of voluntary movement, and their degeneration is associated with Parkinson's disease. Cell replacement therapies, and in particular embryonic stem (ES) cell-derived DA neurons, offer a potential therapeutic venue for Parkinson......, Pitx3, Lmx1b, Engrailed-1, and Engrailed-2. However, none of these factors appear sufficient alone to induce the mature midbrain DA neuron phenotype in ES cell cultures in vitro, suggesting a more complex regulatory network. Here we show that Nurr1 and Pitx3 cooperatively promote terminal maturation...

  7. Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

    Science.gov (United States)

    Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W; Malik, Hassan; Kitteringham, Neil R; Goldring, Chris E; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A

    2015-03-01

    Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  8. Phenotype and specificity of T cells in primary human cytomegalovirus infection during pregnancy: IL-7Rpos long-term memory phenotype is associated with protection from vertical transmission.

    Science.gov (United States)

    Mele, Federico; Fornara, Chiara; Jarrossay, David; Furione, Milena; Arossa, Alessia; Spinillo, Arsenio; Lanzavecchia, Antonio; Gerna, Giuseppe; Sallusto, Federica; Lilleri, Daniele

    2017-01-01

    Congenital human cytomegalovirus (HCMV) infection is the major cause of birth defects and a precise definition of the HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection during pregnancy. In this study, a high throughput method was used to define the frequency of CD4+ and CD8+ T cells specific for four HCMV proteins in the naïve compartment of seronegative subjects and the effector/memory compartments of subjects with primary/remote HCMV infection. The naïve repertoire displayed comparable frequencies of T cells that were reactive with HCMV structural (pp65, gB and the pentamer gHgLpUL128L) and non-structural (IE-1) proteins. Whereas, following natural infection, the majority of effector/memory CD4+ and CD8+ T cells recognized either gB or IE-1, respectively, and pp65. The pattern of T cell reactivity was comparable at early and late stages of infection and in pregnant women with primary HCMV infection transmitting or not transmitting the virus to the fetus. At an early stage of primary infection, about 50% of HCMV-reactive CD4+ T cells were long-term IL-7Rpos memory cells, while 6-12 months later, the frequency of these cells increased to 70%, approaching 100% in remote infections. In contrast, only 10-20% of HCMV-specific CD8+ T cells were long-term memory cells up to 12 months after infection onset, thereafter increasing to 70% in remote infections. Interestingly, a significantly higher frequency of HCMV-specific CD4+ T cells with a long-term IL-7Rpos memory phenotype was observed in non-transmitting compared to transmitting women. These findings indicate that immunodominance in HCMV infection is not predetermined in the naïve compartment, but is the result of virus-host interactions and suggest that prompt control of HCMV infection in pregnancy is associated with the rapid development of long-term IL-7Rpos memory HCMV-specific CD4+ T cells and a low risk of virus transmission to the fetus.

  9. STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

    Science.gov (United States)

    Benito, Cristina; Davis, Catherine M; Gomez-Sanchez, Jose A; Turmaine, Mark; Meijer, Dies; Poli, Valeria; Mirsky, Rhona; Jessen, Kristjan R

    2017-04-19

    After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population. SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal

  10. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    International Nuclear Information System (INIS)

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-01-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2 + , CD3 + , CD4 + or CD2 + , CD3 + , CD8 + ) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by 51 Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype

  11. Phenotypic Diversity of Sickle Cell Disease in Patients with a Double Heterozygosity for Hb S and Hb D-Punjab.

    Science.gov (United States)

    Torres, Lidiane S; Okumura, Jéssika V; Belini-Júnior, Édis; Oliveira, Renan G; Nascimento, Patrícia P; Silva, Danilo G H; Lobo, Clarisse L C; Oliani, Sonia M; Bonini-Domingos, Claudia R

    2016-09-01

    Phenotypic heterogeneity for sickle cell disease is associated to several genetic factors such as genotype for sickle cell disease, β-globin gene cluster haplotypes and Hb F levels. The coinheritance of Hb S (HBB: c.20A > T) and Hb D-Punjab (HBB: c.364G > C) results in a double heterozygosity, which constitutes one of the genotypic causes of sickle cell disease. This study aimed to assess the phenotypic diversity of sickle cell disease presented by carriers of the Hb S/Hb D-Punjab genotype and the Bantu [- + - - - -] haplotype. We evaluated medical records from 12 patients with sickle cell disease whose Hb S/Hb D-Punjab genotype and Bantu haplotype were confirmed by molecular analysis. Hb S and Hb D-Punjab levels were quantified by chromatographic analysis. Mean concentrations of Hb S and Hb D-Punjab were 44.8 ± 2.3% and 43.3 ± 1.8%, respectively. Painful crises were present in eight (66.7%) patients evaluated, representing the most common clinical event. Acute chest syndrome (ACS) was the second most prevalent manifestation, occurring in two individuals (16.7%). Three patients were asymptomatic, while another two exhibited greater diversity of severe clinical manifestations. Medical records here analyzed reported a significant clinical diversity in sickle cell disease ranging from the absence of symptoms to wide phenotypic variety. The sickle cell disease genotype, Bantu haplotype and hemoglobin (Hb) levels did not influence the clinical diversity. Thus, we concluded that the phenotypic variation in sickle cell disease was present within a specific genotype for disease regardless of the β-globin gene cluster haplotypes.

  12. Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

    Directory of Open Access Journals (Sweden)

    Plumas Joel

    2009-01-01

    Full Text Available Abstract Background Gene modified dendritic cells (DC are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG, the gibbon ape leukaemia virus envelope (GaLV or the feline endogenous virus envelope (RD114. At the same time, we evaluated transgene expression (E-GFP reporter gene under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV under the control of phoshoglycerate kinase (PGK and elongation factor-1 (EF1α promoters (28% to 90% of E-GFP+ cells, respectively in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12 described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

  13. ALDH1A3: A Marker of Mesenchymal Phenotype in Gliomas Associated with Cell Invasion.

    Directory of Open Access Journals (Sweden)

    Wenlong Zhang

    Full Text Available Aldehyde dehydrogenases (ALDH is a family of enzymes including 19 members. For now, ALDH activity had been wildly used as a marker of cancer stem cells (CSCs. But biological functions of relevant isoforms and their clinical applications are still controversial. Here, we investigate the clinical significance and potential function of ALDH1A3 in gliomas. By whole-genome transcriptome microarray and mRNA sequencing analysis, we compared the expression of ALDH1A3 in high- and low- grade gliomas as well as different molecular subtypes. Microarray analysis was performed to identify the correlated genes of ALDH1A3. We further used Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG pathways analysis to explore the biological function of ALDH1A3. Finally, by mRNA knockdown we revealed the relationship between ALDH1A3 and the ability of tumor invasion. ALDH1A3 overexpression was significantly associated with high grade as well as the higher mortality of gliomas in survival analysis. ALDH1A3 was characteristically highly expressed in Mesenchymal (Mes subtype gliomas. Moreover, we found that ALDH1A3 was most relevant to extracellular matrix organization and cell adhesion biological process, and the ability of tumor invasion was suppressed after ALDH1A3 knockdown in vitro. In conclusion, ALDH1A3 can serve as a novel marker of Mes phenotype in gliomas with potential clinical prognostic value. The expression of ALDH1A3 is associated with tumor cell invasion.

  14. Red cells with paroxysmal nocturnal hemoglobinuria-phenotype in patients with acute leukemia.

    Science.gov (United States)

    Meletis, John; Terpos, Evangelos; Samarkos, Michalis; Meletis, Christos; Apostolidou, Effie; Komninaka, Veroniki; Anargyrou, Konstantinos; Korovesis, Konstantinos; Mavrogianni, Despina; Variami, Eleni; Viniou, Nora; Konstantopoulos, Konstantinos

    2002-04-01

    CD55 and CD59 are complement regulatory proteins that are linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. They are reduced mainly in paroxysmal nocturnal hemoglobinuria (PNH) and in other hematological disorders. However, there are very few reports in the literature concerning their expression in patients with acute leukemias (AL). We studied the CD55 and CD59 expression in 88 newly diagnosed patients with AL [65 with acute non-lymphoblastic leukemia (ANLL) and 23 with acute lymphoblastic leukemia (ALL)] using the sephacryl gel test, the Ham and sucrose lysis tests and we compared the results with patients' clinical data and disease course. Eight patients with PNH were also studied as controls. Red cell populations deficient in both CD55 and CD59 were detected in 23% of ANLL patients (especially of M(0), M(2) and M(6) FAB subtypes), 13% of ALL and in all PNH patients. CD55-deficient erythrocytes were found in 6 ANLL patients while the expression of CD59 was decreased in only 3 patients with ANLL. No ALL patient had an isolated deficiency of these antigens. There was no correlation between the existence of CD55 and/or CD59 deficiency and the percentage of bone marrow infiltration, karyotype or response to treatment. However no patient with M(3), M(5), M(7) subtype of ANLL and mature B- or T-cell ALL showed a reduced expression of both antigens. The deficient populations showed no alteration after chemotherapy treatment or during disease course. This study provides evidence about the lower expression of CD55 and CD59 in some AL patients and the correlation with their clinical data. The possible mechanisms and the significance of this phenotype are discussed.

  15. Oxygen tension regulates the osteogenic, chondrogenic and endochondral phenotype of bone marrow derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sheehy, Eamon J.; Buckley, Conor T. [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland); Kelly, Daniel J., E-mail: kellyd9@tcd.ie [Trinity Centre for Bioengineering, School of Engineering, Trinity College Dublin, Dublin 2 (Ireland)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Expansion in low oxygen enhances MSC proliferation and osteogenesis. Black-Right-Pointing-Pointer Differentiation in low oxygen enhances chondrogenesis and suppresses hypertrophy. Black-Right-Pointing-Pointer Oxygen can regulate the MSC phenotype for use in tissue engineering applications. -- Abstract: The local oxygen tension is a key regulator of the fate of mesenchymal stem cells (MSCs). The objective of this study was to investigate the effect of a low oxygen tension during expansion and differentiation on the proliferation kinetics as well as the subsequent osteogenic and chondrogenic potential of MSCs. We first hypothesised that expansion in a low oxygen tension (5% pO{sub 2}) would improve both the subsequent osteogenic and chondrogenic potential of MSCs compared to expansion in a normoxic environment (20% pO{sub 2}). Furthermore, we hypothesised that chondrogenic differentiation in a low oxygen environment would suppress hypertrophy of MSCs cultured in both pellets and hydrogels used in tissue engineering strategies. MSCs expanded at 5% pO{sub 2} proliferated faster forming larger colonies, resulting in higher cell yields. Expansion at 5% pO{sub 2} also enhanced subsequent osteogenesis of MSCs, whereas differentiation at 5% pO{sub 2} was found to be a more potent promoter of chondrogenesis than expansion at 5% pO{sub 2}. Greater collagen accumulation, and more intense staining for collagen types I and X, was observed in pellets maintained at 20% pO{sub 2} compared to 5% pO{sub 2}. Both pellets and hydrogels stained more intensely for type II collagen when undergoing chondrogenesis in a low oxygen environment. Differentiation at 5% pO{sub 2} also appeared to inhibit hypertrophy in both pellets and hydrogels, as demonstrated by reduced collagen type X and Alizarin Red staining and alkaline phosphatase activity. This study demonstrates that the local oxygen environment can be manipulated in vitro to either stabilise a

  16. Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

    Science.gov (United States)

    Lee, Sangyeop; Chon, Hyangah; Lee, Jiyoung; Ko, Juhui; Chung, Bong Hyun; Lim, Dong Woo; Choo, Jaebum

    2014-01-15

    We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment. © 2013 Elsevier B.V. All rights reserved.

  17. Single-cell Transcriptional Analysis Reveals Novel Neuronal Phenotypes and Interaction Networks involved In the Central Circadian Clock

    Directory of Open Access Journals (Sweden)

    James Park

    2016-10-01

    Full Text Available Single-cell heterogeneity confounds efforts to understand how a population of cells organizes into cellular networks that underlie tissue-level function. This complexity is prominent in the mammalian suprachiasmatic nucleus (SCN. Here, individual neurons exhibit a remarkable amount of asynchronous behavior and transcriptional heterogeneity. However, SCN neurons are able to generate precisely coordinated synaptic and molecular outputs that synchronize the body to a common circadian cycle by organizing into cellular networks. To understand this emergent cellular network property, it is important to reconcile single-neuron heterogeneity with network organization. In light of recent studies suggesting that transcriptionally heterogeneous cells organize into distinct cellular phenotypes, we characterized the transcriptional, spatial, and functional organization of 352 SCN neurons from mice experiencing phase-shifts in their circadian cycle. Using the community structure detection method and multivariate analytical techniques, we identified previously undescribed neuronal phenotypes that are likely to participate in regulatory networks with known SCN cell types. Based on the newly discovered neuronal phenotypes, we developed a data-driven neuronal network structure in which multiple cell types interact through known synaptic and paracrine signaling mechanisms. These results provide a basis from which to interpret the functional variability of SCN neurons and describe methodologies towards understanding how a population of heterogeneous single cells organizes into cellular networks that underlie tissue-level function.

  18. iTRAQ quantitative proteomics-based identification of cell adhesion as a dominant phenotypic modulation in thrombin-stimulated human aortic endothelial cells.

    Science.gov (United States)

    Wang, Huang-Joe; Chen, Sung-Fang; Lo, Wan-Yu

    2015-05-01

    The phenotypic changes in thrombin-stimulated endothelial cells include alterations in permeability, cell shape, vasomotor tone, leukocyte trafficking, migration, proliferation, and angiogenesis. Previous studies regarding the pleotropic effects of thrombin on the endothelium used human umbilical vein endothelial cells (HUVECs)-cells derived from fetal tissue that does not exist in adults. Only a few groups have used screening approaches such as microarrays to profile the global effects of thrombin on endothelial cells. Moreover, the proteomic changes of thrombin-stimulated human aortic endothelial cells (HAECs) have not been elucidated. HAECs were stimulated with 2 units/mL thrombin for 5h and their proteome was investigated using isobaric tags for the relative and absolute quantification (iTRAQ) and the MetaCore(TM) software. A total of 627 (experiment A) and 622 proteins (experiment B) were quantified in the duplicated iTRAQ analyses. MetaCore(TM) pathway analysis identified cell adhesion as a dominant phenotype in thrombin-stimulated HAECs. Replicated iTRAQ data revealed that "Cell adhesion_Chemokines and adhesion," "Cell adhesion_Histamine H1 receptor signaling in the interruption of cell barrier integrity," and "Cell adhesion_Integrin-mediated cell adhesion and migration" were among the top 10 statistically significant pathways. The cell adhesion phenotype was verified by increased THP-1 adhesion to thrombin-stimulated HAECs. In addition, the expression of ICAM-1, VCAM-1, and SELE was significantly upregulated in thrombin-stimulated HAECs. Several regulatory pathways are altered in thrombin-stimulated HAECs, with cell adhesion being the dominant altered phenotype. Our findings show the feasibility of the iTRAQ technique for evaluating cellular responses to acute stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Effects of HIV infection and ART on phenotype and function of circulating monocytes, natural killer, and innate lymphoid cells.

    Science.gov (United States)

    Nabatanzi, Rose; Cose, Stephen; Joloba, Moses; Jones, Sarah Rowland; Nakanjako, Damalie

    2018-03-15

    HIV infection causes upregulation of markers of inflammation, immune activation and apoptosis of host adaptive, and innate immune cells particularly monocytes, natural killer (NK) and innate lymphoid cells (ILCs). Although antiretroviral therapy (ART) restores CD4 T-cell counts, the persistent aberrant activation of monocytes, NK and ILCs observed likely contributes to the incomplete recovery of T-cell effector functions. A better understanding of the effects of HIV infection and ART on the phenotype and function of circulating monocytes, NK, and ILCs is required to guide development of novel therapeutic interventions to optimize immune recovery.

  20. Phenotypic stability and plasticity in GMP-derived cells as determined by their underlying regulatory network.

    Science.gov (United States)

    Ramírez, Carlos; Mendoza, Luis

    2018-04-01

    Blood cell formation has been recognized as a suitable system to study celular differentiation mainly because of its experimental accessibility, and because it shows characteristics such as hierarchical and gradual bifurcated patterns of commitment, which are present in several developmental processes. Although hematopoiesis has been extensively studied and there is a wealth of molecular and cellular data about it, it is not clear how the underlying molecular regulatory networks define or restrict cellular differentiation processes. Here, we infer the molecular regulatory network that controls the differentiation of a blood cell subpopulation derived from the granulocyte-monocyte precursor (GMP), comprising monocytes, neutrophils, eosinophils, basophils and mast cells. We integrate published qualitative experimental data into a model to describe temporal expression patterns observed in GMP-derived cells. The model is implemented as a Boolean network, and its dynamical behavior is studied. Steady states of the network can be clearly identified with the expression profiles of monocytes, mast cells, neutrophils, basophils, and eosinophils, under wild-type and mutant backgrounds. All scripts are publicly available at https://github.com/caramirezal/RegulatoryNetworkGMPModel. lmendoza@biomedicas.unam.mx. Supplementary data are available at Bioinformatics online.

  1. Murine Lupus Susceptibility Locus Sle2 Activates DNA-Reactive B Cells through Two Sub-Loci with Distinct Phenotypes

    Science.gov (United States)

    Zeumer, Leilani; Sang, Allison; Niu, Haitao; Morel, Laurence

    2010-01-01

    The NZM2410-derived Sle2 lupus susceptibility locus induces an abnormal B cell differentiation which most prominently leads to the expansion of autoreactive B1a cells. We have mapped the expansion of B1a cells to three Sle2 sub-loci, Sle2a, Sle2b, and Sle2c. Sle2 also enhances the breach of B cell tolerance to nuclear antigens in the 56R anti-DNA immunoglobulin transgenic (Tg) model. This study used the Sle2 sub-congenic strains to map the activation of 56R Tg B cells. Sle2c strongly sustained the breach of tolerance and the activation of anti-DNA B cells. The production of Tg-encoded anti-DNA antibodies was more modest in Sle2a expressing mice, but Sle2a was responsible for the recruitment for Tg B cells to the marginal zone, a phenotype that has been found for 56R Tg B cells in mice expressing the whole Sle2 interval. In addition, Sle2a promoted the production of endogenously encoded anti-DNA antibodies. Overall, this study showed that at least two Sle2 genes are involved in the activation of anti-DNA B cells, and excluded more than two-thirds of the Sle2 interval from contributing to this phenotype. This constitutes an important step toward the identification of novel genes that play a critical role in B cell tolerance. PMID:21270826

  2. Synthetic tetracycline-inducible regulatory networks: computer-aided design of dynamic phenotypes

    Directory of Open Access Journals (Sweden)

    Kaznessis Yiannis N

    2007-01-01

    Full Text Available Abstract Background Tightly regulated gene networks, precisely controlling the expression of protein molecules, have received considerable interest by the biomedical community due to their promising applications. Among the most well studied inducible transcription systems are the tetracycline regulatory expression systems based on the tetracycline resistance operon of Escherichia coli, Tet-Off (tTA and Tet-On (rtTA. Despite their initial success and improved designs, limitations still persist, such as low inducer sensitivity. Instead of looking at these networks statically, and simply changing or mutating the promoter and operator regions with trial and error, a systematic investigation of the dynamic behavior of the network can result in rational design of regulatory gene expression systems. Sophisticated algorithms can accurately capture the dynamical behavior of gene networks. With computer aided design, we aim to improve the synthesis of regulatory networks and propose new designs that enable tighter control of expression. Results In this paper we engineer novel networks by recombining existing genes or part of genes. We synthesize four novel regulatory networks based on the Tet-Off and Tet-On systems. We model all the known individual biomolecular interactions involved in transcription, translation, regulation and induction. With multiple time-scale stochastic-discrete and stochastic-continuous models we accurately capture the transient and steady state dynamics of these networks. Important biomolecular interactions are identified and the strength of the interactions engineered to satisfy design criteria. A set of clear design rules is developed and appropriate mutants of regulatory proteins and operator sites are proposed. Conclusion The complexity of biomolecular interactions is accurately captured through computer simulations. Computer simulations allow us to look into the molecular level, portray the dynamic behavior of gene regulatory

  3. Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome

    DEFF Research Database (Denmark)

    Pedersen, Martin Bjerregård; Hamilton-Dutoit, Stephen Jacques; Bendix, Knud

    2014-01-01

    Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome.......Identification of a subset of perpheral T-cell lymphoma, not otherwise specified, characterized by FOXP3-positive regulatory T-cell phenotype, HTLV-1 negativity and poor outcome....

  4. Phenotypic malignant changes and untargeted lipidomic analysis of long-term exposed prostate cancer cells to endocrine disruptors

    International Nuclear Information System (INIS)

    Bedia, Carmen; Dalmau, Núria; Jaumot, Joaquim; Tauler, Romà

    2015-01-01

    Endocrine disruptors (EDs) are a class of environmental toxic molecules able to interfere with the normal hormone metabolism. Numerous studies involve EDs exposure to initiation and development of cancers, including prostate cancer. In this work, three different EDs (aldrin, aroclor 1254 and chlorpyrifos (CPF)) were investigated as potential inducers of a malignant phenotype in DU145 prostate cancer cells after a chronic exposure. Epithelial to mesenchymal transition (EMT) induction, proliferation, migration, colony formation and release of metalloproteinase 2 (MMP-2) were analyzed in 50-day exposed cells to the selected EDs. As a result, aldrin and CPF exposure led to an EMT induction (loss of 16% and 14% of E-cadherin levels, respectively, compared to the unexposed cells). Aroclor and CPF presented an increased migration (134% and 126%, respectively), colony formation (204% and 144%, respectively) and MMP-2 release (137% in both cases) compared to the unexposed cells. An untargeted lipidomic analysis was performed to decipher the lipids involved in the observed transformations. As general results, aldrin exposure showed a global decrease in phospholipids and sphingolipids, and aroclor and CPF showed an increase of certain phospholipids, glycosphingolipids as well as a remarkable increase of some cardiolipin species. Furthermore, the three exposures resulted in an increase of some triglyceride species. In conclusion, some significant changes in lipids were identified and thus we postulate that some lipid compounds and lipid metabolic pathways could be involved in the acquisition of the malignant phenotype in exposed prostate cancer cells to the selected EDs. - Highlights: • Aldrin, aroclor and chlorpyrifos induced an aggressive phenotype in DU145 cells. • An untargeted lipidomic analysis has been performed on chronic exposed cells. • Lipidomic results showed changes in specific lipid species under chronic exposure. • These lipids may have a role in the

  5. Phenotypic malignant changes and untargeted lipidomic analysis of long-term exposed prostate cancer cells to endocrine disruptors

    Energy Technology Data Exchange (ETDEWEB)

    Bedia, Carmen, E-mail: carmen.bedia@idaea.csic.es; Dalmau, Núria, E-mail: nuria.dalmau@idaea.csic.es; Jaumot, Joaquim, E-mail: joaquim.jaumot@idaea.csic.es; Tauler, Romà, E-mail: roma.tauler@idaea.csic.es

    2015-07-15

    Endocrine disruptors (EDs) are a class of environmental toxic molecules able to interfere with the normal hormone metabolism. Numerous studies involve EDs exposure to initiation and development of cancers, including prostate cancer. In this work, three different EDs (aldrin, aroclor 1254 and chlorpyrifos (CPF)) were investigated as potential inducers of a malignant phenotype in DU145 prostate cancer cells after a chronic exposure. Epithelial to mesenchymal transition (EMT) induction, proliferation, migration, colony formation and release of metalloproteinase 2 (MMP-2) were analyzed in 50-day exposed cells to the selected EDs. As a result, aldrin and CPF exposure led to an EMT induction (loss of 16% and 14% of E-cadherin levels, respectively, compared to the unexposed cells). Aroclor and CPF presented an increased migration (134% and 126%, respectively), colony formation (204% and 144%, respectively) and MMP-2 release (137% in both cases) compared to the unexposed cells. An untargeted lipidomic analysis was performed to decipher the lipids involved in the observed transformations. As general results, aldrin exposure showed a global decrease in phospholipids and sphingolipids, and aroclor and CPF showed an increase of certain phospholipids, glycosphingolipids as well as a remarkable increase of some cardiolipin species. Furthermore, the three exposures resulted in an increase of some triglyceride species. In conclusion, some significant changes in lipids were identified and thus we postulate that some lipid compounds and lipid metabolic pathways could be involved in the acquisition of the malignant phenotype in exposed prostate cancer cells to the selected EDs. - Highlights: • Aldrin, aroclor and chlorpyrifos induced an aggressive phenotype in DU145 cells. • An untargeted lipidomic analysis has been performed on chronic exposed cells. • Lipidomic results showed changes in specific lipid species under chronic exposure. • These lipids may have a role in the

  6. Dynamic ubiquitin signaling in cell cycle regulation.

    Science.gov (United States)

    Gilberto, Samuel; Peter, Matthias

    2017-08-07

    The cell division cycle is driven by a collection of enzymes that coordinate DNA duplication and separation, ensuring that genomic information is faithfully and perpetually maintained. The activity of the effector proteins that perform and coordinate these biological processes oscillates by regulated expression and/or posttranslational modifications. Ubiquitylation is a cardinal cellular modification and is long known for driving cell cycle transitions. In this review, we emphasize emerging concepts of how ubiquitylation brings the necessary dynamicity and plasticity that underlie the processes of DNA replication and mitosis. New studies, often focusing on the regulation of chromosomal proteins like DNA polymerases or kinetochore kinases, are demonstrating that ubiquitylation is a versatile modification that can be used to fine-tune these cell cycle events, frequently through processes that do not involve proteasomal degradation. Understanding how the increasing variety of identified ubiquitin signals are transduced will allow us to develop a deeper mechanistic perception of how the multiple factors come together to faithfully propagate genomic information. Here, we discuss these and additional conceptual challenges that are currently under study toward understanding how ubiquitin governs cell cycle regulation. © 2017 Gilberto and Peter.

  7. Shear Stress Induces Phenotypic Modulation of Vascular Smooth Muscle Cells via AMPK/mTOR/ULK1-Mediated Autophagy.

    Science.gov (United States)

    Sun, Liqian; Zhao, Manman; Liu, Aihua; Lv, Ming; Zhang, Jingbo; Li, Youxiang; Yang, Xinjian; Wu, Zhongxue

    2018-03-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) is involved in the pathophysiological processes of the intracranial aneurysms (IAs). Although shear stress has been implicated in the proliferation, migration, and phenotypic conversion of VSMCs, the molecular mechanisms underlying these events are currently unknown. In this study, we investigated whether shear stress(SS)-induced VSMC phenotypic modulation was mediated by autophagy involved in adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway. The results show that shear stress could inhibit the expression of key VSMC contractile genes and induce pro-inflammatory/matrix-remodeling genes levels, contributing to VSMCs phenotypic switching from a contractile to a synthetic phenotype. More importantly, Shear stress also markedly increased the levels of the autophagy marker microtubule-associated protein light chain 3-II (LC3II), Beclin-1, and p62 degradation. The autophagy inhibitor 3-methyladenine (3-MA) significantly blocked shear-induced phenotypic modulation of VSMCs. To further explore the molecular mechanism involved in shear-induced autophagy, we found that shear stress could activate AMPK/mTOR/ULK1 signaling pathway in VSMCs. Compound C, a pharmacological inhibitor of AMPK, significantly reduced the levels of p-AMPK and p-ULK, enhanced p-mTOR level, and finally decreased LC3II and Beclin-1 level, which suggested that activated AMPK/mTOR/ULK1 signaling was related to shear-mediated autophagy. These results indicate that shear stress promotes VSMC phenotypic modulation through the induction of autophagy involved in activating the AMPK/mTOR/ULK1 pathway.

  8. Dynamic and diverse changes in the functional properties of vascular smooth muscle cells in pulmonary hypertension.

    Science.gov (United States)

    Stenmark, Kurt R; Frid, Maria G; Graham, Brian B; Tuder, Rubin M

    2018-03-15

    Pulmonary hypertension (PH) is the end result of interaction between pulmonary vascular tone and a complex series of cellular and molecular events termed 'vascular remodelling'. The remodelling process, which can involve the entirety of pulmonary arterial vasculature, almost universally involves medial thickening, driven by increased numbers and hypertrophy of its principal cellular constituent, smooth muscle cells (SMCs). It is noted, however that SMCs comprise heterogeneous populations of cells, which can exhibit markedly different proliferative, inflammatory, and extracellular matrix production changes during remodelling. We further consider that these functional changes in SMCs of different phenotype and their role in PH are dynamic and may undergo significant changes over time (which we will refer to as cellular plasticity); no single property can account for the complexity of the contribution of SMC to pulmonary vascular remodelling. Thus, the approaches used to pharmacologically manipulate PH by targeting the SMC phenotype(s) must take into account processes that underlie dominant phenotypes that drive the disease. We present evidence for time- and location-specific changes in SMC proliferation in various animal models of PH; we highlight the transient nature (rather than continuous) of SMC proliferation, emphasizing that the heterogenic SMC populations that reside in different locations along the pulmonary vascular tree exhibit distinct responses to the stresses associated with the development of PH. We also consider that cells that have often been termed 'SMCs' may arise from many origins, including endothelial cells, fibroblasts and resident or circulating progenitors, and thus may contribute via distinct signalling pathways to the remodelling process. Ultimately, PH is characterized by long-lived, apoptosis-resistant SMC. In line with this key pathogenic characteristic, we address the acquisition of a pro-inflammatory phenotype by SMC that is essential

  9. Data in support of dyslipidemia-associated alterations in B cell subpopulations frequency and phenotype during experimental atherosclerosis

    Directory of Open Access Journals (Sweden)

    Héctor Rincón-Arévalo

    2016-06-01

    Full Text Available Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient (apoE−/− mice fed or not with high-fat diet (HFD, by flow cytometry. Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosis (DOI: 10.1016/j.atherosclerosis.2015.12.022, article reference: ATH14410 [1]. The data contained in this article shows and supports that mice with advanced atherosclerosis have a variety of alterations in frequency and phenotype of B cell subsets, most of which associated with dyslipidemia.

  10. Data in support of dyslipidemia-associated alterations in B cell subpopulations frequency and phenotype during experimental atherosclerosis

    Science.gov (United States)

    Rincón-Arévalo, Héctor; Castaño, Diana; Villa-Pulgarín, Janny; Rojas, Mauricio; Vásquez, Gloria; Correa, Luis A.; Ramírez-Pineda, José R.; Yassin, Lina M.

    2016-01-01

    Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient (apoE−/−) mice fed or not with high-fat diet (HFD), by flow cytometry. Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosis (DOI: 10.1016/j.atherosclerosis.2015.12.022, article reference: ATH14410) [1]. The data contained in this article shows and supports that mice with advanced atherosclerosis have a variety of alterations in frequency and phenotype of B cell subsets, most of which associated with dyslipidemia. PMID:27081674

  11. A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.

    Science.gov (United States)

    Vinardell, Tatiana; Sheehy, Eamon J; Buckley, Conor T; Kelly, Daniel J

    2012-06-01

    Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-β3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional 28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (∼2.8% w/w) and collagen (∼1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption, as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation

  12. Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

    Science.gov (United States)

    Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

    2008-02-01

    Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

  13. Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells.

    Science.gov (United States)

    Semeraro, F; Ammollo, C T; Esmon, N L; Esmon, C T

    2014-10-01

    Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction. To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS). PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test. Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma. Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones. © 2014 International Society on Thrombosis and Haemostasis.

  14. A single amino acid substitution controls DAF-dependent phenotype of echovirus 11 in rhabdomyosarcoma cells.

    Science.gov (United States)

    Novoselov, Alexey V; Rezaykin, Alexey V; Sergeev, Alexander G; Fadeyev, Fedor A; Grigoryeva, Julia V; Sokolova, Zoya I

    2012-06-01

    Decay accelerating factor (DAF, CD55) is used by DAF-dependent (Daf+) variants of echovirus 11 (EV11) as a primary cellular receptor. The interaction of EV11 with DAF is completely reversible, therefore DAF-dependent variants require an unidentified coreceptor to initiate uncoating. Daf- variants of EV11, which do not interact with DAF, use an alternative primary cellular receptor. The aim of this study was to test the hypothesis whether the coreceptor, which is necessary for the uncoating of DAF-dependent variants, may act as an alternative primary receptor for the Daf- variants of EV11. By using the model of the two closely related daf+ and daf- clones of EV11 in rhabdomyosarcoma (RD) cell line, it was shown that a single amino acid substitution in the capsid protein VP2 could control the expression of the DAF-dependent phenotype. Anti-DAF monoclonal antibody has blocked the infection of RD cells by the DAF-dependent daf+ clone, but not by the daf- clone of EV11. Since the structural proteins of the two clones differed only in the receptor binding site for DAF, the unidentified non-DAF primary receptor for the daf- clone might have the same conformation as the uncoating coreceptor required for the daf+ clone. Despite the difference in primary receptors, both daf+ and daf- clones were equally inhibited by a monoclonal antibody to beta2-microglobulin. The monoclonal antibody B9.12.1 to class I human leukocyte antigen molecules showed no inhibitory effect in regards to either clone. The hypothesis of convergent intracellular traffic of Daf+ and Daf- variants of EV11 is discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

    Institute of Scientific and Technical Information of China (English)

    Zhi-Bo Wang; Xiaoqing Zhang; Xue-Jun Li

    2013-01-01

    Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease.Here,we developed a closely representative cell model of SMA by knocking down the disease-determining gene,survival motor neuron (SMN),in human embryonic stem cells (hESCs).Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons.Notably,the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated.Furthermore,these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-A7 (lacking exon 7)knockdown,and were specific to spinal motor neurons.Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes,including specific axonal defects and motor neuron loss.Finally,knockdown of SMNFL led to excessive mitochondrial oxidative stress in human motor neuron progenitors.The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine,a potent antioxidant,which prevented disease-related apoptosis and subsequent motor neuron death.Thus,we report here the successful establishment of an hESC-based SMA model,which exhibits disease gene isoform specificity,cell type specificity,and phenotype reversibility.Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.

  16. Bone Cells Dynamics during Peri-Implantitis: a Theoretical Analysis

    Directory of Open Access Journals (Sweden)

    Maria Helena Fernandes

    2016-09-01

    Full Text Available Objectives: The present manuscript aims a detailed characterization of the bone cells dynamics during physiological bone remodelling and, subsequently, to address the cellular and molecular mechanisms that play a fundamental role in the immune-inflammatory-induced uncoupled bone remodelling observed in peri-implantitis. Results: An intimate relationship between the immune system and bone is acknowledged to be determinant for bone tissue remodelling and integrity. Due to the close interaction of immune and bone cells, the two systems share a number of surface receptors, cytokines, signalling pathways and transcription factors that are involved in mutual regulatory mechanisms. This physiological equilibrium is disturbed in pathological conditions, as verified in peri-implantitis establishment and development. Activation of the innate and adaptive immune response, challenged by the local bacterial infection, induces the synthesis of high levels of a variety of pro- and anti-inflammatory cytokines that disturb the normal functioning of the bone cells, by uncoupling bone resorption and formation, ending up with a net alveolar bone loss and subsequent implant failure. Most data points to an immune-inflammatory induced osteoclast differentiation and function, as the major underlying mechanism to the uncoupled bone resorption to bone formation. Further, the disturbed functioning of osteoblasts, reflected by the possible expression of a fibro-osteoblastic phenotype, may also play a role. Conclusions: Alveolar bone loss is a hallmark of peri-implantitis. A great deal of data is still needed on the cellular and humoral crosstalk in the context of an integrated view of the osteoimmunologic interplay occurring in the peri-implantitis environment subjacent to the bone loss outcome.

  17. Dynamic cell culture system (7-IML-1)

    Science.gov (United States)

    Cogoli, Augusto

    1992-01-01

    This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

  18. Structural dynamics of the cell nucleus

    Science.gov (United States)

    Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons. PMID:21738832

  19. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    DEFF Research Database (Denmark)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M

    2012-01-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been...... by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite...... and differentiation to osteoblasts, adipocytes and chondrocytes....

  20. Characterization of the metabolic phenotype of rapamycin-treated CD8+ T cells with augmented ability to generate long-lasting memory cells.

    Directory of Open Access Journals (Sweden)

    Shan He

    Full Text Available BACKGROUND: Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized. DESIGN AND METHODS: Using lymphocytic choriomeningitis virus (LCMV peptide gp33-specific CD8(+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo. RESULTS: Antigen-activated CD8(+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS. These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells. CONCLUSIONS: Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

  1. Low interleukin-2 concentration favors generation of early memory T cells over effector phenotypes during chimeric antigen receptor T-cell expansion.

    Science.gov (United States)

    Kaartinen, Tanja; Luostarinen, Annu; Maliniemi, Pilvi; Keto, Joni; Arvas, Mikko; Belt, Heini; Koponen, Jonna; Loskog, Angelica; Mustjoki, Satu; Porkka, Kimmo; Ylä-Herttuala, Seppo; Korhonen, Matti

    2017-06-01

    Adoptive T-cell therapy offers new options for cancer treatment. Clinical results suggest that T-cell persistence, depending on T-cell memory, improves efficacy. The use of interleukin (IL)-2 for in vitro T-cell expansion is not straightforward because it drives effector T-cell differentiation but does not promote the formation of T-cell memory. We have developed a cost-effective expansion protocol for chimeric antigen receptor (CAR) T cells with an early memory phenotype. Lymphocytes were transduced with third-generation lentiviral vectors and expanded using CD3/CD28 microbeads. The effects of altering the IL-2 supplementation (0-300 IU/mL) and length of expansion (10-20 days) on the phenotype of the T-cell products were analyzed. High IL-2 levels led to a decrease in overall generation of early memory T cells by both decreasing central memory T cells and augmenting effectors. T memory stem cells (T SCM , CD95 + CD45RO - CD45RA + CD27 + ) were present variably during T-cell expansion. However, their presence was not IL-2 dependent but was linked to expansion kinetics. CD19-CAR T cells generated in these conditions displayed in vitro antileukemic activity. In summary, production of CAR T cells without any cytokine supplementation yielded the highest proportion of early memory T cells, provided a 10-fold cell expansion and the cells were functionally potent. The number of early memory T cells in a T-cell preparation can be increased by simply reducing the amount of IL-2 and limiting the length of T-cell expansion, providing cells with potentially higher in vivo performance. These findings are significant for robust and cost-effective T-cell manufacturing. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  2. Phenotype of dendritic cells generated in the presence of non-small cell lung cancer antigens - preliminary report.

    Directory of Open Access Journals (Sweden)

    Dariusz Sagan

    2009-01-01

    Full Text Available Therapeutic outcomes of definitively treated non-small-cell lung cancer (NSCLC are unacceptably poor. It has been proposed that the manipulation of dendritic cells (DCs as a "natural" vaccine adjuvant may prove to be a particularly effective way to stimulate antitumor immunity. Presently, there is no standardized methodology for preparing vaccines and many questions concerning the optimal source and type of antigens as well as maturation state and activity of DCs are still unsolved. The study population comprised of ten patients with histologically confirmed NSCLC (mean age: 67.63 +/- 6.15 years. Resected small tumor pieces were placed in tissue culture dishes containing different growth factors in order to obtain pure cancer cells. Seven days after the operation, the PBMC were collected and monocytes were purified by the adherence to culture dishes. Monocytes were cultured in RPMI 1640 medium supplemented with 10% of autologous plasma in the presence of rhIL-4 and rhGM-CSF to generate immature autologous (DCs. TNF-alpha with or without tumor cells' lysate were added to maturation of DCs. After 7 days of culture, DCs were harvested and the expression of CD1a, CD83, CD80, CD86 and HLA-DR antigens were analyzed by flow cytometry. We discovered higher (p=0.07 percentage of semimature DCs in tumor cell lysate culture in comparison with TNF-alpha culture (21.22 +/- 16.82% versus 11.27 +/- 11.64%. The expression of co-stimulatory and maturation markers (CD86, CD83 and HLA-DR was higher on DCs from the culture with tumor cell lysate compared with TNF-alpha culture as a control. Specimen of NSCLC's culture prepared in this way could generate differences in DCs phenotype, which may have an influence on the therapeutic and protective antitumor immunity of the vaccine. Our research seems to be the next step in the development of DC-based vaccine. We are going to continue the investigation to start the preparation of a pattern of immunological vaccine

  3. A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro

    Directory of Open Access Journals (Sweden)

    Lewis Fiona C

    2012-08-01

    Full Text Available Abstract Background Human embryonic stem cells (hESCs represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF feeder-layers and exogenous protein media supplementation. Results These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP, which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages. Conclusions PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

  4. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Genz, Berit [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Thomas, Maria [Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart (Germany); Pützer, Brigitte M. [Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Rostock (Germany); Siatkowski, Marcin; Fuellen, Georg [Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock (Germany); Vollmar, Brigitte [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany); Abshagen, Kerstin, E-mail: kerstin.abshagen@uni-rostock.de [Institute for Experimental Surgery, Rostock University Medical Center, Rostock (Germany)

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells.

  5. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

    International Nuclear Information System (INIS)

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M.; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-01-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. - Highlights: • We performed adenoviral overexpression of Lhx2 in primary hepatic stellate cells. • Hepatic stellate cells expressed stem cell markers during cultivation. • Cell migration and contractility was slightly hampered upon Lhx2 overexpression. • Lhx2 overexpression did not affect stem cell character of hepatic stellate cells

  6. Canine Mammary Cancer Stem Cells are Radio- and Chemo-Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype

    International Nuclear Information System (INIS)

    Pang, Lisa Y.; Cervantes-Arias, Alejandro; Else, Rod W.; Argyle, David J.

    2011-01-01

    Canine mammary carcinoma is the most common cancer among female dogs and is often fatal due to the development of distant metastases. In humans, solid tumors are made up of heterogeneous cell populations, which perform different roles in the tumor economy. A small subset of tumor cells can hold or acquire stem cell characteristics, enabling them to drive tumor growth, recurrence and metastasis. In veterinary medicine, the molecular drivers of canine mammary carcinoma are as yet undefined. Here we report that putative cancer stem cells (CSCs) can be isolated form a canine mammary carcinoma cell line, REM134. We show that these cells have an increased ability to form tumorspheres, a characteristic of stem cells, and that they express embryonic stem cell markers associated with pluripotency. Moreover, canine CSCs are relatively resistant to the cytotoxic effects of common chemotherapeutic drugs and ionizing radiation, indicating that failure of clinical therapy to eradicate canine mammary cancer may be due to the survival of CSCs. The epithelial to mesenchymal transition (EMT) has been associated with cancer invasion, metastasis, and the acquisition of stem cell characteristics. Our results show that canine CSCs predominantly express mesenchymal markers and are more invasive than parental cells, indicating that these cells have a mesenchymal phenotype. Furthermore, we show that canine mammary cancer cells can be induced to undergo EMT by TGFβ and that these cells have an increased ability to form tumorspheres. Our findings indicate that EMT induction can enrich for cells with CSC properties, and provide further insight into canine CSC biology

  7. Canine Mammary Cancer Stem Cells are Radio- and Chemo-Resistant and Exhibit an Epithelial-Mesenchymal Transition Phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Lisa Y., E-mail: lisa.pang@ed.ac.uk; Cervantes-Arias, Alejandro; Else, Rod W.; Argyle, David J. [Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh, Easter Bush, Midlothian, EH25 9RG (United Kingdom)

    2011-03-30

    Canine mammary carcinoma is the most common cancer among female dogs and is often fatal due to the development of distant metastases. In humans, solid tumors are made up of heterogeneous cell populations, which perform different roles in the tumor economy. A small subset of tumor cells can hold or acquire stem cell characteristics, enabling them to drive tumor growth, recurrence and metastasis. In veterinary medicine, the molecular drivers of canine mammary carcinoma are as yet undefined. Here we report that putative cancer stem cells (CSCs) can be isolated form a canine mammary carcinoma cell line, REM134. We show that these cells have an increased ability to form tumorspheres, a characteristic of stem cells, and that they express embryonic stem cell markers associated with pluripotency. Moreover, canine CSCs are relatively resistant to the cytotoxic effects of common chemotherapeutic drugs and ionizing radiation, indicating that failure of clinical therapy to eradicate canine mammary cancer may be due to the survival of CSCs. The epithelial to mesenchymal transition (EMT) has been associated with cancer invasion, metastasis, and the acquisition of stem cell characteristics. Our results show that canine CSCs predominantly express mesenchymal markers and are more invasive than parental cells, indicating that these cells have a mesenchymal phenotype. Furthermore, we show that canine mammary cancer cells can be induced to undergo EMT by TGFβ and that these cells have an increased ability to form tumorspheres. Our findings indicate that EMT induction can enrich for cells with CSC properties, and provide further insight into canine CSC biology.

  8. Diagnosis of renal cell cancer by dynamic MRI

    International Nuclear Information System (INIS)

    Togami, Izumi; Kitagawa, Takahiro; Katoh, Katsuya

    1992-01-01

    Dynamic MRI was performed in 15 cases (16 lesions) of renal cell cancer. The enhanced pattern of the tumor was mainly evaluated and findings were compared with these of dynamic CT and renal angiography. Enhanced patterns on dynamic MRI and dynamic CT were similar, but each phase on dynamic MRI tended to be prolonged compared with dynamic CT. Many hypervascular tumors on renal angiography had prominent enhancement in an early phase on dynamic MRI, but there was no prominent enhancement in cases with tumor thrombi in the renal vein or IVC. All hypovascular tumors were enhanced to some degree without exception on dynamic MRI. Dynamic MRI is considered to be useful for the evaluation of the characterization, especially vascularity, of renal cell cancer, but we should pay attention to the differential diagnosis from other tumor in atypical cases because its enhanced patterns are various on dynamic MRI. (author)

  9. Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Mao, Lizheng [Jiangsu Asialand Biomed-Technology Co. Ltd., Changzhou, Jiangsu 213164 (China); Deng, Linhong, E-mail: dlh@cczu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, and Bioengineering College, Chongqing University, Shapingba, Chongqing 400030 (China); Changzhou Key Laboratory of Respiratory Medical Engineering, Institute of Biomedical Engineering and Health Sciences, Changzhou University, Changzhou, Jiangsu 213164 (China)

    2016-11-15

    A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes

  10. Overexpression of soluble ADAM33 promotes a hypercontractile phenotype of the airway smooth muscle cell in rat

    International Nuclear Information System (INIS)

    Duan, Yiyuan; Long, Jiaoyue; Chen, Jun; Jiang, Xuemei; Zhu, Jian; Jin, Yang; Lin, Feng; Zhong, Jun; Xu, Rong; Mao, Lizheng; Deng, Linhong

    2016-01-01

    A disintegrin and metalloproteinase 33 (ADAM33) has been identified as a susceptibility gene for asthma, but details of the causality are not fully understood. We hypothesize that soluble ADAM33 (sADAM33) overexpression can alter the mechanical behaviors of airway smooth muscle cells (ASMCs) via regulation of the cell's contractile phenotype, and thus contributes to airway hyperresponsiveness (AHR) in asthma. To test this hypothesis, we either overexpressed or knocked down the sADAM33 in rat ASMCs by transfecting the cells with sADAM33 coding sequence or a small interfering RNA (siRNA) that specifically targets the ADAM33 disintegrin domain, and subsequently assessed the cells for stiffness, contractility and traction force, together with the expression level of contractile and proliferative phenotype markers. We also investigated whether these changes were dependent on Rho/ROCK pathway by culturing the ASMCs either in the absence or presence of ROCK inhibitor (H1152). The results showed that the ASMCs with sADAM33 overexpression were stiffer and more contractile, generated greater traction force, exhibited increased expression levels of contractile phenotype markers and markedly enhanced Rho activation. Furthermore these changes were largely attenuated when the cells were cultured in the presence of H-1152. However, the knock-down of ADAM33 seemed insufficient to influence majority of the mechanical behaviors of the ASMCs. Taken together, we demonstrated that sADAM33 overexpression altered the mechanical behaviors of ASMCs in vitro, which was most likely by promoting a hypercontractile phenotype transition of ASMCs through Rho/ROCK pathway. This revelation may establish the previously missing link between ADAM33 expression and AHR, and also provide useful insight for targeting sADAM33 in asthma prevention and therapy. - Highlights: • sADAM33 overexpression enhances the stiffness, traction force and contractility of ASMCs. • sADAM33 overexpression promotes a

  11. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  12. Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

    Science.gov (United States)

    Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

    2016-01-01

    We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

  13. Neurospheres induced from bone marrow stromal cells are multipotent for differentiation into neuron, astrocyte, and oligodendrocyte phenotypes

    International Nuclear Information System (INIS)

    Suzuki, Hidenori; Taguchi, Toshihiko; Tanaka, Hiroshi; Kataoka, Hideo; Li Zhenglin; Muramatsu, Keiichi; Gondo, Toshikazu; Kawai, Shinya

    2004-01-01

    Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed β-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system

  14. Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems

    OpenAIRE

    Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason F.; Ng, Cherie T.; Krogan, Nevan; Sharan, Roded; Ideker, Trey

    2016-01-01

    Summary Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology?s hierarchical structure, we organize genotype ...

  15. In vitro cytokine production and phenotype expression by blood mononuclear cells from umbilical cords, children and adults

    DEFF Research Database (Denmark)

    Müller, K; Zak, M; Nielsen, S

    1996-01-01

    Age related differences in immunological reactions include variations in the in vitro functions of blood mononuclear cells (MNC). In an attempt to understand the mechanism behind these differences we examined age related differences in the phenotype profiles of MNC in parallel with the in vitro......, and unmeasurable levels in cord blood MNC. Flow cytometry analysis of the phenotypic distribution of MNC revealed age related differences in the expression of CD3, CD4, CD8, CD14, CD19, CD45RA, CD45R0, CD2, LFA-1, ICAM-1 and LFA-3. Correlation studies did not indicate that the observed differences in cytokine....... In conclusion, the study provides evidence of age related differences in the production of TNF alpha, IL-6 and IFNg among neonates, children and adults. These differences may to some extent be caused by differences in the expression of cell surface molecules involved in cellular interactions and signalling....

  16. Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells.

    Science.gov (United States)

    Genz, Berit; Thomas, Maria; Pützer, Brigitte M; Siatkowski, Marcin; Fuellen, Georg; Vollmar, Brigitte; Abshagen, Kerstin

    2014-11-01

    Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. T-bet and Eomes are differentially linked to the exhausted phenotype of CD8+ T cells in HIV infection.

    Directory of Open Access Journals (Sweden)

    Marcus Buggert

    2014-07-01

    Full Text Available CD8(+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8(+ T cell differentiation, T-bet and Eomesodermin (Eomes, have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4, functional characteristics and memory differentiation of CD8(+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8(+ T cells was elevated in chronically infected individuals and highly associated with a T-bet(dimEomes(hi expressional profile. Interestingly, both resting and activated HIV-specific CD8(+ T cells in chronic infection were almost exclusively T-bet(dimEomes(hi cells, while CMV-specific CD8(+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-bet(dimEomes(hi virus-specific CD8(+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector characteristics. The transitional and exhausted phenotype of HIV-specific CD8(+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8(+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8(+ T cells to control the viral replication post-ART cessation.

  18. A population of human brain cells expressing phenotypic markers of more than one lineage can be induced in vitro to differentiate into mesenchymal cells

    International Nuclear Information System (INIS)

    Rieske, Piotr; Augelli, Brian J.; Stawski, Robert; Gaughan, John; Azizi, S. Ausim; Krynska, Barbara

    2009-01-01

    Proliferating astrocytic cells from germinal, as well as mature areas of brain parenchyma, have the characteristics of neural stem/progenitor cells and are capable of generating both neurons and glia. We previously reported that primary fetal human brain cells, designated as Normal Human Astrocytes (NHA), expressed, in addition to GFAP, Vimentin and Nestin, low levels of βIII-Tubulin, an early neuronal marker, and differentiated into neurons and astrocytes in vitro. Here, we showed that primary NHA cells co-express low levels of mesenchymal markers Fibronectin and Collagen-1 in culture. These cells transitioned into mesenchymal-like cells when cultured in adherent conditions in serum containing media. The mesenchymal-like derivatives of these cells were characterized based on their morphological changes, high expression of Vimentin and extracellular matrix (ECM) proteins, Collagen-1 and Fibronectin, and decline of neural markers. When incubated in osteogenic and adipogenic induction media, the mesenchymal-like cells differentiated into osteoblasts and adipocytes. Furthermore, NHA cells express markers of neural crest cells, SOX-10 and p75. These data support the idea of ectoderm-derived mesenchymal lineages. These findings suggest that a population of primitive fetal brain cells with neural/neural crest/mesenchymal phenotype, resembles the remarkable phenotypic plasticity of neural crest cells, and differentiates into adipocytes and osteocytes under the influence of environmental factors

  19. High-resolution phenotypic profiling of natural products-induced effects on the single-cell level

    KAUST Repository

    Kremb, Stephan Georg

    2017-03-15

    Natural products (NPs) are highly evolved molecules making them a valuable resource for new therapeutics. Here we demonstrate the usefulness of broad-spectrum phenotypic profiling of NP-induced perturbations on single cells with imaging-based High-Content Screening to inform on physiology, mechanisms-of-actions, and multi-level toxicity. Our technology platform aims at broad applicability using a comprehensive marker panel with standardized settings streamlined towards an easy implementation in laboratories dedicated to natural products research.

  20. PAF-receptor is preferentially expressed in a distinct synthetic phenotype of smooth muscle cells cloned from human internal thoracic artery: Functional implications in cell migration

    International Nuclear Information System (INIS)

    Stengel, Dominique; O'Neil, Caroline; Brocheriou, Isabelle; Karabina, Sonia-Athina; Durand, Herve; Caplice, Noel M.; Pickering, J. Geoffrey; Ninio, Ewa

    2006-01-01

    Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation

  1. Clinical phenotypes and the biological parameters of Congolese patients suffering from sickle cell anemia: A first report from Central Africa.

    Science.gov (United States)

    Mikobi, Tite M; Lukusa Tshilobo, Prosper; Aloni, Michel N; Akilimali, Pierre Z; Mvumbi-Lelo, Georges; Mbuyi-Muamba, Jean Marie

    2017-11-01

    The influence of phenotype on the clinical course and laboratory features of sickle cell anemia (SCA) is rarely described in sub-Saharan Africa. A cross-sectional study was conducted in Kinshasa. A clinical phenotype score was built up. The following definitions were applied: asymptomatic clinical phenotype (ACP; score≤5), moderate clinical phenotype (MCP; score between 6 and 15), and severe clinical phenotype (SCP; score≥16). ANOVA test were used to compare differences among categorical variables. We have studied 140 patients. The mean body mass index (BMI) value of three groups was lower (Sickle cell patients with ACP have a high mean steady-state hemoglobin concentration compared to those with MCP and SCP (Psickle cell anemia clinical and biological variability in our midst. © 2017 Wiley Periodicals, Inc.

  2. Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line.

    Science.gov (United States)

    Vassbotn, F S; Ostman, A; Langeland, N; Holmsen, H; Westermark, B; Heldin, C H; Nistér, M

    1994-02-01

    Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

  3. Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

    Directory of Open Access Journals (Sweden)

    Akira Shimamoto

    Full Text Available Werner syndrome (WS is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs. In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

  4. 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.

  5. Reprogramming suppresses premature senescence phenotypes of Werner syndrome cells and maintains chromosomal stability over long-term culture.

    Science.gov (United States)

    Shimamoto, Akira; Kagawa, Harunobu; Zensho, Kazumasa; Sera, Yukihiro; Kazuki, Yasuhiro; Osaki, Mitsuhiko; Oshimura, Mitsuo; Ishigaki, Yasuhito; Hamasaki, Kanya; Kodama, Yoshiaki; Yuasa, Shinsuke; Fukuda, Keiichi; Hirashima, Kyotaro; Seimiya, Hiroyuki; Koyama, Hirofumi; Shimizu, Takahiko; Takemoto, Minoru; Yokote, Koutaro; Goto, Makoto; Tahara, Hidetoshi

    2014-01-01

    Werner syndrome (WS) is a premature aging disorder characterized by chromosomal instability and cancer predisposition. Mutations in WRN are responsible for the disease and cause telomere dysfunction, resulting in accelerated aging. Recent studies have revealed that cells from WS patients can be successfully reprogrammed into induced pluripotent stem cells (iPSCs). In the present study, we describe the effects of long-term culture on WS iPSCs, which acquired and maintained infinite proliferative potential for self-renewal over 2 years. After long-term cultures, WS iPSCs exhibited stable undifferentiated states and differentiation capacity, and premature upregulation of senescence-associated genes in WS cells was completely suppressed in WS iPSCs despite WRN deficiency. WS iPSCs also showed recapitulation of the phenotypes during differentiation. Furthermore, karyotype analysis indicated that WS iPSCs were stable, and half of the descendant clones had chromosomal profiles that were similar to those of parental cells. These unexpected properties might be achieved by induced expression of endogenous telomerase gene during reprogramming, which trigger telomerase reactivation leading to suppression of both replicative senescence and telomere dysfunction in WS cells. These findings demonstrated that reprogramming suppressed premature senescence phenotypes in WS cells and WS iPSCs could lead to chromosomal stability over the long term. WS iPSCs will provide opportunities to identify affected lineages in WS and to develop a new strategy for the treatment of WS.

  6. Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas.

    Science.gov (United States)

    Arai, Eri; Gotoh, Masahiro; Tian, Ying; Sakamoto, Hiromi; Ono, Masaya; Matsuda, Akio; Takahashi, Yoriko; Miyata, Sayaka; Totsuka, Hirohiko; Chiku, Suenori; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Matsumoto, Kenji; Yamada, Tesshi; Yoshida, Teruhiko; Kanai, Yae

    2015-12-01

    CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p = 1.427 × 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10(-6))" and "Spindle assembly and chromosome separation (p = 9.260 × 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs.

  7. HIV infection is associated with preservation of MAIT cells in the lungs but alteration of their phenotype and T cell receptor repertoire

    DEFF Research Database (Denmark)

    Wong, E. B.; Xulu, B.; Prakadan, S.

    2016-01-01

    Tuberculosis remains the leading cause of death in HIV-positive people. A better understanding of the impact of HIV on lung immunity may lead to novel immunotherapeutic interventions. MAIT cells are tissue-homing donor-unrestricted T cells with broad anti-microbial activity. HIV infection causes ...... to determine the mechanisms underlying the altered phenotypes of lung-resident MAITs and whether these can be targeted to improve anti-microbial lung immunity in people living with HIV....

  8. B-1a transitional cells are phenotypically distinct and are lacking in mice deficient in IκBNS

    Science.gov (United States)

    Pedersen, Gabriel K.; Àdori, Monika; Khoenkhoen, Sharesta; Dosenovic, Pia; Beutler, Bruce; Karlsson Hedestam, Gunilla B.

    2014-01-01

    B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93+IgM+CD5+) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93+IgM+CD5− cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS. PMID:25228759

  9. Dental Pulp Cells Isolated from Teeth with Superficial Caries Retain an Inflammatory Phenotype and Display an Enhanced Matrix Mineralization Potential

    Directory of Open Access Journals (Sweden)

    Reem El-Gendy

    2017-04-01

    Full Text Available We have isolated dental pulp cells (DPCs from three healthy (hDPCs and three carious (cDPCs donors and shown that compared to hDPCs cells isolated from superficial carious lesions show higher clonogenic potential; show an equivalent proportion of cells with putative stem cell surface markers; show enhanced matrix mineralization capability; have enhanced angiogenic marker expression and retain the inflammatory phenotype in vitro characteristic of superficial caries lesions in vivo. Our findings suggest that cDPCs may be used for further investigation of the cross talk between inflammatory, angiogenic and mineralization pathways in repair of carious pulp. In addition cells derived from carious pulps (almost always discarded may have potential for future applications in mineralized tissue repair and regeneration.

  10. Novel effector phenotype of Tim-3+ regulatory T cells leads to enhanced suppressive function in head and neck cancer patients.

    Science.gov (United States)

    Liu, Zhuqing; McMichael, Elizabeth L; Shayan, Gulidanna; Li, Jing; Chen, Kevin; Srivastava, Raghvendra M; Kane, Lawrence P; Lu, Binfeng; Ferris, Robert L

    2018-04-30

    Regulatory T (Treg) cells are important suppressive cells among tumor infiltrating lymphocytes (TIL). Treg express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear. CD4+CTLA-4+CD25high Treg were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3+ and Tim-3- TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene expression profiling and NanoString analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg.  Results: Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit naïve T cell proliferation than Tim-3- Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39 and IFN-γ receptor. Exogenous IFN-γ treatment could partially reverse the suppressive function of Tim-3+ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs. Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T cell proliferation despite high PD-1 expression.  IFN-γ induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. Copyright ©2018, American Association for Cancer Research.

  11. Phenotypic and Functional Changes Induced in Hematopoietic Stem/Progenitor Cells After Gamma-Ray Radiation Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D. [Institute of Emerging Diseases and Innovative Therapies, Functional Bioengineering Laboratory, Commissariat a l' Energie Atomique (CEA), Evry (France); Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D. [Institut National de la Sante et de la Recherche Medicale (INSERM) U733 (Unite Mixte de Recherche) - UMR INSERM CEA Paris XI (France); Vaigot, P. [Institute of Cellular and Molecular Radiation Biology, Department of Genetic Instability, Recombination and Repair, Commissariat a l' Energie Atomique, Fontenay-aux-Roses (France); Vaigot, P. [UMR 217, UMR-CEA-Centre National de la Recherche Scientifique (France); Barroca, V. [Laboratory of Gametogenesis, Apoptosis, Genotoxicity, Institute of Cellular and Molecular Radiation Biology, Commissariat a l' Energie Atomique, Fontenay-aux-Roses (France); Barroca, V. [Institut National de la Sante et de la Recherche Medicale U566 - UMR INSERM-CEA-PARIS VII (France); Leboulch, Ph. [Genetics Division, Brigham and Women' s Hospital and Harvard Medical School, Boston, Massachusetts (US)

    2009-07-01

    Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding 'side population (SP)' early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SPSK cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit{sup +}Sca-1{sup +}Lin{sup -/low} (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150{sup +}Flk2{sup -} and CD150{sup -}Flk2{sup +} cells, respectively. CD150{sup +} KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not age matched controls, allowed engraftment and significant hematopoietic contribution from transplanted con-genic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice. (authors)

  12. Phenotypic and Functional Changes Induced in Hematopoietic Stem/Progenitor Cells After Gamma-Ray Radiation Exposure

    International Nuclear Information System (INIS)

    Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D.; Simonnet, A.J.; Nehme, J.; Leboulch, Ph.; Tronik-Le Roux, D.; Vaigot, P.; Vaigot, P.; Barroca, V.; Barroca, V.; Leboulch, Ph.

    2009-01-01

    Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding 'side population (SP)' early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SPSK cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit + Sca-1 + Lin -/low (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150 + Flk2 - and CD150 - Flk2 + cells, respectively. CD150 + KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not age matched controls, allowed engraftment and significant hematopoietic contribution from transplanted con-genic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice. (authors)

  13. Expression dynamics of self-renewal factors for spermatogonial stem cells in the mouse testis.

    Science.gov (United States)

    Sakai, Mizuki; Masaki, Kaito; Aiba, Shota; Tone, Masaaki; Takashima, Seiji

    2018-04-16

    Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are bona fide self-renewal factors for spermatogonial stem cells (SSCs). Although GDNF is indispensable for the maintenance of SSCs, the role of FGF2 in the testis remains to be elucidated. To clarify this, the expression dynamics and regulatory mechanisms of Fgf2 and Gdnf in the mouse testes were analyzed. It is well known that Sertoli cells express Gdnf, and its receptor is expressed in a subset of undifferentiated spermatogonia, including SSCs. However, we found that Fgf2 was mainly expressed in the germ cells and its receptors were expressed not only in the cultured spermatogonial cell line, but also in testicular somatic cells. Aging, hypophysectomy, retinoic acid treatment, and testicular injury induced distinct Fgf2 and Gdnf expression dynamics, suggesting a difference in the expression mechanism of Fgf2 and Gdnf in the testis. Such differences might cause a dynamic fluctuation of Gdnf/Fgf2 ratio depending on the intrinsic/extrinsic cues. Considering that FGF2-cultured spermatogonia exhibit more differentiated phenotype than those cultured with GDNF, FGF2 might play a role distinct from that of GDNF in the testis, despite the fact that both factors are self-renewal factor for SSC in vitro.

  14. Phenotypical and functional characterization of double-negative (CD4-CD8-) alpha beta T-cell receptor positive cells from an immunodeficient patient

    DEFF Research Database (Denmark)

    Illum, N; Ralfkiaer, E; Pallesen, G

    1991-01-01

    We have characterized CD4-CD8- double-negative (DN) alpha beta TCR+ T cells from a patient with immunodeficiency, lymphocytosis, lymphadenopathy, and hepatosplenomegaly. The majority of peripheral blood lymphocytes were DN alpha beta TCR+ T cells as evaluated by FACS and biochemical analysis...... (MoAbs) indicated a polyclonal T-cell expansion. Thymic biopsy showed normal histology, whereas lymph node biopsy samples showed altered histological and immunohistological patterns with markedly expanded paracortical areas containing the DN T cells of the same phenotype as found in peripheral blood T...

  15. Phenotype and cell proliferation activity of duct-like structures in human sublingual glands: a histological and immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Elen de Souza TOLENTINO

    2015-06-01

    Full Text Available There are several age-related microscopic changes in the salivary glands, including the increase in the number of duct-like structures (DLS. However, the true origin and the phenotype of the DLS are not known. Objective To evaluate the phenotype and the cell proliferation index of the DLS of human sublingual glands. Material and Methods Sixty sublingual glands obtained from human cadavers were divided into two groups - 0-30 and 61-90 years old. The phenotype was estimated by immunostaining for cytokeratin 19 (CK 19 and the S-100 protein as well as by the presence of mucin and glycogen. The cell proliferation index was determined by the Ki-67 antibody. The histochemical techniques used periodic acid-Schiff (PAS and Alcian Blue. In each captured microscopic field, the DLS were counted to establish a percentage for the staining profile. The statistical analysis was accomplished using Student's t-test, the Mann-Whitney test and Pearson's correlation coefficient (p<0.05. Results Comparing both groups, only CK 19 showed a statistically significant difference (p=0.033, with the strongest expression in the elderly group. There was no significant difference between PAS and Alcian Blue (p=0.270. In both groups, the immunostaining for CK 19 was stronger than that for S-100 (p=0.004;p<0.001, but there was no correlation between the two immunomarkers (ρ=-0.163; p=0.315. There was no immunostaining for Ki-67. Conclusions DLS demonstrate a ductal phenotypic profile and do not present cell proliferation activity. DLS may represent a regressive process arising from acini or represent the result of metaplasia.

  16. A dynamical model for plant cell wall architecture formation.

    NARCIS (Netherlands)

    Mulder, B.M.; Emons, A.M.C.

    2001-01-01

    We discuss a dynamical mathematical model to explain cell wall architecture in plant cells. The highly regular textures observed in cell walls reflect the spatial organisation of the cellulose microfibrils (CMFs), the most important structural component of cell walls. Based on a geometrical theory

  17. Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

    Energy Technology Data Exchange (ETDEWEB)

    Sheard, Michael A., E-mail: msheard@chla.usc.edu [Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Ghent, Matthew V., E-mail: mattghent@gmail.com [Department of Pathology, Keck School of Medicine, University of Southern California, Health Sciences Campus, Los Angeles, CA 90089 (United States); Cabral, Daniel J., E-mail: dcabral14@gmail.com [Cancer Center and Departments of Cell Biology & Biochemistry, Pharmacology & Neuroscience, Internal Medicine and Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (United States); Lee, Joanne C., E-mail: joannebarnhart@gmail.com [Cancer Center and Departments of Cell Biology & Biochemistry, Pharmacology & Neuroscience, Internal Medicine and Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (United States); Khankaldyyan, Vazgen, E-mail: khangaldian@yahoo.com [Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Ji, Lingyun, E-mail: lingyun.ji@med.usc.edu [Developmental Therapeutics Program, USC-CHLA Institute for Pediatric Clinical Research, Division of Hematology-Oncology, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Wu, Samuel Q., E-mail: swu@chla.usc.edu [Medical Genetics, Children' s Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027 (United States); Kang, Min H., E-mail: min.kang@ttuhsc.edu [Cancer Center and Departments of Cell Biology & Biochemistry, Pharmacology & Neuroscience, Internal Medicine and Pediatrics, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430 (United States); and others

    2015-05-15

    Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo. - Highlights: • Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function. • Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death. • The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent.

  18. Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

    International Nuclear Information System (INIS)

    Sheard, Michael A.; Ghent, Matthew V.; Cabral, Daniel J.; Lee, Joanne C.; Khankaldyyan, Vazgen; Ji, Lingyun; Wu, Samuel Q.; Kang, Min H.

    2015-01-01

    Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival, expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo. - Highlights: • Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function. • Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death. • The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent

  19. Human Adult Stem Cells Maintain a Constant Phenotype Profile Irrespective of Their Origin, Basal Media, and Long Term Cultures

    Directory of Open Access Journals (Sweden)

    Indumathi Somasundaram

    2015-01-01

    Full Text Available The study aims to identify the phenotypic marker expressions of different human adult stem cells derived from, namely, bone marrow, subcutaneous fat, and omentum fat, cultured in different media, namely, DMEM-Low Glucose, Alpha-MEM, DMEM-F12 and DMEM-KO and under long term culture conditions (>P20. We characterized immunophenotype by using various hematopoietic, mesenchymal, endothelial markers, and cell adhesion molecules in the long term cultures (Passages-P1, P3, P5, P9, P12, P15, and P20. Interestingly, data revealed similar marker expression profiles irrespective of source, basal media, and extensive culturing. This demonstrates that all adult stem cell sources mentioned in this study share similar phenotypic marker and all media seem appropriate for culturing these sources. However, a disparity was observed in the markers such as CD49d, CD54, CD117, CD29, and CD106, thereby warranting further research on these markers. Besides the aforesaid objective, it is understood from the study that immunophenotyping acts as a valu